The ultrafast laser direct writing 3D lithography is presented by covering its physical and technological working principles, current state-of-the-art and potential for advanced (nano-)printing of diverse materials ranging from biocompatible, biodegradable and renewable organics to amorphous, ceramic and crystalline inorganics.

Objective The objective of this study was to assess a novel 3D microstructured scaffold seeded with allogeneic chondrocytes (cells) in a rabbit osteochondral defect model. Design Direct laser writing lithography in pre-polymers was employed to fabricate custom silicon-zirconium containing hybrid organic-inorganic (HOI) polymer SZ2080 scaffolds of a predefined morphology. Hexagon-pored HOI scaffolds were seeded with chondrocytes (cells), and tissue-engineered cartilage biocompatibility, potency, efficacy, and shelf-life in vitro was assessed by morphological, ELISA (enzyme-linked immunosorbent assay) and PCR (polymerase chain reaction) analysis. Osteochondral defect was created in the weight-bearing area of medial femoral condyle for in vivo study. Polymerized fibrin was added to every defect of 5 experimental groups. Cartilage repair was analyzed after 6 months using macroscopical (Oswestry Arthroscopy Score [OAS]), histological, and electromechanical quantitative potential (QP) scores. Collagen scaffold (CS) was used as a positive comparator for in vitro and in vivo studies. Results Type II collagen gene upregulation and protein secretion was maintained up to 8 days in seeded HOI. In vivo analysis revealed improvement in all scaffold treatment groups. For the first time, electromechanical properties of a cellular-based scaffold were analyzed in a preclinical study. Cell addition did not enhance OAS but improved histological and QP scores in HOI groups. Conclusions HOI material is biocompatible for up to 8 days in vitro and is supportive of cartilage formation at 6 months in vivo. Electromechanical measurement offers a reliable quality assessment of repaired cartilage.

An ultrafast laser assisted mesoscale lithography will be introduced by presenting its technological principles, current state-of-the-art and potential in 3D printing of diverse materials ranging from biocompatible, biodegradable and renewable organics to amorphous, ceramic and crystalline inorganics. Its applications towards prototyping and producing bio-medical implants, micro-optics and nano-photonics as well as creating micro-fluidic sensors will be shown. A special emphasis on the development and applications of microfabricated structures for life-sciences will be given, namely customization of laser direct write lithography-made 3D scaffolds for optimized in vivo outcome. Furthermore, the possibility to employ the technique for precision additive manufacturing out of plant-based resins and pure inorganics will be demonstrated. Finally, some unique functional properties of selected prototypes will be provided in detail validating their high efficiency performance.

In this talk we will present results on stimuli-responsive reversible deformations in polymeric microstructures fabricated using direct laser writing in pre-polymers (DLW-PP) technique. This microstructure behavior can be employed for micro-actuation applications in MEMS as well as designing new passive chemical sensors for microfluidic applications via hybrid (additive-subtractive) microfabrication technique. In this talk we will present our recent results in this field introducing few new concepts: a novel all-optical readout chemical sensor suitable for microfluidic applications, micromechanical components acting as flow control mechanisms based on bi-polymeric structures and solvent-sensitive micromechanical plug for microchannels.

INTRODUCTION:In complex clinical conditions when physiological bone regeneration is insufficient, there is a need to develop synthetic material-based scaffolds. The morphologic properties of porous scaffolds are of crucial importance. The dimensional accuracy of 3D printed scaffolds can be affected by a variety of factors. MATERIALS AND METHODS:Three groups of 3D printed scaffolds were investigated: PLA1 (pure polylactic acid) printed with an FDM Ultimaker Original printer, PLA2 and composite PLA/hydroxyapatite (PLA/HAp) scaffolds printed with a Pharaoh XD 20. PLA/HAp filament was created with hot-melt extrusion (HME) equipment. The morphology of the prepared scaffolds was investigated with SEM, micro-CT and superimposition techniques, gravimetric and liquid displacement methods. RESULTS:Layer heights of PLA1 scaffolds varied the most. PLA1 scaffold volume statistically significantly differed from PLA2 (p < 0.001) and PLA/HAp (p < 0.01) groups. Filament composition had no effect on the volumes of the scaffolds printed with the Pharaoh XD 20 printer (p > 0.05). The total porosity of printed PLA/HAp scaffolds deviated the least from the original STL model. CONCLUSIONS:This study showed that PLA/10% HAp filament fabricated with HME and printed with FFF 3D printer produced equal or even better accuracy of printed scaffolds than scaffolds printed with pure PLA filament. Further research is needed to analyze the effect of HAp on 3D scaffold morphology, accuracy, mechanical and biologic properties.

The investigation of biobased systems as photocurable resins for optical 3D printing has attracted great attention in recent years; therefore, novel vanillin acrylate-based resins were designed and investigated. Cross-linked polymers were prepared by radical photopolymerization of vanillin derivatives (vanillin dimethacrylate and vanillin diacrylate) using ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate as photoinitiator. The changes of rheological properties were examined during the curing with ultraviolet/visible irradiation to detect the influences of solvent, photoinitiator, and vanillin derivative on cross-linking rate and network formation. Vanillin diacrylate-based polymers had higher values of yield of insoluble fraction, thermal stability, and better mechanical properties in comparison to vanillin dimethacrylate-based polymers. Moreover, the vanillin diacrylate polymer film showed a significant antimicrobial effect, only a bit weaker than that of chitosan film. Thermal and mechanical properties of vanillin acrylate-based polymers were comparable with those of commercial petroleum-derived materials used in optical 3D printing. Also, vanillin diacrylate proved to be well-suited for optical printing as was demonstrated by employing direct laser writing 3D lithography and microtransfer molding techniques.

Materials obtained from renewable sources are emerging to replace the starting materials of petroleum-derived plastics. They offer easy processing, fulfill technological, functional and durability requirements at the same time ensuring increased bio-compatibility, recycling, and eventually lower cost. On the other hand, optical 3D printing (O3DP) is a rapid prototyping tool (and an additive manufacturing technique) being developed as a choice for efficient and low waste production method, yet currently associated with mainly petroleum-derived resins. Here we employ a single bio-based resin derived from soy beans, suitable for O3DP in the scales from nano- to macro-dimensions, which can be processed even without the addition of photoinitiator. The approach is validated using both state-of-the art laser nanolithography setup as well as a widespread table-top 3D printer - sub-micrometer accuracy 3D objects are fabricated reproducibly. Additionally, chess-like figures are made in an industrial line commercially delivering small batch production services. Such concept is believed to make a breakthrough in rapid prototyping by switching the focus of O3DP to bio-based resins instead of being restricted to conventional petroleum-derived photopolymers.

Direct laser writing 3D lithography in pre-polymers was employed to microstructure custom 3D silicon-zirconium hybrid organic-inorganic polymer SZ2080 scaffolds (HOI) of varying morphology for cartilage repair in a preclinical xenogeneic model. Scaffolds were fabricated to contain tetragonal and hexagonal pores, followed by pore scaling of 1.5 and 2 times. HOI scaffolds were seeded with human chondrocytes (cells) and biocompatibility was analyzed in vitro. Tissue engineered cartilage (TEC) potency, efficacy and shelf-life in vitro was assessed by morphological, biomechanical, metabolic activity, cell count, ELISA and PCR analysis. Optimal HOI scaffold was implanted in a long-term preclinical osteochondral defect of immunodeficient rat model and analyzed for the translated efficacy in experimental groups. Collagen scaffold was a positive comparator for in vitro and in vivo studies. Treatment efficacy was evaluated after 3 months using standardized macroscopical and histological scores. Biocompatibility was superior in tetragon-pored scaffold (HOI-T) compared to hexagon-pored HOI in vitro. Cartilage tissue formation in HOI with tetragonal pores scaled 1.5 times was comparable to HOI-T at least for up to 7 days in vitro. HOI-T with and without cells improved cartilage repair and were comparable to collagen scaffold in vivo at 3-months follow-up.

In this Letter, we report on design and realization of solvent-sensitive microstructures based on three-dimensional periodic lattices fabricated in a polymeric photoresist. Sensing is based on reversible size change in polymeric microstructures upon immersion in wetting and non-wetting solvents. Its readout is achieved purely optically by observing modification of a Moiré pattern formed by grating-like deformable and rigid polymeric structures. A compact micro-sensor using these principles was realized using a direct laser writing technique in the photoresist. High sensitivity and easy optical readout of the sensor were demonstrated. In the future, sensors based on similar principles may find applications in microfluidic systems, such as lab-on-a-chip.

Background More than 2 million bone transplant procedures are carried out each year, making bone the second most commonly transplanted tissue in the world. 3D structured and individually fabricated bone scaffolds are promising treatment strategy, which would allow to shorten the time and lower the extent of the surgery. Cell-laid mineralised extracellular matrix (ECM) was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Aim/Hypothesis 3D printed PLA HAP and their decellularized scaffolds have a potential to be used as graft substitutes in bone tissue regeneration. Material and Methods There were 6 groups in the study (n = 8 gp)- negative control, Geistlich Bio-Oss®, pure polylactic acid (PLA), PLA hydroxyapatite (HAP), PLA HAP cellularized with dental pulp stem cells and their decellularized scaffolds. Scaffolds were fabricated using FFF 3D printer. The filament for printing was produced by Filabot extruder system. Dental pulp stem cells were isolated from dental pulp of incisors of adult Wistar rats. All materials were implanted in critical-size Wistar rat's calvarial defect model in vivo to evaluate materials’ osteoregenerative potential. The defects were evaluated by micro-computed tomography and histological analysis eight weeks after surgery. All procedures were approved by License of Animal Research Ethics Committee No G2-40, 2016-03-18. Shapiro-Wilk test was used to test for normality in groups. For normally distributed data parametric statistics data analysis methods were used and for non-normally distributed data – nonparametric. Results Histometric measurements showed that 3D printed PLA scaffolds had more pronounced inflammation reaction during biodegradation, however scaffolds with HAP showed appropriate inflammatory responses. Micro-CT results showed no significant difference between different scaffold groups (P > 0.05), however PLA scaffolds displayed poorer results (2.63 ± 1.28 mm3) in new bone formation. Nevertheless, decellularized PLA HAP scaffolds had more pronounced osteoregenerative potential (4.05 ± 1.48 mm3) compared to other experimental groups, close to Geistlich Bio-Oss® results (4.04 ± 0.44 mm3). There was a significant difference between the (P < 0.05) gender groups in PLA and PLA HAP cellularized with dental pulp stem cells groups. Conclusion and Clinical Implications Within the limits of this study we concluded that 3D printed scaffolds with HAP improve biodegradation. Their decellularized scaffolds have great promise within the field of tissue engineering and may be used as graft substitutes in reconstructive surgery.

In this work we reveal an influence of polarization of the laser beam on polymerization in direct laser writing. It was experimentally found that the width of suspended lines fabricated in SZ2080, OrmoComp and PETA (pentaerythritol triacrylate) pre-polymers directly depends on the incident polarization and is largest when the angle between the electric field vector and the sample translation direction is alpha = 90 degrees and the smallest when alpha = 0 degrees. The size of polymerized structures is consistent with theoretical simulations based on vectorial Debye theory. Experiments were performed by using average laser power corresponding to the middle value of the fabrication window. Polarization was found to be affecting feature sizes while structuring various widespread photoresists, the observed variation was material dependent and measured from 5 to 22% in the line-width. The performed study proves that polarization can be used as a variable parameter for fine tuning of the voxel's aspect ratio.

3D meso-scale structures that can reach up to centimeters in overall size but retain micro- or nano-features, proved to be promising in various science fields ranging from micro-mechanical metamaterials to photonics and bio-medical scaffolds. In this work we present synchronization of the linear and galvano scanners for efficient femtosecond 3D optical printing of objects at the meso-scale (from sub-μm to sub-cm spanning five orders of magnitude). In such configuration the linear stages provide stitch-free structuring at nearly limitless (up to tens-of-cm) working area, while galvo-scanners allow to achieve translation velocities in the range of mm/s-cm/s without sacrificing nano-scale positioning accuracy and preserving undistorted shape of the final print. The principle behind this approach is demonstrated, proving its inherent advantages in comparison to separate use of only linear stages or scanners. The printing rate is calculated in terms voxels/s, showcasing the capability to maintain an optimal feature size while increasing throughput. Full capabilities of this approach are demonstrated by fabricating structures that reach millimeters in size but still retain μm-scale features: scaffolds for cell growth, microlenses and photonic crystals. All this is combined into a benchmark structure: a meso-butterfly. Provided results show that synchronization of two scan modes is crucial for the end goal of industrial-scale implementation of this technology and makes the laser printing well aligned with similar approaches in nanofabrication by electron and ion beams.

We present a way to utilize 3D polymeric microstructures, fabricated by direct laser writing in pre-polymers, for smart materials’ applications. The structures undergo swelling or shrinkage induced by interaction with liquids which can be converted into different types of deformations (elongation, bending, etc.) by careful choice of the 3D geometry. Several architectural designs of the polymeric structures are proposed for sensing and actuation applications. A non-uniform sensitivity of an object to the surrounding medium is achieved by tweaking the geometry of the structures. Also, a novel design of the chemical sensor based on 3D periodic lattice produced Moiré pattern imaging is presented.

Poly(glycerol sebacate) (PGS) has been utilised in numerous biomaterial applications over recent years. This elastomeric and rapidly degradable polymer is cytocompatible and suited to various applications in soft tissue engineering and drug delivery. Although PGS is simple to synthesise as an insoluble prepolymer, it requires the application of high temperatures for extended periods of time to produce an insoluble matrix. This places limitations on the processing capabilities of PGS and its possible applications. Here, we present a photocurable form of PGS with improved processing capabilities: PGS-methacrylate (PGS-M). By methacrylating the secondary hydroxyl groups of the glycerol units in the PGS prepolymer chains, the material was rendered photocurable and, in combination with a photoinitiator, crosslinked rapidly on exposure to UV light at ambient temperatures. The polymer’s molecular weight and the degree of methacrylation could be controlled independently and the mechanical properties of the crosslinked material tailored. The polymer also displayed rapid degradation under physiological conditions and cytocompatibility with various primary cell types. As a demonstration of the processing capabilities of PGS-M, µm scale 3D scaffold structures were fabricated using 2-photon polymerisation and used for 3D cell culture. The tunable properties of PGS-M coupled with its enhanced processing capabilities make the polymer an attractive potential biomaterial for various future applications.

We investigate reversible deformations of polymeric microstructures fabricated using direct laser writing three-dimensional lithography upon immersion in various solvents. Swelling and shrinkage of sub-micrometre size features are induced by interaction with surrounding solvent and such deformations can be exploited to create larger structures whose size, shape, and other structural parameters depend on the surroundings. We describe diffractive optical elements, micro-mechanical sensors and also hybrid deformable structures, that can be used to implement micro-actuation, micro-sensing, and other functionalities highly sought for micro-optical, micromechanical, and micro-fluidic systems.

We present the results of fabricating micro-structured macro-constructs for tissue engineering applications by using direct laser writing in pre-polymers (DLW-PP). The limiting factors for rapid construction of millimeter sized scaffolds, including the choice of material, software and hardware, as well as geometry, are discussed. We demonstrate that by using the hybrid inorganic-organic pre-polymer SZ2080 and employing a laser system which can combine the movement of linear motion stages with scanners, the structuring throughput reaching up to 51300 μm3/s can be achieved maintaining structural elements with 15 μm resolution. This allows to construct up to 30 scaffolds overnight with measurements reaching 1515×1515×195 μm3 – big enough for a surgeon to handle – and enables serial fabrication of such structures required for statistical tissue formation studies. Also, the versatility of DLW-PP technique is demonstrated by presenting the fabricated scaffolds with different pore shapes and sizes as well as manufactured out of pure and photo-sensitized SZ2080 material.

Summary form only given. A direct laser writing 3D nanolithography is a popular technique for fabrication of microstructures for a wide spectrum of scientific and industrial applications [1]. However, a systematic study of polarization control in light-assisted additive manufacturing of polymers is still limited. In glass patterning by nano-gratings, the polarisation control is essential for defining local form birefringence and patterning of optical anisotropy [2]. Here we reveal influence of polarisation on polymerisation in direct laser writing.

Direct laser writing in pre-polymers (DLW-PP) employing ultrashort pulses is a well-established technique for creating 3D microstructured scaffolds for in vitro tissue-engineering studies [1]. This is predetermined by the wide range of processable materials, full control of the 3D microarchitecture, scaling flexibility, and other characteristics. Despite all that, demonstrations of in vivo capabilities of DLW-PP manufactured scaffolds are still very scarce [2, 3]. This is mainly due to the point-by-point fabrication nature resulting in a relatively long manufacturing time of one structure, specifically if millimeter-sized objects which could be conveniently handled by a surgeon are needed. However, a growing interest in the development of this technique, especially for tissue engineering applications, suggests that it might one day become a commonly used method for the creation of patient-specific macro-scale microstructured scaffolds.

An approach employing ultrafast laser hybrid subtractive-additive microfabrication combining ablation, 3D nanolithography and welding is proposed for the realization of Lab-On-Chip (LOC) device. Single amplified Yb:KGW fs-pulsed laser source is shown to be suitable for fabricating microgrooves in glass slabs, polymerization of fine-meshes filter out of hybrid organic-inorganic photopolymer SZ2080 inside them, and, lastly, sealing the whole chip with cover glass into a single monolithic piece. The created microfluidic device proved its particle sorting function by separating 1 μm and 10 μm polystyrene spheres in a mixture. All together, this shows that fs-laser microfabrication technology is a flexible and versatile tool for the manufacturing of mesoscale multi-material LOC devices.

We present a novel hybrid glass-polymer micromechanical sensor by combining two femtosecond laser direct writing processes: laser illumination followed by chemical etching of glass and two-photon polymerization. This incorporation of techniques demonstrates the capability of combining mechanical deformable devices made of silica with an integrated polymer structure for passive chemical sensing application. We demonstrate that such a sensor could be utilized for investigating the elastic properties of polymeric microstructures fabricated via the two-photon polymerization technique. Moreover, we show that polymeric microstructure stiffness increases when immersed in organic liquids.