Virginija Bukelskiene

3D microporous scaffolds manufactured via combination of fused filament fabrication and direct laser writing ablation

A 3D printing fused filament fabrication (FFF) approach has been implemented for the creation of microstructures having an internal 3D microstructure geometry. These objects were produced without any sacrificial structures or additional support materials, just by precisely tuning the nozzle heating, fan cooling and translation velocity parameters. The manufactured microporous structures out of polylactic acid (PLA) had fully controllable porosity (20%–60%) and consisted of desired volume pores (~0.056 μm3). The prepared scaffolds showed biocompatibility and were suitable for the primary stem cell growth. In addition, direct laser writing (DLW) ablation was employed to modify the surfaces of the PLA structures, drill holes, as well as shape the outer geometries of the created objects. The proposed combination of FFF printing with DLW offers successful fabrication of 3D microporous structures with functionalization capabilities, such as the modification of surfaces, the generation of grooves and microholes and cutting out precisely shaped structures (micro-arrows, micro-gears). The produced structures could serve as biomedical templates for cell culturing, as well as biodegradable implants for tissue engineering. The additional micro-architecture is important in connection with the cell types used for the intention of cell growing. Moreover, we show that surface roughness can be modified at the nanoscale by immersion into an acetone bath, thus increasing the hydrophilicity. The approach is not limited to biomedical applications, it could be employed for the manufacturing of bioresorbable 3D microfluidic and micromechanic structures.

Micro-structured polymer scaffolds fabricated by direct laser writing for tissue engineering

This work presents the latest results on direct laser writing of polymeric materials for tissue engineering applications. A femtosecond Yb:KGW laser (300 fs, 200 kHz, 515 nm) was used as a light source for non-linear lithography. Fabrication was implemented in various photosensitive polymeric materials, such as: hybrid organic-inorganic sol-gel based on silicon-zirconium oxides, commercial ORMOCER® class photoresins. These materials were structured via multi-photon polymerization technique with submicron resolution. Porous three-dimensional scaffolds for artificial tissue engineering were fabricated with constructed system and were up to several millimeters in overall size with 10 to 100 μm internal pores. Biocompatibility of the used materials was tested in primary rabbit muscle-derived stem cell culture in vitro and using laboratory rats in vivo. This interdisciplinary study suggests that proposed technique and materials are suitable for tissue engineering applications.

Holographic lithography for biomedical applications

Fabrication of scaffolds for cell growth with appropriate mechanical characteristics is top-most important for successful creation of tissue. Due to ability of fast fabrication of periodic structures with a different period, the holographic lithography technique is a suitable tool for scaffolds fabrication. The scaffolds fabricated by holographic lithography can be used in various biomedical investigations such as the cellular adhesion, proliferation and viability. These investigations allow selection of the suitable material and geometry of scaffolds which can be used in creation of tissue. Scaffolds fabricated from di-acrylated poly(ethylene glycol) (PEG-DA-258) over a large area by holographic lithography technique are presented in this paper. The PEG-DA scaffolds fabricated by holographic lithography showed good cytocompatibility for rabbit myogenic stem cells. It was observed that adult rabbit muscle-derived myogenic stem cells grew onto PEG-DA scaffolds. They were attached to the pillars and formed cell-cell interactions. It demonstrates that the fabricated structures have potential to be an interconnection channel network for cell-to-cell interactions, flow transport of nutrients and metabolic waste as well as vascular capillary ingrowth. These results are encouraging for further development of holographic lithography by improving its efficiency for microstructuring three-dimensional scaffolds out of biodegradable hydrogels

Role of MAP kinases in nitric oxide induced muscle-derived adult stem cell apoptosis.

Apoptosis in heart failure has been intensively investigated in vitro and in vivo. Stem cells have therapeutic value in the direct treatment of diseases, including cardiovascular disease. The main drawback of stem cell therapy is their poor survival in the diseased tissues. Since intracellular mitogen‐activated protein kinases (MAPKs) actively participate in the regulation of cell survival and of proapoptotic signals, the ability to manipulate the mechanisms of MAPKs activation in myogenic stem cells might increase the survival of transplanted stem cells. Our results clearly demonstrate sustained activation of all three MAPKs, ERK, JNK and p38 in myogenic stem cells after exposure to the NO inducer, NOC‐18. Inhibition of MAPKs phosphorylation by specific inhibitors revealed the anti‐apoptotic role of MAPKs in myogenic stem cells.

Long‐term muscle‐derived cell culture: multipotency and susceptibility to cell death stimuli

Improvement in the yield of adult organism stem cells, and the ability to manage their differentiation and survival potential are the major goals in their application in regenerative medicine and in the adult stem cell research. We have demonstrated that adult rabbit muscle‐derived cell lines with an unlimited proliferative potential in vitro can differentiate into myogenic, osteogenic, adipogenic and neurogenic lineages. Studies of cell survival in vitro showed that differentiated cells, except neurogenic ones, are more resistant to apoptosis inducers compared to proliferating cells. Resistance to death signals correlated with the level of protein kinase AKT phosphorylation. Skeletal muscle‐derived cell lines can be multipurpose tools in therapy. Enhanced resistance of differentiated cells to certain types of damage shows their potential for long‐term survival and maintenance in an organism.

Lithographic microfabrication of biocompatible polymers for tissue engineering and lab-on-a-chip applications

In this work, a combination of Direct Laser Writing (DLW), PoliDiMethylSiloxane (PDMS) soft lithography and UV lithography was used to create cm- scale microstructured polymer scaolds for cell culture experiments out of dierent biocompatible materials: novel hybrid organic-inorganic SZ2080, PDMS elastomer, biodegradable PEG- DA-258 and SU-8. Rabbit muscle-derived stem cells were seeded on the fabricated dierent periodicity scaolds to evaluate if the relief surface had any eect on cell proliferation. An array of microlenses was fabricated using DLW out of SZ2080 and replicated in PDMS and PEG-DA-258, showing good potential applicability of the used techniques in many other elds like micro- and nano- uidics, photonics, and MicroElectroMechanical Systems (MEMS). The synergetic employment of three dierent fabrication techniques allowed to produce desired objects with low cost, high throughput and precision as well as use materials that are dicult to process by other means (PDMS and PEG-DA-258). DLW is a relatively slow fabrication method, since the object has to be written point-by-point. By applying PDMS soft lithography, we were enabled to replicate laser-fabricated scaolds for stem cell growth and micro-optical elements for lab-on-a-chip applications with high speed, low cost and good reproducible quality.

Direct laser fabrication of composite material 3D microstructured scaffolds

Summary form only given. We present direct laser fabrication of 3D microstructured scaffolds consisting out of a few polymeric materials owning different biological properties. Direct laser writing in photo/thermo-sensitive materials using ultra short light pulses of high repetition laser provides unmatched flexibility in controllable 3D microstructuring in a variety of bio-materials [1], as well as manufacturing throughput empowers overall structure size of more than 1 mm3, making it an attractive method to fabricate scaffolds for cell studies and tissue engineering applications [2, 3]. In this work, a femtosecond direct laser writing system was supplemented by a machine vision [4] to relocate the sample between different fabrication steps.

The effect of larger than cell diameter polylactic acid surface patterns on osteogenic differentiation of rat dental pulp stem cells: Effect of surface patterns on DPSC osteogenic differentiation

Topography of the scaffold is one of the most important factors defining the quality of artificial bone. However, the production of precise micro- and nano-structured scaffolds, which is known to enhance osteogenic differentiation, is expensive and time-consuming. Meanwhile, little is known about macro-patterns (larger than cell diameter) effect on cell fate, while this kind of structures would significantly facilitate the manufacturing of artificial skeleton. Therefore, this research is focused on polylactic acid scaffolds macro-pattern impact on rats dental pulp stem cells (DPSCs) morphology, proliferation, and osteogenic differentiation. For this study, two types of scaffolds were 3D printed: wavy and porous. Wavy scaffolds consisted of 188 μm wide joined threads, meaning that cells might have been curved on the filament as well as compressed in the groove. Porous scaffolds were designed to avoid groove formation and consisted of 500 μm threads, arranged in the woodpile manner, forming 300 μm diameter pores. We found that both macro-surfaces influenced DPSC morphology compared to control. As a consequence, enhanced DPSC proliferation and increased osteogenic differentiation potential was registered in cells grown on these scaffolds. Finally, our results showed that the construction of an artificial bone did not necessarily require the precise structuring of the scaffold, because both types of macro-topographic PLA scaffolds were sufficient enough to induce spontaneous DPSC osteogenic differentiation.

Laser microstructured 3D polymeric biocompatible implants

In this report we present experimental results on biocompatibility based on stem cell growth experiments in vitro as well as reaction of living organism to polymer implants in vivo of femtosecond laser 3D micro/nanostructurable photopolymers. A synergetic study on materials for rapid 3D scaffold fabrication having micrometer features and being centimeter in size, their biocompatibility in vitro and in vivo was done. The systematic study was performed providing consistent information which is important for further progress in cell growth and tissue engineering experiments. The chosen materials where of four different classes: well known biocompatible hybrid ORMOCER (Ormocore b59, Micro Resist) [1], widely used biodegradable di-acrylated poly(ethylene)glycol (PEG-DA-258, Sigma-Aldrich) [2], pure acrylate AKRE (SR368, Sartomer) [3] and novel high quality laser structurable material ORMOSIL (SZ2080, FORTH) [4]. All of the materials were evaluated by their suitability for femtosecond laser structuring, which is well established as a technique enabling rapid and flexible production of 3D micro/nanostructures. All photopolymers could be 3D structured with < 1 µm resolution and up to cm in overall sizes, thus materializing the computer models of the scaffolds with required pore sizes and porosities. The typical dimensions of scaffolds were 5 × 5 × 0.5 mm3 discs with 25 µm pore sizes and 40–60% porosity.