Laser Bioprinting Research Articles

Effect of laser bioprinting on growth characteristics of yeasts

Laser-induced forward transfer (LIFT) can be helpful for bioprinting of microbial cells, but data on its impact on the microbial physiology are scarce. The aim of study was to investigate the LIFT effect on eukaryotic microorganisms (yeasts) growth characteristics. Candida albicans, Lipomyces lipofer and Saitozyma podzolica yeasts were printed on solid and liquid nutrient media and compared with traditional methods of inoculation. The laser bioprinting affects growth patterns of yeasts, leading to formation of subpopulations with different growth characteristics. The data obtained highlight necessity of studies of the LIFT effect on microorganisms’ physiology.

Bioprinting with 3-µm laser pulses

Laser-Induced Forward Transfer (LIFT) technology has now found its wide application in the field of bioprinting. Laser bioprinting allows the spatial transfer of living micro-objects (cells, microorganisms) in a gel medium using the energy of a laser pulse. Typically, to effectively absorb laser pulse energy, a thin metal layer is deposited on the donor substrate. The disadvantages of this approach are the appearance of cytotoxic metal nanoparticles during laser bioprinting and the need for continuous movement of the donor substrate. The article proposes a method that allows moving to absorption film-free bioprinting using Q-switched Er:YAG nanosecond laser operating at a wavelength of 2.94 µm. This wavelength is effectively absorbed in a top ten-micron hydrogel layer, allowing for a cleaner (without nanoparticles from absorbing layer) and simpler (without the need to constantly move the donor substrate from pulse to pulse) laser bioprinting process. Using strain Escherichia coli as an example, the suitability of the proposed method for successful laser bioprinting is demonstrated. A model describing in detail the processes occurring in the hydrogel layer is presented.

Materials and Applications of 3D Printing Technology in Dentistry: An Overview.

This narrative review aims to provide an overview of the mechanisms of 3D printing, the dental materials relevant to each mechanism, and the possible applications of these materials within different areas of dentistry. Subtopics within 3D printing technology in dentistry were identified and divided among five reviewers. Electronic searches of the Medline (PubMed) database were performed with the following search keywords: 3D printing, digital light processing, stereolithography, digital dentistry, dental materials, and a combination of the keywords. For this review, only studies or review papers investigating 3D printing technology for dental or medical applications were included. Due to the nature of this review, no formal evidence-based quality assessment was performed, and the search was limited to the English language without further restrictions. A total of 64 articles were included. The significant applications, applied materials, limitations, and future directions of 3D printing technology were reviewed. Subtopics include the chronological evolution of 3D printing technology, the mechanisms of 3D printing technologies along with different printable materials with unique biomechanical properties, and the wide range of applications for 3D printing in dentistry. This review article gives an overview of the history and evolution of 3D printing technology, as well as its associated advantages and disadvantages. Current 3D printing technologies include stereolithography, digital light processing, fused deposition modeling, selective laser sintering/melting, photopolymer jetting, powder binder, and 3D laser bioprinting. The main categories of 3D printing materials are polymers, metals, and ceramics. Despite limitations in printing accuracy and quality, 3D printing technology is now able to offer us a wide variety of potential applications in different fields of dentistry, including prosthodontics, implantology, oral and maxillofacial, orthodontics, endodontics, and periodontics. Understanding the existing spectrum of 3D printing applications in dentistry will serve to further expand its use in the dental field. Three-dimensional printing technology has brought about a paradigm shift in the delivery of clinical care in medicine and dentistry. The clinical use of 3D printing has created versatile applications which streamline our digital workflow. Technological advancements have also paved the way for the integration of new dental materials into dentistry.

Laser-induced forward transfer based laser bioprinting in biomedical applications.

Bioprinting is an emerging field that utilizes 3D printing technology to fabricate intricate biological structures, including tissues and organs. Among the various promising bioprinting techniques, laser-induced forward transfer (LIFT) stands out by employing a laser to precisely transfer cells or bioinks onto a substrate, enabling the creation of complex 3D architectures with characteristics of high printing precision, enhanced cell viability, and excellent technical adaptability. This technology has found extensive applications in the production of biomolecular microarrays and biological structures, demonstrating significant potential in tissue engineering. This review briefly introduces the experimental setup, bioink ejection mechanisms, and parameters relevant to LIFT bioprinting. Furthermore, it presents a detailed summary of both conventional and cutting-edge applications of LIFT in fabricating biomolecule microarrays and various tissues, such as skin, blood vessels and bone. Additionally, the review addresses the existing challenges in this field and provides corresponding suggestions. By contributing to the ongoing development of this field, this review aims to inspire further research on the utilization of LIFT-based bioprinting in biomedical applications.

Laser Bioprinting with Cell Spheroids: Accurate and Gentle.

Laser printing with cell spheroids can become a promising approach in tissue engineering and regenerative medicine. However, the use of standard laser bioprinters for this purpose is not optimal as they are optimized for transferring smaller objects, such as cells and microorganisms. The use of standard laser systems and protocols for the transfer of cell spheroids leads either to their destruction or to a significant deterioration in the quality of bioprinting. The possibilities of cell spheroids printing by laser-induced forward transfer in a gentle mode, which ensures good cell survival ~80% without damage and burns, were demonstrated. The proposed method showed a high spatial resolution of laser printing of cell spheroid geometric structures at the level of 62 ± 33 µm, which is significantly less than the size of the cell spheroid itself. The experiments were performed on a laboratory laser bioprinter with a sterile zone, which was supplemented with a new optical part based on the Pi-Shaper element, which allows for forming laser spots with different non-Gaussian intensity distributions. It is shown that laser spots with an intensity distribution profile of the "Two rings" type (close to Π-shaped) and a size comparable to a spheroid are optimal. To select the operating parameters of laser exposure, spheroid phantoms made of a photocurable resin and spheroids made from human umbilical cord mesenchymal stromal cells were used.

Progress in bioprinting technology for tissue regeneration.

In recent years, due to the increase in diseases that require organ/tissue transplantation and the limited donor, on the other hand, patients have lost hope of recovery and organ transplantation. Regenerative medicine is one of the new sciences that promises a bright future for these patients by providing solutions to repair, improve function, and replace tissue. One of the technologies used in regenerative medicine is three-dimensional (3D) bioprinters. Bioprinting is a new strategy that is the basis for starting a global revolution in the field of medical sciences and has attracted much attention. 3D bioprinters use a combination of advanced biology and cell science, computer science, and materials science to create complex bio-hybrid structures for various applications. The capacity to use this technology can be demonstrated in regenerative medicine to make various connective tissues, such as skin, cartilage, and bone. One of the essential parts of a 3D bioprinter is the bio-ink. Bio-ink is a combination of biologically active molecules, cells, and biomaterials that make the printed product. In this review, we examine the main bioprinting strategies, such as inkjet printing, laser, and extrusion-based bioprinting, as well as some of their applications.

344Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity.

Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons.

Laser-assisted bioprinting of microorganisms with hydrogel microdroplets: peculiarities of Ascomycota and Basidiomycota yeast transfer.

Laser-assisted bioprinting of microbial cells by hydrogel microdroplets is a rapidly developing and promising field that can contribute to solving a number of issues in microbiology and biotechnology. To date, most research on the use of laser bioprinting for microorganism manipulation and sorting has focused on prokaryotes; the bioprinting of eukaryotic microorganisms is much less understood. The use of hydrogel allows solving two fundamental problems: creating comfortable environments for living microorganisms and imparting the necessary rheological properties of the gel for the stable transfer of microdroplets of a preset size. Two main problems were solved in this article. First, the parameters of the hydrogel based on hyaluronic acid and laser fluence to ensure stable transfer of single drops are selected. Second, possible differences in the bioprinting by hyaluronic acid hydrogel microdroplets with yeasts of various taxonomy (Ascomycota vs Basidiomycota), which form and do not form polysaccharide capsules and evaluated. We have performed laser induced forward transfer of 8 yeast species (Goffeauzyma gilvescens, Lipomyces lipofer, Lipomyces starkey, Pichia manshurica, Saitozyma podzolica, Schwanniomyces occidentalis var. occidentalis, Sterigmatosporidium polymorphum, Vanrija humicola) and assessed its viability based on colony formation on the nutrient medium. It is shown that after laser-induced transfer in hydrogel microdroplets the mean viability rate was 77% with some strains showing relatively high viability rates exceeding 90%. Effect of capsules presence on colony formation after laser bioprinting was not revealed. Differences in laser transfer of the yeast of various phyla were found-basidiomycetes formed a greater number of colonies than ascomycetes. The causes and mechanisms of these effects require detailed studies. The data obtained contributes to the knowledge about the bioprinting of eukaryotic microorganisms and can be useful in the studies of single microbial cells and inter-organism interactions.

Theoretical and Experimental Assay of Shock Experienced by Yeast Cells during Laser Bioprinting.

Laser-induced forward transfer (LIFT) is a useful technique for bioprinting using gel-embedded cells. However, little is known about the stresses experienced by cells during LIFT. This paper theoretically and experimentally explores the levels of laser pulse irradiation and pulsed heating experienced by yeast cells during LIFT. It has been found that only 5% of the cells in the gel layer adjacent to the absorbing Ti film should be significantly heated for fractions of microseconds, which was confirmed by the fact that a corresponding population of cells died during LIFT. This was accompanied by the near-complete dimming of intracellular green fluorescent protein, also observed in response to heat shock. It is shown that microorganisms in the gel layer experience laser irradiation with an energy density of ~0.1–6 J/cm2. This level of irradiation had no effect on yeast on its own. We conclude that in a wide range of laser fluences, bioprinting kills only a minority of the cell population. Importantly, we detected a previously unobserved change in membrane permeability in viable cells. Our data provide a wider perspective on the effects of LIFT-based bioprinting on living organisms and might provide new uses for the procedure based on its effects on cell permeability.

Laser Bioprinting of Cells Using UV and Visible Wavelengths: A Comparative DNA Damage Study.

Laser-based techniques for printing cells onto different substrates with high precision and resolution present unique opportunities for contributing to a wide range of biomedical applications, including tissue engineering. In this study, laser-induced forward transfer (LIFT) printing was employed to rapidly and accurately deposit patterns of cancer cells in a non-contact manner, using two different wavelengths, 532 and 355 nm. To evaluate the effect of LIFT on the printed cells, their growth and DNA damage profiles were assessed and evaluated quantitatively over several days. The damaging effect of LIFT-printing was thoroughly investigated, for the first time at a single cell level, by counting individual double strand breaks (DSB). Overall, we found that LIFT was able to safely print patterns of breast cancer cells with high viability with little or no heat or shear damage to the cells, as indicated by unperturbed growth and negligible gross DNA damage.

Laser bioprinting without donor plate

A promising laser bioprinting technology based on the effect of laser-induced direct transfer is rapidly developing and is already actively used in biotechnology and medicine today. Typically, such technologies use donor plates with an absorbent coating, from which gel microdroplets with living cells and microorganisms are transferred to an acceptor substrate under the action of a laser pulse. This article proposes a new bioprinting technology that makes it possible to transfer microdroplet not only without an absorbing coating, but also without the donor plate itself. The essence of this method is that the transfer to the surface of the acceptor is carried out by a series of nanosecond pulse with a wavelength of 3 μm directly from the surface of a liquid with microorganisms. The mechanism of a liquid jet and microdroplets formation upon absorption of a laser pulse in a thin surface liquid layer is discussed in detail. It has been shown that microorganisms transferred using the new bioprinting technology are viable and can be successfully cultivated on a dense nutrient medium.

АДДИТИВНЫЕ ТЕХНОЛОГИИ 3D-БИОПЕЧАТИ ДЛЯ РЕГЕНЕРАТИВНОЙ МЕДИЦИНЫ

The development of tissue engineering in recent years is associated with the use of additive technologies for the production of transplants and their application in regenerative medicine. 3D bioprinting opens up great prospects for obtaining complex, physiologically three-dimensional tissue structures and makes it possible to design and manufacture such structures with control over their size and function. The review considers three main methods of three-dimensional bioprinting: extrusion, droplet and laser bioprinting, each of which has its own advantages and disadvantages. The resulting tissue constructs, created using 3D bioprinting, open up new possibilities in tissue engineering and regenerative medicine for researchers.

3D Tissue Constructs from the Ground Up for Game Changing New Applications in Tissue Engineering and Regenerative Medicine

In view of the very expensive modern healthcare system, sudden loss or failure of organs and tissues could pose a very difficult and costly medical problem to patients. Further, the limited supply of organs globally that a patient can afford for replacement in the event of an organ failure makes the problem even more challenging and complicated. These medical and healthcare challenges have triggered research and developments into tissue engineering to advance the field of regenerative medicine. Especially, the research focus has been on the design, development and optimization of a cell-scaffold-microenvironment to promote the regeneration of various types of tissue including skin, cartilage, bone, tendon and cardiac tissue, to name a few. Studies have been undertaken to produce functional three-dimensional (3D) tissue substitutes or constructs that are based on bio scaffolds from the ground up. To this end, bioprinting strategies have been considered for fabrication of complex 3D functional living tissues or artificial organs. Here, we describe some notable advances in laser bioprinting enabled tissue engineering, which is a rapidly emerging field in 3D bio fabrication technology for applications in regenerative medicine.

On the Shape of Metal Nanoparticles in Laser Printing With Gel Microdroplets

Laser bioprinting is a promising method for biotechnology and medicine. This letter presents an experimental study of metal nanoparticles formed through the destruction of the absorbing Au or Ti films of the donor slide. It is shown that nanosecond laser action leads to the destruction of the film and the removal of its material with the formation of a hole with melted edges. A sharp increase in temperature above the melting point of the metal in the region of the hole leads to the destruction of the material with the formation of submicron nanoparticles. As a consequence, in the case of a gold film, nanoparticles formed due to material destruction are predominantly spherical. In using a titanium film, rectangular nanoparticles equal to the initial film thickness are formed.

Capillary-like Formations of Endothelial Cells in Defined Patterns Generated by Laser Bioprinting.

Bioprinting is seen as a promising technique for tissue engineering, with hopes of one day being able to produce whole organs. However, thick tissue requires a functional vascular network, which naturally contains vessels of various sizes, down to capillaries of ~10 µm in diameter, often spaced less than 200 µm apart. If such thick tissues are to be printed, the vasculature would likely need to be printed at the same time, including the capillaries. While there are many approaches in tissue engineering to produce larger vessels in a defined manner, the small capillaries usually arise only in random patterns by sprouting from the larger vessels or from randomly distributed endothelial cells. Here, we investigated whether the small capillaries could also be printed in predefined patterns. For this purpose, we used a laser-based bioprinting technique that allows for the combination of high resolution and high cell density. Our aim was to achieve the formation of closed tubular structures with lumina by laser-printed endothelial cells along the printed patterns on a surface and in bioprinted tissue. This study shows that such capillaries are directly printable; however, persistence of the printed tubular structures was achieved only in tissue with external stimulation by other cell types.

Metal Nanoparticles in Laser Bioprinting.

Laser bioprinting is a promising method for applications in biotechnology, tissue engineering, and regenerative medicine. It is based on a microdroplet transfer from a donor slide induced by laser pulse heating of a thin metal absorption film covered with a layer of hydrogel containing living cells (bioink). Due to the presence of the metal absorption layer, some debris in the form of metal nanoparticles is printed together with bioink microdroplets. In this article, experimental investigations of the amount of metal nanoparticles formed during the laser bioprinting process and transported in bioink microdroplets are performed. As metal absorption layers, Ti films with the thickness in the range of 25–400 nm, produced by magnetron spattering, were applied. Dependences of the volume of bioink microdroplets and the amount of Ti nanoparticles within them on the laser pulse fluence were obtained. It has been experimentally found that practically all nanoparticles remain in the hydrogel layer on the donor slide during bioprinting, with only a small fraction of them transferred within the microdroplet (0.5% to 2.5%). These results are very important for applications of laser bioprinting since the transferred metal nanoparticles can potentially affect living systems. The good news is that the amount of such nanoparticles is very low to produce any negative effect on the printed cells.

Consideration of requirements for materials for different bioprinting methods

The object of research is the characteristics of the materials used in the bioprinting process. One of the biggest problems in the field of bioprinting is the materials used for printing organs, in particular, the lack of mechanical properties of these materials, such as strength, elasticity, ductility, wear resistance, and the like. They are essential to achieve the stabilization of printed structures. During the study, the requirements for materials used in the technology of three-dimensional bioprinting, including hydrogels, were discussed. Three main methods were considered (extrusion bioprinting, drip bioprinting, laser bioprinting), for each of which separate requirements for materials are put forward. Comparative assessment of these materials for different types of printing techniques are obtained. It is also determined that the extrusion printing technique is the most used for this direction of use, however, there remains the problem of the viability of living cells through the force of the bias stress, which occurs when the substance is squeezed out from the side of the nozzle walls. It is determined that the main requirements are the ability to gel, low surface tension, wettability and viscosity of the substance. Through understanding and structured information, it is possible to provide biological connections for better cellular interactions and improve the nutrient medium for the creation of physiologically relevant, functional tissues that can be engrafted by the human body after implantation. With such initial data, it is possible to develop new materials and improve existing ones that would meet all these requirements. By identifying the key problem, new ways of solving it can be developed. The above problems are some of the main reasons why researchers are still far from the bioprinting of clinically significant functional organs. Nonetheless, thanks to the new development, bioprinting will become a key technology for future tissue engineering, regenerative medicine and pharmaceuticals.

Evolution of Shock-Induced Pressure in Laser Bioprinting

Laser bioprinting with gel microdroplets that contain living cells is a promising method for use in microbiology, biotechnology, and medicine. Laser engineering of microbial systems (LEMS) technology by laser-induced forward transfer (LIFT) is highly effective in isolating difficult-to-cultivate and uncultured microorganisms, which are essential for modern bioscience. In LEMS the transfer of a microdroplet of a gel substrate containing living cell occurs due to the rapid heating under the tight focusing of a nanosecond infrared laser pulse onto thin metal film with the substrate layer. During laser transfer, living organisms are affected by temperature and pressure jumps, high dynamic loads, and several others. The study of these factors’ role is important both for improving laser printing technology itself and from a purely theoretical point of view in relation to understanding the mechanisms of LEMS action. This article presents the results of an experimental study of bubbles, gel jets, and shock waves arising in liquid media during nanosecond laser heating of a Ti film obtained using time-resolving shadow microscopy. Estimates of the pressure jumps experienced by microorganisms in the process of laser transfer are performed: in the operating range of laser energies for bioprinting LEMS technology, pressure jumps near the absorbing film of the donor plate is about 30 MPa. The efficiency of laser pulse energy conversion to mechanical post-effects is about 10%. The estimates obtained are of great importance for microbiology, biotechnology, and medicine, particularly for improving the technologies related to laser bioprinting and the laser engineering of microbial systems.

3D Bioprinting: Print the Future of Periodontics

3D Bioprinting is a pioneering technology in the field of regenerative medicine that enables the fabrication of living tissues using the living cells by the printing process. Since, periodontitis has become more prevalent disease among the population; there is a need for increased periodontal regenerative procedures to restore normal healthy periodontium for the patients. Regenerative procedures require placement of tissues that are biocompatible, bioresorbabale, promotes cell growth and proliferation in restoring the defect. Such structures can be made from this 3D bioprinting technology which uses several bioinks and various bioprinting methods such as autonomous self-assembly, extrusion- based, laser bioprinting etc., to print the tissues. Every method has their own pros and cons, hence having a detailed knowledge of the method aids in improvising the techniques further. In summary, we have a detailed discussion of this 3D bioprinting technology in this review and additionally, it throws light on using various bioprinting strategies and materials to improve the field of periodontal tissue engineering further in our clinical practices.

Разрушение пленки золота при моделировании процесса лазерной биопечати

Laser printing with gel microdroplets is a promising method for biotechnology and medicine. During printing, a nanosecond laser pulse is absorbed in a thin metal film of a donor substrate with a covered gel layer, which leads to its heating, partial destruction of the film, and transfer of a gel microdroplet to the acceptor substrate. In this work, the dynamics of destruction of a film with a thickness of 50 nm is studied. It is shown that when threshold is exceeded, the gold film peels off from the glass plate. A further increase in the laser fluence occurs to the formation of a orifice in the film. The results obtained are of interest for improving the technology of laser bioprinting.