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Cartilage damage typically starts at its surface, either due to wear or trauma. Treatment of these superficial defects is important in preventing degradation and osteoarthritis (OA). Biomaterials currently used for deep cartilage defects lack appropriate properties for this application. Therefore, we investigated photo-crosslinked methacrylamide-modified gelatin (gelMA) as a candidate for treatment of surface defects. It allows for liquid application, filling of surface defects and forming a protective layer after UV-crosslinking, thereby keeping therapeutic cells in place. GelMA and photo-initiator (Li-TPO) concentration were optimized for application as a carrier to create a favourable environment for human articular chondrocytes (hAC). Primary hAC were used in passages 3 and 5, encapsulated into two different gelMA concentrations (7.5 wt% (soft) and 10 wt% (stiff)) and cultivated for 3 weeks with TGF-β3 (0, 1 and 10 ng/mL). Higher TGF-β3 concentrations induced spherical cell morphology independent of gelMA stiffness, while low TGF-β3 concentrations only induced rounded morphology in stiff gelMA. Gene expression did not vary across gel stiffnesses. As a functional model gelMA was loaded with two different cell types (hAC and/or human adipose-derived stem cells (ASC/TERT1) and applied to human osteochondral osteoarthritic plugs. GelMA attached to the cartilage, smoothened the surface and retained cells in place. Resistance against shear forces was tested using a tribometer, simulating normal human gait and revealing maintained cell viability. In conclusion gelMA is a versatile, biocompatible material with good bonding capabilities to cartilage matrix, allowing sealing and smoothening of superficial cartilage defects while simultaneously delivering therapeutic cells for tissue regeneration. This article is protected by copyright. All rights reserved.

Since its inception, tissue engineering and regenerative medicine (TERM) has been relying on either scaffold-based or scaffold-free strategies. Recent reports outlined the possibility of a synergistic, convergence approach, referred to as the third TERM strategy, which could alleviate bottlenecks of the two previous options. This strategy requires the fabrication of highly porous microscaffolds, allowing to create single spheroids within each of them. The resulting tissue units can then be combined and used as modular building blocks for creating tissue constructs through a bottom-up self-assembly. Such strategy can have a significant impact for the future of TERM, but so far, no reports have assessed its feasibility in detail. This work reports a first systematic study, which includes a comparison of the in vitro behavior of tissue units based on adipose derived stem cell spheroids cultured within microscaffolds versus conventional spheroids. We first proved that the presence of the microscaffold neither impairs the cells 'ability to form spheroids nor impacts their viability. Importantly, the fusiogenic and the differentiation potential (i.e. chondrogenesis and osteogenesis), which are important features for cellularized building blocks to be used in TERM, are preserved when spheroids are cultured within microscaffolds. Significant benefits of microscaffold-based tissue units include the enhanced cell retention, the decreased compaction and the better control over the size observed when larger tissue constructs are formed through self-assembly. The proof of concept study presented here demonstrates the great potential offered by those microsize tissue units to be used as building blocks for directed tissue self-assembly. STATEMENT OF SIGNIFICANCE: One of the most exciting and recent advances in tissue engineering and regenerative medicine (TERM) is to combine together multiple micro-size cellularized units, which are able to self-assemble altogether to recreate larger tissue constructs. In this work, we produce such modules by forming single spheroids within highly porous microscaffolds, and study how this new microenvironment impacts on the spheroid's behavior and stemness potential. This work highlights as well that such novel route is enabled by two-photon polymerization, which is an additive manufacturing technique offering high spatial resolution down to 100 nm. These findings provide a first scientific evidence about the utilization of hybrid spheroid microscaffold-based tissue units with great perspective as a modular tool for TERM.

‘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 µm 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.

Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl hydrogels hold an impressive track record. However, over the past decade, increasing attention is being paid to thiol-ene photo-click chemistry to obtain hydrogel networks benefitting from a faster reactivity (i.e. seconds vs minutes) along with superior biocompatibility and processability. In order to exploit this photo-click chemistry, often an ene-functionality (e.g.. Norbornene) is introduced onto gelatin followed by crosslinking in the presence of a multifunctional thiol (e.g.. DTT). To date, very limited research has been performed on the influence of the applied thiolated crosslinker on the final hydrogel properties. Therefore, the present work assesses the influence of different thiolated crosslinkers on the crosslinking kinetics, mechanical properties and biological performance of the hydrogels upon encapsulation of primary adipose tissue-derived stem cells in which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerisation which indicated, in addition to differences in processing window and swelling ratio, a previously unreported phenomenon. At high intensities (i.e. ≥ 150 mW), the laser results in cleavage of the gelatin backbone even in the absence of distinct photo-cleavable functionalities. This can have potential to introduce channels or softer regions in gels to result in zones characterized by different degradation speeds or the formation of blood vessels. Consequently, the present study can be used to provide guidance towards tailoring the thiol-ene system towards the desired applications. Creative Commons Attribution license.

In this work we present the design, synthesis and systematic investigation of the optical properties of symmetric triphenylamine (TPA)-substituted thiophenes. The use of electron-donating (–OMe, –tBu, –Me, –TMS), -neutral (–H) or -withdrawing (–F, –CN, –SO2Me) substituents gives rise to D–A–D based two-photon absorption (2PA) chromophores. The photophysical properties of these compounds, including one-photon absorption and 2PA using two-photon-excited fluorescence, were investigated in different organic solvents with varying polarity. The maximum 2PA cross sections prove to be strongly dependent on the nature of the TPA substituent and range between ∼173 GM (Goeppert-Mayer units) and 379 GM. Although most of the investigated substances also exhibit high fluorescence quantum yields, two-photon absorption screening tests of an acrylate monomer formulation revealed the efficiency of these materials as 2PA photoinitiators. These results are supported by quantum chemical calculations of the spin density distribution indicating that the mechanism of polymerization initiation using acrylate monomer is favored by strong localization of the unpaired electrons in the triplet state on the C2 carbon of the thiophene moiety.

How to pack materials into well-defined volumes efficiently has been a longstanding question of interest to physicists, material scientists, and mathematicians as these materials have broad applications ranging from shipping goods in commerce to seeds in agriculture and to spheroids in tissue engineering. How many marbles or gumball candies can you pack into a jar? Although these seem to be idle questions they have been studied for centuries and have recently become of greater interest with their broadening applications in science and medicine. Here, we study a similar problem where we try to pack cells into a spherical porous buckyball structure. The experimental limitations are short of the theoretical maximum packing density due to the microscale of the structures that the cells are being packed into. We show that we can pack more cells into a confined micro-structure (buckyball cage) by employing acoustofluidic activation and their hydrodynamic effect at the bottom of a liquid‐carrier chamber compared to randomly dropping cells onto these buckyballs by gravity. Although, in essence, cells would be expected to achieve a higher maximum volume fraction than marbles in a jar, given that they can squeeze and reshape and reorient their structure, the packing density of cells into the spherical buckyball cages are far from this theoretical limit. This is mainly dictated by the experimental limitations of cells washing away as well as being loaded into the chamber.

Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.

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.

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for biofabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs. STATEMENT OF SIGNIFICANCE: Gelatin and more specifically gelatin-methacryloyl has emerged to become one of the gold standard materials as an extracellular matrix mimic in the field of biofabrication. However, also other modification strategies have been elaborated to take advantage of a plethora of crosslinking chemistries. Therefore, a review paper focusing on the different modification strategies and processing of gelatin is presented. Particular attention is paid to the underlying chemistry along with the benefits and drawbacks of each type of crosslinking chemistry. The different strategies were classified based on their basic crosslinking mechanism including chain- or step-growth polymerization. Within the step-growth classification, a further distinction is made between click chemistries as well as other strategies. The influence of these modifications on the physical gelation and processing conditions including mechanical properties is presented. Additionally, substantial attention is put to the applied photoinitiators and the different biofabrication technologies including inkjet, deposition or light-based technologies.

Directed tissue self-assembly or bottom-up modular approach in tissue biofabrication is an attractive and potentially superior alternative to a classic top-down solid scaffold-based approach in tissue engineering. For example, rapidly emerging organ printing technology using self-assembling tissue spheroids as building blocks is enabling computer-aided robotic bioprinting of three-dimensional (3D) tissue constructs. However, achieving proper material properties while maintaining desirable geometry and shape of 3D bioprinted tissue engineered constructs using directed tissue self-assembly, is still a challenge. Proponents of directed tissue self-assembly see the solution of this problem in developing methods of accelerated tissue maturation and/or using sacrificial temporal supporting of removable hydrogels. In the meantime, there is a growing consensus that a third strategy based on the integration of a directed tissue self-assembly approach with a conventional solid scaffold-based approach could be a pot...

Cells respond to topographical, mechanical and biochemical characteristics of the surrounding environment. Capability to reconstruct these factors individually, and also acting in accord, would facilitate systematic investigations of a multitude of related biological and tissue engineering questions. The subject of the present review is a group of technologies allowing realization of customized cell-culture matrices. These methods utilize photochemistry induced by multiphoton absorption and are carried out using essentially identical equipment. Fabrication of 2D microstructured substrates, complex 3D scaffolds, containing actively induced topographies, and immobilization of biomolecules in a spatially defined manner was demonstrated with these techniques. The reviewed reports indicate that multiphoton processing is a promising technology platform for the development of standard biomimetic microenvironments for 3D cell culture.

Correction for ‘Towards efficient initiators for two-photon induced polymerization: fine tuning of the donor/acceptor properties’ by Brigitte Holzer et al., Mol. Syst. Des. Eng., 2019, DOI: 10.1039/c8me00101d.

In this study, a novel rapid prototyping technology was used to fabricate scaphoid and lunate bone prostheses, two carpal bones that are prone to avascular necrosis. Carpal prostheses were fabricated with an Envisiontec Perfactory SXGA stereolithography system using Envisiontec eShell 200 photocurable polymer. Fabrication was guided using 3-D models, which were generated using Mimics software (Materialise NV, Leuven, Belgium) from patient computer tomography data. The prostheses were fabricated in a layer-by-layer manner; approximately 50-microm thick layers were observed in the prostheses. Hardness and Young's modulus values of polymerized eShell 200 material were 93.8 +/- 7.25 MPa and 3050 +/- 90 MPa, respectively. The minimum compressive force required for fracture was 1360 N for the scaphoid prosthesis and 1248 N for the lunate prosthesis. Polymerized Envisiontec eShell material exhibited high human neonatal epidermal keratinocyte cell viability rate in an MTT assay. The results of this study indicate that small bone prostheses fabricated by stereolithography using eShell 200 polymer may have suitable geometry, mechanical properties, and cytocompatibility properties for in vivo use.

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young's modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

We report on recent advances in the fabrication of three-dimensional (3D) scaffolds for tissue engineering and regenerative medicine constructs using a two-photon polymerization technique (2PP). 2PP is a novel CAD/CAM technology allowing the fabrication of any computer-designed 3D structure from a photosensitive polymeric material. The flexibility of this technology and the ability to precisely define 3D construct geometry allows issues associated with vascularization and patient-specific tissue fabrication to be directly addressed. The fabrication of reproducible scaffold structures by 2PP is important for systematic studies of cellular processes and better understanding of in vitro tissue formation. In this study, 2PP was applied for the generation of 3D scaffold-like structures, using the photosensitive organic–inorganic hybrid polymer ORMOCER® (ORganically MOdified CERamics) and epoxy-based SU8 materials. By comparing the proliferation rates of cells grown on flat material surfaces and under control conditions, it was demonstrated that ORMOCER® and SU8 are not cytotoxic. Additional tests show that the DNA strand breaking of GFSHR-17 granulosa cells was not affected by the presence of ORMOCER®. Furthermore, gap junction conductance measurements revealed that ORMOCER® did not alter the formation of cell–cell junctions, critical for functional tissue growth. The possibilities of seeding 3D structures with cells were analysed. These studies demonstrate the great potential of 2PP technique for the manufacturing of scaffolds with controlled topology and properties. Copyright © 2008 John Wiley & Sons, Ltd.

Materials used in ossicular replacement prostheses must demonstrate appropriate biological compatibility, acoustic transmission, stability, and stiffness properties. Prostheses prepared using Teflon®, titanium, Ceravital and other conventional materials have demonstrated several problems, including migration, perforation of the tympanic membrane, difficulty in shaping the prostheses, and reactivity with the surrounding tissues. We have used two-photon polymerization for rapid prototyping of Ormocer® middle-ear bone replacement prostheses. Ormocer® surfaces fabricated using two-photon polymerization exhibited acceptable cell viability and cell growth profiles. The Ormocer® prosthesis was able to be inserted and removed from the site of use in the frozen human head without fracture. Our results demonstrate that two-photon polymerization is able to create ossicular replacement prostheses and other medical devices with a larger range of sizes, shapes and materials than other microfabrication techniques.

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.

Three-dimensional microstructured medical devices, including microneedles and tissue engineering scaffolds, were fabricated by two photon induced polymerization of Ormocer organic-inorganic hybrid materials. Femtosecond laser pulses from a titanium:sapphire laser were used to break chemical bonds on Irgacure 369 photoinitiator within a small focal volume. The radicalized starter molecules reacted with Ormocer US-S4 monomers to create radicalized polymolecules. The desired structures are fabricated by moving the laser focus in three dimensions using a galvano-scanner and a micropositioning system. Ormocer surfaces fabricated using two photon induced polymerization demonstrated acceptable cell viability and cell growth profiles against B35 neuroblast-like cells and HT1080 epithelial-like cells. Lego-like interlocking tissue engineering scaffolds and microneedle arrays with unique geometries were created using two photon induced polymerization. These results suggest that two photon induced polymerization is able to create medical microdevices with a larger range of sizes, shapes, and materials than chemical isotropic etching, injection molding, reactive ion etching, surface micromachining, bulk micromachining, polysilicon micromolding, lithography-electroforming-replication, or other conventional microfabrication techniques.

A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.

Hydrogels are extensively explored as scaffolding materials for 2D/3D cell culture and tissue engineering. Owing to the substantial complexity of tissues, it is increasingly important to develop 3D biomimetic hydrogels with user-defined architectures and controllable biological functions. To this end, one promising approach is to utilize photolithography-based additive manufacturing technologies (AMTs) in combination with photosensitive hydrogels. We here review recent advances in photolithography-based additive manufacturing of 3D hydrogels for tissue engineering applications. Given the importance of materials selection, we firstly give an overview of water-soluble photoinitiators for single- and two-photon polymerization, photopolymerizable hydrogel precursors and light-triggered chemistries for hydrogel formation. Through the text we discuss the design considerations of hydrogel precursors and synthetic approaches to polymerizable hydrogel precursors of synthetic and natural origins. Next, we shift to how photopolymerizable hydrogels could integrate with photolithography-based AMTs for creating well-defined hydrogel structures. We illustrate the working-principles of both single- and two-photon lithography and case studies of their applications in tissue engineering. In particular, two-photon lithography is highlighted as a powerful tool for 3D functionalization/construction of hydrogel constructs with μm-scale resolution. Within the text we also explain the chemical reactions involved in two-photon-induced biofunctionalization and polymerization. In the end, we summarize the limitations of available hydrogel systems and photolithography-based AMTs as well as a future outlook on potential optimizations.

Tissue engineering is a biomedical technology of artificial development of living three-dimensional human tissues and organs. Tissue engineering is based on two distinct premises. First more conventional approach uses solid biodegradable porous scaffolds as a temporal supporting framework for living cells attachment and sequential formation of three-dimensional tissue whereas second emerging approach is a solid scaffold-free directed tissue self-assembly with using tissue spheroids or microtissues as building blocks. In this paper novel hybrid approach or so-called third strategy in tissue engineering which combines advantages of first and second approaches is presented. The novel concept of lockyballs or tissue spheroids encaged into interlockable synthetic microscaffolds is described. Published version

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.

Hydrogels are polymeric materials with water contents similar to that of soft tissues. Due to their biomimetic properties, they have been extensively used in various biomedical applications including cell encapsulation for tissue engineering. The utilization of photopolymers provides a possibility for the temporal and spatial controlling of hydrogel cross-links. We produced three-dimensional (3-D) hydrogel scaffolds by means of the two-photon polymerization (2PP) technique. Using a highly efficient water-soluble initiator, photopolymers with up to 80 wt.% water were processed with high precision and reproducibility at a writing speed of 10 mm/s . The biocompatibility of the applied materials was verified using Caenorhabditis elegans as living test organisms. Furthermore, these living organisms were successfully embedded within a 200×200×35 μm 3 hydrogel scaffold. As most biologic tissues exhibit a window of transparency at the wavelength of the applied femtosecond laser, it is suggested that 2PP is promising for an in situ approach. Our results demonstrate the feasibility of and potential for bio-fabricating 3-D tissue constructs in the micrometre-range via near-infrared lasers in direct contact with a living organism.

Microneedles are small-scale devices that may be used for drug delivery and biosensing. In this study, the forces required for mechanical failure, the modes of mechanical failure, as well as the mechanisms for microneedle penetration into porcine skin were examined. Microneedles produced from the acrylate-based polymer e-Shell 200 using an indirect rapid prototyping approach involving two-photon polymerization and poly(dimethylsiloxane) micromolding were found to possess sufficient strength for penetration of porcine skin. The failure forces were an order of magnitude greater than the forces necessary for full insertion into the skin. Bending was the most common form of failure; an increasing aspect ratio and a decreasing tip diameter were associated with lower failure forces. Video captured during skin penetration revealed that microneedle penetration into the skin occurred by means of a series of insertions and not by means of a single insertion event. Images obtained during and after skin penetration confirmed microneedle penetration of skin as well as transdermal delivery of lucifer yellow dye. These findings shed insight into the mechanisms of microneedle penetration and failure, facilitating design improvements for polymer microneedles.

In the present work, 3D CAD scaffolds for tissue engineering applications were developed starting from methacrylamide-modified gelatin (GelMOD) using two-photon polymerization (2PP). The scaffolds were cross-linked employing the biocompatible photoinitiator Irgacure 2959. Because gelatin is derived from collagen (i.e., the main constituent of the ECM), the developed materials mimic the cellular microenvironment from a chemical point of view. In addition, by applying the 2PP technique, structural properties of the cellular microenvironment can also be mimicked. Furthermore, in vitro degradation assays indicated that the enzymatic degradation capability of gelatin is preserved for the methacrylamide-modified derivative. An in depth morphological analysis of the 2PP-fabricated scaffolds demonstrated that the parameters of the CAD model are reproduced with great precision, including the ridge-like surface topography on the order of 1.5 μm. The developed scaffolds showed an excellent stability in culture medium. In a final part of the present work, the suitability of the developed scaffolds for tissue engineering applications was verified. The results indicated that the applied materials are suitable to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Upon applying osteogenic stimulation, the seeded cells differentiated into the anticipated lineage. Energy dispersive X-ray (EDX) analysis showed the induced calcification of the scaffolds. The results clearly indicate that 2PP is capable of manufacturing precisely constructed 3D tissue engineering scaffolds using photosensitive polymers as starting material.

The natural cell environment is characterized by complex three-dimensional structures, which contain features at multiple length scales. Many in vitro studies of cell behavior in three dimensions rely on the availability of artificial scaffolds with controlled three-dimensional topologies. In this paper, we demonstrate fabrication of three-dimensional scaffolds for tissue engineering out of poly(ethylene glycol) diacrylate (PEGda) materials by means of two-photon polymerization (2PP). This laser nanostructuring approach offers unique possibilities for rapid manufacturing of three-dimensional structures with arbitrary geometries. The spatial resolution dependence on the applied irradiation parameters is investigated for two PEGda formulations, which are characterized by molecular weights of 302 and 742. We demonstrate that minimum feature sizes of 200 nm are obtained in both materials. In addition, an extensive study of the cytotoxicity of the material formulations with respect to photoinitiator type and photoinitiator concentration is undertaken. Aqueous extracts from photopolymerized PEGda samples indicate the presence of water-soluble molecules, which are toxic to fibroblasts. It is shown that sample aging in aqueous medium reduces the cytotoxicity of these extracts; this mechanism provides a route for biomedical applications of structures generated by 2PP microfabrication and photopolymerization technologies in general. Finally, a fully biocompatible combination of PEGda and a photoinitiator is identified. Fabrication of reproducible scaffold structures is very important for systematic investigation of cellular processes in three dimensions and for better understanding of in vitro tissue formation. The results of this work suggest that 2PP may be used to polymerize poly(ethylene glycol)-based materials into three-dimensional structures with well-defined geometries that mimic the physical and biological properties of native cell environments.

Microneedle devices for transdermal delivery of nanoscale pharmacologic agents were fabricated out of organically-modified ceramic (Ormocer) materials using two photon polymerization. Out-of-plane hollow microneedle arrays with various aspect ratios were fabricated using this rapid prototyping process. Human epidermal keratinocyte (HEK) viability on Ormocer surfaces fabricated using two photon polymerization was similar to that on control surfaces. Nanoindentation studies were performed to determine hardness and Young's modulus values for Ormocer materials. Microneedies were shown to enable more rapid distribution of the PEG-amine quantum dot solution to the deep epidermis and dermis layers of porcine skin than topical administration. Our results suggest that two photon polymerization may be used to create microneedle arrays for transdermal delivery of nanoscale pharmacologic agents.

One promising option for transdermal delivery of protein- and nucleic acid-based pharmacologic agents involves the use of microneedles. However, microneedle-generated pores may allow microorganisms to penetrate the stratum corneum layer of the epidermis and cause local or systemic infection. In this study, microneedles with antimicrobial functionality were fabricated using two photon polymerization-micromolding and pulsed laser deposition. The antibacterial activity of the silver-coated organically modified ceramic (Ormocer®) microneedles was demonstrated using an agar diffusion assay. Human epidermal keratinocyte (HEK) viability on the Ormocer® surfaces coated with silver was similar to that on uncoated Ormocer® surfaces. This study indicates that coating microneedles with silver thin films using pulsed laser deposition is a useful and novel approach for creating microneedles with antimicrobial functionality.

The use of microneedles for transdermal drug delivery is limited due to the risk of infection associated with formation of channels through the stratum corneum layer of the epidermis. The risk of infection associated with use of microneedles may be reduced by imparting these devices with antimicrobial properties. In this study, a photopolymerization-micromolding technique was used to fabricate microneedle arrays from a photosensitive material containing polyethylene glycol 600 diacrylate, gentamicin sulfate, and a photoinitiator. Scanning electron microscopy indicated that the photopolymerization-micromolding process produced microneedle arrays that exhibited good microneedle-to-microneedle uniformity. An agar plating assay revealed that microneedles fabricated with polyethylene glycol 600 diacrylate containing 2 mg mL(-1) gentamicin sulfate inhibited growth of Staphylococcus aureus bacteria. Scanning electron microscopy revealed no platelet aggregation on the surfaces of platelet rich plasma-exposed undoped polyethylene glycol 600 diacrylate microneedles and gentamicin-doped polyethylene glycol 600 diacrylate microneedles. These efforts will enable wider adoption of microneedles for transdermal delivery of pharmacologic agents.

Background: Microneedle-mediated drug delivery is a promising method for transdermal delivery of insulin, incretin mimetics, and other protein-based pharmacologic agents for treatment of diabetes mellitus. One factor that has limited clinical application of conventional microneedle technology is the poor fracture behavior of microneedles that are created using conventional materials and methods. In this study polymer microneedles for transdermal delivery were created using a two-photon polymerization (2PP) microfabrication and subsequent polydimethylsiloxane (PDMS) micromolding process. Methods: Solid microneedle arrays, fabricated by means of 2PP, were used to create negative molds from PDMS. Using these molds microneedle arrays were subsequently prepared by molding eShell 200, a photo-reactive acrylate-based polymer that exhibits water and perspiration resistance. Results: The eShell 200 microneedle array demonstrated suitable compressive strength for use in transdermal drug delivery applications. Human epidermal keratinocyte viability on the eShell 200 polymer surfaces was similar to that on polystyrene control surfaces. In vitro studies demonstrated that eShell 200 microneedle arrays fabricated using the 2PP microfabrication and PDMS micromolding process technique successfully penetrated human stratum corneum and epidermis. Conclusions: Our results suggest that a 2PP microfabrication and subsequent PDMS micromolding process may be used to create microneedle structures with appropriate structural, mechanical, and biological properties for transdermal drug delivery of insulin and other protein-based pharmacologic agents for treatment of diabetes mellitus.

In the present work, the two-photon polymerization (2PP) technique was applied to develop precisely defined biodegradable 3D tissue engineering scaffolds. The scaffolds were fabricated via photopolymerization of gelatin modified with methacrylamide moieties. The results indicate that the gelatin derivative (GelMod) preserves its enzymatic degradation capability after photopolymerization. In addition, the developed scaffolds using 2PP support primary adipose-derived stem cell (ASC) adhesion, proliferation and differentiation into the anticipated lineage.

Surface modification of vascular implants, like stents, using polymer coatings is an often employed and efficient method. Prevalent, a biodegradable polymer is applied as coating, which additionally functions as local drug delivery system. Handicap of nearly all body foreign polymeric surfaces are inflammatory response, granulation and low reendothelialization. To improve wound healing we modified the surface of the often used biodegradable polyester poly(e- caprolactone) (PCL). Therefore, we activated the surface by means of wet chemical and plasma chemical methods to create terminal amino groups. These groups were used to bind acetylsalicylic acid (ASS) as model drug to reduce inflammatory reactions. This surface loading functions as an additionally drug reservoir beside a possible incorporated bulk drug. Furthermore, the Vascular Endothelial Growth Factor (VEGF) was coupled to the activated surface to enhance a fast reendothelialization of implants which reduces risk of thrombosis, chronic inflammatory body response and granulation. The surface activation and modifications were analyzed by FTIR-ATR-spectroscopy and contact angle measurements. Additionally, amino groups were detected by fluorescence measurements after the reaction of the amino functionalized surfaces with 4-chloro-7-nitrobenzo-2-oxa-1,3- diazole (NBD-CL). The release of surface bonded ASS was analyzed by HPLC. Coupled VEGF was analyzed by using an indirect ELISA.

Two-photon polymerization technique was applied to generate three-dimensional (3D) scaffold-like structures using the photosensitive organic—inorganic hybrid polymer ORMOCER®. The structures were studied with respect to potential applications as scaffold for tissue engineering. Cell counting and comet assay, respectively, demonstrated that doubling time and DNA strand breaks of CHO cells, GFSHR-17 granulosa cells, GM-7373 endothelial cells, and SH-SY5Y neuroblastoma cells were not affected by ORMOCER®. ORMOCER®related alteration of formation of tissue specific cell-to-cell adhesions like gap junctions was ruled out by double whole-cell patch-clamp technique. Additionally, growth of cells on the vertical surfaces of 3D structures composed of ORMOCER®is shown.

Two-photon polymerization (2PP) is a novel technology for the fabrication of complex three-dimensional (3D) microstructures. The number of applications employing this technology is rapidly increasing, and includes the fabrication of three-dimensional photonic crystals [1-4], medical devices, and scaffolds for tissue engineering [5, 6]. We have used 2PP to fabricate microneedle arrays with various geometries. These devices provide a unique approach for transdermal delivery of nucleic acid- and protein-based pharmacologic agents. Many of issues associated with conventional intravenous drug administration, including pain to the patient, trauma at the injection site, and difficulty in providing sustained release of a pharmacological agent, may be eliminated by applying the microneedles. The effect of microneedle geometry (e.g., tip sharpness and aspect ratio) on skin penetration was examined. Our results indicate that microneedles created using 2PP technique are suitable for in vivo use, and integration with next generation MEMS- and NEMS-based drug delivery devices.

Investigations of two-photon polymerization (TPP) with sub-100 nm in the structuring resolution are presented by using photosensitive sol-gel material. The high photosensitivity of this material allows for TPP using a large variety in laser pulse durations covering a range between sub-10 fs and ≈140 fs. In this study, the authors demonstrate TPP structuring to obtain sub-100 nm in resolution by different approaches, namely, by adding a cross-linker to the material and polymerization with sub-10 fs short pulses. Additionally, a simulation and model based characterization method for periodic sub-100 nm structures was implemented and applied in an experimental white light interference Fourier-Scatterometry setup.

One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.