Inkjet Bioprinting Research Articles

Human gelatin-based composite hydrogels for osteochondral tissue engineering and their adaptation into bioinks for extrusion, inkjet, and digital light processing bioprinting

The investigation of novel hydrogel systems allows for the study of relationships between biomaterials, cells, and other factors within osteochondral tissue engineering. Three-dimensional (3D) printing is a popular research method that can allow for further interrogation of these questions via the fabrication of 3D hydrogel environments that mimic tissue-specific, complex architectures. However, the adaptation of promising hydrogel biomaterial systems into 3D-printable bioinks remains a challenge. Here, we delineated an approach to that process. First, we characterized a novel methacryloylated gelatin composite hydrogel system and assessed how calcium phosphate and glycosaminoglycan additives upregulated bone- and cartilage-like matrix deposition and certain genetic markers of differentiation within human mesenchymal stem cells (hMSCs), such as RUNX2 and SOX9. Then, new assays were developed and utilized to study the effects of xanthan gum and nanofibrillated cellulose, which allowed for cohesive fiber deposition, reliable droplet formation, and non-fracturing digital light processing (DLP)-printed constructs within extrusion, inkjet, and DLP techniques, respectively. Finally, these bioinks were used to 3D print constructs containing viable encapsulated hMSCs over a 7 d period, where DLP printed constructs facilitated the highest observed increase in cell number over 7 d (∼2.4×). The results presented here describe the promotion of osteochondral phenotypes via these novel composite hydrogel formulations, establish their ability to bioprint viable, cell-encapsulating constructs using three different 3D printing methods on multiple bioprinters, and document how a library of modular bioink additives affected those physicochemical properties important to printability.

Assessment of Angiogenesis and Cell Survivability of an Inkjet Bioprinted Biological Implant in an Animal Model.

The rapidly growing field of tissue engineering hopes to soon address the shortage of transplantable tissues, allowing for precise control and fabrication that could be made for each specific patient. The protocols currently in place to print large-scale tissues have yet to address the main challenge of nutritional deficiencies in the central areas of the engineered tissue, causing necrosis deep within and rendering it ineffective. Bioprinted microvasculature has been proposed to encourage angiogenesis and facilitate the mobility of oxygen and nutrients throughout the engineered tissue. An implant made via an inkjet printing process containing human microvascular endothelial cells was placed in both B17-SCID and NSG-SGM3 animal models to determine the rate of angiogenesis and degree of cell survival. The implantable tissues were made using a combination of alginate and gelatin type B; all implants were printed via previously published procedures using a modified HP inkjet printer. Histopathological results show a dramatic increase in the average microvasculature formation for mice that received the printed constructs within the implant area when compared to the manual and control implants, indicating inkjet bioprinting technology can be effectively used for vascularization of engineered tissues.

Integrating melt electrowriting and inkjet bioprinting for engineering structurally organized articular cartilage

Successful cartilage engineering requires the generation of biological grafts mimicking the structure, composition and mechanical behaviour of the native tissue. Here melt electrowriting (MEW) was used to produce arrays of polymeric structures whose function was to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjet printing was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (50 mm × 50 mm) cartilage implants containing over 3,500 cellular aggregates in under 15 min. After 8 weeks in culture, a 50-fold increase in the compressive stiffness of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.

Jetting droplets on semi-solid substrate for biological applications

A droplet exits the inkjet orifice during the jetting phase and subsequently lands on a substrate in the impact phase. This article highlights the journey of a jetted ink, beginning with droplet formation followed by drop impact on varying substrates such as solid, liquid and semi-solid. In bioprinting where the viability of cells plays a pivotal role in the success of bioprinted constructs, the key is to implement strategies that reduce cell deformation during and after the ejection of cell-laden droplets in the droplet-based printing processes such as inkjet bioprinting. The event post impact (i.e. spreading, evaporation and absorption) lie key factors in improving jetted cells while also tuning hydrogel substrates properties that support cell growth. With various strategies and approaches in inkjet printing, there lies opportunities through designing materials and innovative platforms using inkjet printing for biological studies.

Improving printability of hydrogel-based bio-inks for thermal inkjet bioprinting applications via saponification and heat treatment processes.

Material jetting bioprinting is a highly promising three-dimensional (3D) bioprinting technique that facilitates drop-on-demand (DOD) deposition of biomaterials and cells at pre-defined positions with high precision and resolution. A major challenge that hinders the prevalent use of the material jetting bioprinting technique is due to its limited range of printable hydrogel-based bio-inks. As a proof-of-concept, further modifications were made to gelatin methacrylate (GelMA), a gold-standard bio-ink, to improve its printability in a thermal inkjet bioprinter (HP Inc. D300e Digital Dispenser). A two-step modification process comprising saponification and heat treatment was performed; the GelMA bio-ink was first modified via a saponification process under highly alkali conditions to obtain saponified GelMA (SP-GelMA), followed by heat treatment via an autoclaving process to obtain heat-treated SP-GelMA (HSP-GelMA). The bio-ink modification process was optimized by evaluating the material properties of the GelMA bio-inks via rheological characterization, the bio-ink crosslinking test, nuclear magnetic resonance (NMR) spectroscopy and the material swelling ratio after different numbers of heat treatment cycles (0, 1, 2 and 3 cycles). Lastly, size-exclusion chromatography with multi-angle light scattering (SEC-MALS) was performed to determine the effect of heat treatment on the molecular weight of the bio-inks. In this work, the 4% H2SP-GelMA bio-inks (after 2 heat treatment cycles) demonstrated good printability and biocompatibility (in terms of cell viability and proliferation profile). Furthermore, thermal inkjet bioprinting of the modified hydrogel-based bio-ink (a two-step modification process comprising saponification and heat treatment) via direct/indirect cell patterning is a facile approach for potential fundamental cell-cell and cell-material interaction studies.

Novel Combinatorial Strategy Using Thermal Inkjet Bioprinting, Chemotherapy, and Radiation on Human Breast Cancer Cells; an In-Vitro Cell Viability Assessment

Background: Breast cancer (BC) continues to have the second highest mortality amongst women in the United States after lung cancer. For 2021, the American Cancer Association predicted 281,550 new invasive breast cancer cases besides 49,290 new cases of non-invasive breast cancer and 43,600 deaths from the metastatic disease. A treatment modality is radiation therapy, which is given for local control as well as palliation of patient symptoms. The initial step of new drug development is in-vitro cell studies, which help describe new drug properties and toxicities. However, these models are not optimal, and better ones have yet to be determined. This study uses bioprinting technology to elucidate the sensitivity of tumor cells to the combination of palbociclib (PD) and letrozole (Let) treatment. We hypothesize that this technology could serve as a model to predict treatment outcomes more efficiently. Methods: The breast cancer cell lines MCF7 and MDA-MB-231 as well as the normal breast epithelial cell line, MCF-10A, were treated with PD-Let with and without radiotherapy (RT), and cell viability was compared in pairwise fashion for thermally inkjet bioprinted (TIB) and manually seeded (MS) cells. Results: In absence of radiation, the TIB MCF7 cells have 2.5 times higher viability than manually seeded (MS) cells when treated with 100 µM palbociclib and 10 µM letrozole, a 36% higher viability when treated with 50 µM palbociclib and 10 µM letrozole, and an 8% higher viability when treated with 10 µM palbociclib and 10 µM letrozole. With 10 Gy of radiation, TIB cells had a 45% higher survival rate than MS cells at the lowest palbociclib concentration and a 29% higher survival rate at the intermediate palbociclib concentration. Without radiation treatment, at a concentration of 10 μM PD-Let, TIB MDA-MB-231 cells show a 8% higher viability than MS cells when treated with 10 µM PD and 10 µM Let; at higher drug concentrations, the differences disappeared, but some 1.7% of the TIB MDA-MB-231 cells survived exposure to 150 μM of PD + 10 μM letrozole vs. none of the MS cells. These cells are more radiation sensitive than the other cell lines tested and less sensitive to the combo drug treatments. We observed an 18% higher survival of TIB MCF-10A cells without radiation treatment when exposed to 10 μM PD + 10 μM Let but no difference in cell survival between the two groups when radiation was applied. Independent of growth conditions, TIB cells did not show more resistance to radiation treatment than MS cells, but a higher resistance to the combo treatment was observed, which was most pronounced in the MCF-7 cell line. Conclusion: Based on these results, we suggest that TIB used in in-vitro models could be a feasible strategy to develop and/or test new anticancer drugs.

Bioprinting of Stem Cells in Multimaterial Scaffolds and Their Applications in Bone Tissue Engineering.

Bioprinting stem cells into three-dimensional (3D) scaffolds has emerged as a new avenue for regenerative medicine, bone tissue engineering, and biosensor manufacturing in recent years. Mesenchymal stem cells, such as adipose-derived and bone-marrow-derived stem cells, are capable of multipotent differentiation in a 3D culture. The use of different printing methods results in varying effects on the bioprinted stem cells with the appearance of no general adverse effects. Specifically, extrusion, inkjet, and laser-assisted bioprinting are three methods that impact stem cell viability, proliferation, and differentiation potential. Each printing method confers advantages and disadvantages that directly influence cellular behavior. Additionally, the acquisition of 3D bioprinters has become more prominent with innovative technology and affordability. With accessible technology, custom 3D bioprinters with capabilities to print high-performance bioinks are used for biosensor fabrication. Such 3D printed biosensors are used to control conductivity and electrical transmission in physiological environments. Once printed, the scaffolds containing the aforementioned stem cells have a significant impact on cellular behavior and differentiation. Natural polymer hydrogels and natural composites can impact osteogenic differentiation with some inducing chondrogenesis. Further studies have shown enhanced osteogenesis using cell-laden scaffolds in vivo. Furthermore, selective use of biomaterials can directly influence cell fate and the quantity of osteogenesis. This review evaluates the impact of extrusion, inkjet, and laser-assisted bioprinting on adipose-derived and bone-marrow-derived stem cells along with the effect of incorporating these stem cells into natural and composite biomaterials.

Laser-based bioprinting for multilayer cell patterning in tissue engineering and cancer research.

3D printing, or additive manufacturing, is a process for patterning functional materials based on the digital 3D model. A bioink that contains cells, growth factors, and biomaterials are utilized for assisting cells to develop into tissues and organs. As a promising technique in regenerative medicine, many kinds of bioprinting platforms have been utilized, including extrusion-based bioprinting, inkjet bioprinting, and laser-based bioprinting. Laser-based bioprinting, a kind of bioprinting technology using the laser as the energy source, has advantages over other methods. Compared with inkjet bioprinting and extrusion-based bioprinting, laser-based bioprinting is nozzle-free, which makes it a valid tool that can adapt to the viscosity of the bioink; the cell viability is also improved because of elimination of nozzle, which could cause cell damage when the bioinks flow through a nozzle. Accurate tuning of the laser source and bioink may provide a higher resolution for reconstruction of tissue that may be transplanted used as an in vitro disease model. Here, we introduce the mechanism of this technology and the essential factors in the process of laser-based bioprinting. Then, the most potential applications are listed, including tissue engineering and cancer models. Finally, we present the challenges and opportunities faced by laser-based bioprinting.

Advances in tissue engineering of vasculature through three-dimensional bioprinting.

A significant challenge facing tissue engineering is the fabrication of vasculature constructs which contains vascularized tissue constructs to recapitulate viable, complex and functional organs or tissues, and free-standing vascular structures potentially providing clinical applications in the future. Three-dimensional (3D) bioprinting has emerged as a promising technology, possessing a number of merits that other conventional biofabrication methods do not have. Over the last decade, 3D bioprinting has contributed a variety of techniques and strategies to generate both vascularized tissue constructs and free-standing vascular structures. This review focuses on different strategies to print two kinds of vasculature constructs, namely vascularized tissue constructs and vessel-like tubular structures, highlighting the feasibility and shortcoming of the current methods for vasculature constructs fabrication. Generally, both direct printing and indirect printing can be employed in vascularized tissue engineering. Direct printing allows for structural fabrication with synchronous cell seeding, while indirect printing is more effective in generating complex architecture. During the fabrication process, 3D bioprinting techniques including extrusion bioprinting, inkjet bioprinting and light-assisted bioprinting should be selectively implemented to exert advantages and obtain the desirable tissue structure. Also, appropriate cells and biomaterials matter a lot to match various bioprinting techniques and thus achieve successful fabrication of specific vasculature constructs. The 3D bioprinting has been developed to help provide various fabrication techniques, devoting to producing structurally stable, physiologically relevant, and biologically appealing constructs. However, although the optimization of biomaterials and innovation of printing strategies may improve the fabricated vessel-like structures, 3D bioprinting is still in the infant period and has a great gap between in vitro trials and in vivo applications. The article reviews the present achievement of 3D bioprinting in generating vasculature constructs and also provides perspectives on future directions of advanced vasculature constructs fabrication.

The Role of Concentration on Drop Formation and Breakup of Collagen, Fibrinogen, and Thrombin Solutions during Inkjet Bioprinting.

The influence of protein concentration on drop formation and breakup of aqueous solutions of fibrous proteins collagen and fibrinogen and globular protein thrombin in different concentration regimes has been investigated during drop-on-demand (DOD) inkjet printing. The capillary-driven thinning and breakup of dilute collagen, fibrinogen, and thrombin solutions, the solutions in which protein molecules are far away from each other, are predominantly resisted by inertial force. Although the capillary-driven thinning and breakup of semidilute unentangled collagen and fibrinogen solutions, the solutions in which protein molecules begin to interpenetrate each other, are predominantly resisted by inertial force on the initial onset of necking, the breakup of droplets is delayed because of the resistance of elastic force. The resistance of viscous force to the necking and breakup of both the dilute and semidilute unentangled protein solutions is negligible. Aggregates or subvisible particles (between 1 and 100 μm) constantly disrupt the formation of droplets for the semidilute unentangled protein solutions, even when their inverse Ohnesorge number (Z) is within the printability range of 4 ≤ Z ≤ 14. Although aggregates are present in the dilute protein solutions, they do not disrupt the formation of droplets.

Inkjet Bioprinting of Biomaterials.

The inkjet technique has the capability of generating droplets in the picoliter volume range, firing thousands of times in a few seconds and printing in the noncontact manner. Since its emergence, inkjet technology has been widely utilized in the publishing industry for printing of text and pictures. As the technology developed, its applications have been expanded from two-dimensional (2D) to three-dimensional (3D) and even used to fabricate components of electronic devices. At the end of the twentieth century, researchers were aware of the potential value of this technology in life sciences and tissue engineering because its picoliter-level printing unit is suitable for depositing biological components. Currently inkjet technology has been becoming a practical tool in modern medicine serving for drug development, scaffold building, and cell depositing. In this article, we first review the history, principles and different methods of developing this technology. Next, we focus on the recent achievements of inkjet printing in the biological field. Inkjet bioprinting of generic biomaterials, biomacromolecules, DNAs, and cells and their major applications are introduced in order of increasing complexity. The current limitations/challenges and corresponding solutions of this technology are also discussed. A new concept, biopixels, is put forward with a combination of the key characteristics of inkjet printing and basic biological units to bring a comprehensive view on inkjet-based bioprinting. Finally, a roadmap of the entire 3D bioprinting is depicted at the end of this review article, clearly demonstrating the past, present, and future of 3D bioprinting and our current progress in this field.

Thermal Bioprinting Causes Ample Alterations of Expression of LUCAT1, IL6, CCL26, and NRN1L Genes and Massive Phosphorylation of Critical Oncogenic Drug Resistance Pathways in Breast Cancer Cells.

Bioprinting technology merges engineering and biological fields and together, they possess a great translational potential, which can tremendously impact the future of regenerative medicine and drug discovery. However, the molecular effects elicited by thermal inkjet bioprinting in breast cancer cells remains elusive. Previous studies have suggested that bioprinting can be used to model tissues for drug discovery and pharmacology. We report viability, apoptosis, phosphorylation, and RNA sequence analysis of bioprinted MCF7 breast cancer cells at separate timepoints post-bioprinting. An Annexin A5-FITC apoptosis stain was used in combination with flow cytometry at 2 and 24 h post-bioprinting. Antibody arrays using a Human phospho-MAPK array kit was performed 24 h post-bioprinting. RNA sequence analysis was conducted in samples collected at 2, 7, and 24 h post-bioprinting. The post-bioprinting cell viability averages were 77 and 76% at 24 h and 48 h, with 31 and 64% apoptotic cells at 2 and 24 h after bioprinting. A total of 21 kinases were phosphorylated in the bioprinted cells and 9 were phosphorylated in the manually seeded controls. The RNA seq analysis in the bioprinted cells identified a total of 12,235 genes, of which 9.7% were significantly differentially expressed. Using a ±2-fold change as the cutoff, 266 upregulated and 206 downregulated genes were observed in the bioprinted cells, with the following 5 genes uniquely expressed NRN1L, LUCAT1, IL6, CCL26, and LOC401585. This suggests that thermal inkjet bioprinting is stimulating large scale gene alterations that could potentially be utilized for drug discovery. Moreover, bioprinting activates key pathways implicated in drug resistance, cell motility, proliferation, survival, and differentiation.

Tissue engineering of retina through high resolution 3-dimensional inkjet bioprinting

The mammalian retina contains multiple cellular layers, each carrying out a specific task. Such a controlled organization should be considered as a crucial factor for designing retinal therapies. The maintenance of retinal layered complexity through the use of scaffold-free techniques has recently emerged as a promising approach for clinical ocular tissue engineering. In an attempt to fabricate such layered retinal model, we are proposing herein a unique inkjet bioprinting system applied to the deposition of a photoreceptor cells (PRs) layer on top of a bioprinted retinal pigment epithelium (RPE), in a precise arrangement and without any carrier material. The results showed that, after bioprinting, both RPE and PRs were well positioned in a layered structure and expressed their structural markers, which was further demonstrated by ZO1, MITF, rhodopsin, opsin B, opsin R/G and PNA immunostaining, three days after bioprinting. We also showed that considerable amounts of human vascular endothelial growth factors (hVEGF) were released from the RPE printed layer, which confirmed the formation of a functional RPE monolayer after bioprinting. Microstructures of bioprinted cells as well as phagocytosis of photoreceptor outer segments by apical RPE microvilli were finally established through transmission electron microscopy (TEM) imaging. In summary, using this carrier-free bioprinting method, it was possible to develop a reasonable in vitro retina model for studying some sight-threatening diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

Inkjet Bioprinting on Parchment Paper for Hit Identification from Small Molecule Libraries.

In this study, an inkjet bioprinting-based high-throughput screening (HTS) system was designed and applied for the first time to a catecholpyrimidine-based small molecule library to find hit compounds that inhibit c-Jun NH2-terminal kinase1 (JNK1). JNK1 kinase, inactivated MAPKAPK2, and specific fluorescent peptides along with bioink were printed on parchment paper under optimized printing conditions that did not allow rapid evaporation of printed media based on Triton-X and glycerol. Subsequently, different small compounds were printed and tested against JNK1 kinase to evaluate their degree of phosphorylation inhibition. After printing and incubation, fluorescence intensities from the phosphorylated/nonphosphorylated peptide were acquired for the % phosphorylation analysis. The IM50 (inhibitory mole 50) value was determined as 1.55 × 10–15 mol for the hit compound, 22. Thus, this work demonstrated that inkjet bioprinting-based HTS can potentially be adopted for the drug discovery process using small molecule libraries, and cost-effective HTS can be expected to be established based on its low nano- to picoliter printing volume.

Bioprinting for Liver Transplantation.

Bioprinting techniques can be used for the in vitro fabrication of functional complex bio-structures. Thus, extensive research is being carried on the use of various techniques for the development of 3D cellular structures. This article focuses on direct writing techniques commonly used for the fabrication of cell structures. Three different types of bioprinting techniques are depicted: Laser-based bioprinting, ink-jet bioprinting and extrusion bioprinting. Further on, a special reference is made to the use of the bioprinting techniques for the fabrication of 2D and 3D liver model structures and liver on chip platforms. The field of liver tissue engineering has been rapidly developed, and a wide range of materials can be used for building novel functional liver structures. The focus on liver is due to its importance as one of the most critical organs on which to test new pharmaceuticals, as it is involved in many metabolic and detoxification processes, and the toxicity of the liver is often the cause of drug rejection.

Engineering inkjet bioprinting processes toward translational therapies

Bioprinting is the assembly of three-dimensional (3D) tissue constructs by layering cell-laden biomaterials using additive manufacturing techniques, offering great potential for tissue engineering and regenerative medicine. Such a process can be performed with high resolution and control by personalized or commercially available inkjet printers. However, bioprinting's clinical translation is significantly limited due to process engineering challenges. Upstream challenges include synthesis, cellular incorporation, and functionalization of "bioinks," and extrusion of print geometries. Downstream challenges address sterilization, culture, implantation, and degradation. In the long run, bioinks must provide a microenvironment to support cell growth, development, and maturation and must interact and integrate with the surrounding tissues after implantation. Additionally, a robust, scaleable manufacturing process must pass regulatory scrutiny from regulatory bodies such as U.S. Food and Drug Administration, European Medicines Agency, or Australian Therapeutic Goods Administration for bioprinting to have a real clinical impact. In this review, recent advances in inkjet-based 3D bioprinting will be presented, emphasizing on biomaterials available, their properties, and the process to generate bioprinted constructs with application in medicine. Current challenges and the future path of bioprinting and bioinks will be addressed, with emphasis in mass production aspects and the regulatory framework bioink-based products must comply to translate this technology from the bench to the clinic.

Effects of printing conditions on cell distribution within microspheres during inkjet-based bioprinting

Inkjet-based bioprinting have been widely employed in a variety of applications in tissue engineering and drug screening and delivery. The typical bioink used in inkjet bioprinting consists of biological materials and living cells. During inkjet bioprinting, the cell-laden bioink is ejected out from the inkjet dispenser to form microspheres with cells encapsulated. The cell distribution within microspheres is defined as the distribution of cell number within the microspheres. The paper focuses on the effects of polymer concentration, excitation voltage, and cell concentration on the cell distribution within microspheres during inkjet printing of cell-laden bioink. The normal distribution has been utilized to fit the experimental results to obtain the mean and standard deviation of the distribution. It is found that the cell distribution within the microspheres increases with the increase of the cell concentration, sodium alginate concentration, and the excitation voltage.

High resolution bioprinting of multi-component hydrogels

Materials capable of directing cell fate by providing spatially-graded mechanical and biomolecular cues are critically important in the reconstitution of living matter. Herein, we report a multi-component inkjet bioprinting method that allows for spatially varying composition and network properties in cell-instructive glycosaminoglycan (GAG)-based biohybrid and pure poly(ethylene glycol) hydrogels with unprecedented (50 μm) resolution. The principle relies on the covalent crosslinking of different polymeric precursors through a very rapid bio-orthogonal Michael type addition scheme adjusted in ways to occur during the fusion of bio-ink droplets prior to and upon contact with the target. Exemplary data show that chemotactic molecular gradients produced by this approach within printed GAG-gels of defined zonal architecture can effectively direct migratory activity and morphogenesis of embedded human bone-marrow derived mesenchymal stem cells. The introduced methodology is expected to enable a new, holistic level of control over reductionistic tissue and organoid models.

Thermal inkjet bioprinting triggers the activation of the VEGF pathway in human microvascular endothelial cells in vitro

One biofabrication process that has gained tremendous momentum in the field of tissue engineering and regenerative medicine is cell-printing or most commonly bioprinting. We have shown that thermal inkjet bioprinted human microvascular endothelial cells were recruited or otherwise involved in the formation of microvasculature to form graft-host anastomoses upon implantation. The present study aims to quantify and characterize the expression and activation of specific cytokines and kinases in vitro. Morphological characteristics demonstrate elongated protrusions of TIB-HMVECs at 5–6 times the size of manually pipetted cells. Moreover, annexin V-FITC and propidium iodide apoptosis assay via flow cytometry demonstrated a 75% apoptosis among printed cells as compared to among control cells. Cell viability at a 3 d incubation period was significantly higher for printed cells as compared to control. Milliplex magnetic bead panels confirmed significant overexpression of HSP70, IL-1α, VEGF-A, IL-8, and FGF-1 of printed cells compared to control. In addition, a Human phospho-kinase array displayed a significant over activation of the heat-shock proteins HSP27 and HSP60 of printed cells compared to the manually seeded cells. Collectively, it is suggested that the massive appearance of capillary blood vessels upon implantation that has been reported elsewhere may be due to the activation of the HSP-NF-κB pathway to produce VEGF. This cell activation may be used as a new strategy for vascularization of tissue engineered constructs which are in high demand in regenerative medicine applications.

Physics of bioprinting

Bioprinters are being extensively used for different applications in life sciences and medicine in general and more specifically in regenerative medicine, tissue, and organ fabrication. The technology has matured from its purely academic origin owing to the involvement of materials science, engineering, biology, and physics, as well as commercial entities. Nevertheless, despite the progress in the science and the understanding of the mechanisms underlying the various bioprinting technologies, further efforts are needed to develop more quantitative strategies. In particular, predictive modeling is necessary to optimize the printing parameters and thus enhance the quality of the final products. Here, we review the physics that underpins the most commonly employed approaches, such as extrusion, laser-based, and inkjet bioprinting. We provide an overview of the relevant parameters, their inter-relationships, and the equations that govern the various printing processes and thus allow for their optimization. We present our perspective on the field and views on future strategies for its further advancement. Our intention with this review is to provide the practitioners of bioprinting with additional tools to enhance the quantitative aspects of their work and move the technology beyond its early, mostly “trial and error” character.