Bioprinted Tissues Research Articles

Formulation and evaluation of a bioink composed of alginate, gelatin, and nanocellulose for meniscal tissue engineering.

1The necessity to preserve meniscal function prompts the research and development of novel treatment options, like three-dimensional (3D) bioprinting. However, bioinks for meniscal 3D bioprinting have not been extensively explored. Therefore, in this study, a bioink composed of alginate, gelatin, and carboxymethylated cellulose nanocrystal (CCNC) was formulated and evaluated. Firstly, bioinks with varying concentrations of the aforementioned components were subjected to rheological analysis (amplitude sweep test, temperature sweep test, and rotation). The optimal bioink formulation of 4.0% gelatin, 0.75% alginate, and 1.4% CCNC dissolved in 4.6% D-mannitol was further used for printing accuracy analysis, followed by 3D bioprinting with normal human knee articular chondrocytes (NHAC-kn). The encapsulated cells' viability was > 98%, and collagen II expression was stimulated by the bioink. The formulated bioink is printable, stable under cell culture conditions, biocompatible, and able to maintain the native phenotype of chondrocytes. Aside from meniscal tissue bioprinting, it is believed that this bioink could serve as a basis for the development of bioinks for various tissues.

Ethical challenges with 3D bioprinted tissues and organs

3D Bioprinting is fast advancing to offer capabilities to process living cells into geometrically and functionally complex tissue and organ substitutes. As bioprinted constructs are making their way into clinic, the bioprinting community needs to consider the responsible innovation and translation of the bioprinted tissues and organs.

Periodontitis: An Oral Disease with Severe Consequences.

Periodontitis, being a multifactorial disorder is found to be the most common oral disease denoted by the inflammation of gingiva and resorption of tooth supporting alveolar bone. The disease being closely linked with fast life style and determined by unhygienic behavioural factors, the internal milieu of oral cavity and formation of plaque biofilm on the dental and gingival surfaces. Porphyromonas gingivalis, being the major keystone pathogen of the periodontal biofilm evokes host immune responses that causes damage of gingival tissues and resorption of bones. The biofilm associated microbial community progressively aggravates the condition resulting in chronic inflammation and finally tooth loss. The disease often maintains bidirectional relationship with different systemic, genetic, autoimmune, immunodeficiency diseases and even psychological disorders. The disease can be diagnosed and predicted by various genetic, radiographic and computer-aided design (CAD) & computer-aided engineering (CAE) and artificial neural network (ANN). The elucidation of genetic background explains the inheritance of the disease. The therapeutic approaches commonly followed include mechanical removal of dental plaque with the use of systemic antibiotics. Awareness generation amongst local people, adoption of good practice of timely tooth brushing preferably with fluoride paste or with nanoconjugate pastes will reduce the chance of periodontal plaque formation. Modern tissue engineering technology like 3D bioprinting of periodontal tissue may help in patient specific flawless regeneration of tooth structures and associated bones.

Culture of 3D bioprinted bone constructs requires an increased fluid dynamic stimulation

In vitro flow-induced mechanical stimulation of developing bone tissue constructs has been shown to favor mineral deposition in scaffolds seeded with cells directly exposed to the fluid flow. However, the effect of fluid dynamic parameters, such as shear stress (SS), within 3D bioprinted constructs is still unclear. Thus, this study aimed at correlating the SS levels and the mineral deposition in 3D bioprinted constructs, evaluating the possible dampening effect of the hydrogel. Human mesenchymal stem cells (hMSCs) were embedded in 3D bioprinted porous structures made of alginate and gelatin. 3D bioprinted constructs were cultured in an osteogenic medium assessing the influence of different flow rates (0, 0.7 and 7 ml/min) on calcium and collagen deposition through histology, and bone volume (BV) through micro-computed tomography. Uniform distribution of calcium and collagen was observed in all groups. Nevertheless, BV significantly increased in perfused groups as compared to static control, ranging from 0.35±0.28 mm3, 11.90±8.74 mm3 and 25.81±5.02 mm3 at week 3 to 2.28±0.78 mm3, 22.55±2.45 mm3 and 46.05±5.95 mm3 at week 6 in static, 0.7 and 7 ml/min groups, respectively. SS values on construct fibers in the range 10-100 mPa in 7 ml/min samples were twice as high as those in 0.7 ml/min samples showing the same trend of BV. The obtained results suggest that it is necessary to enhance the flow-induced mechanical stimulation of cell-embedding hydrogels to increase the amount of mineral deposited by hMSCs, compared to what is generally reported for the development of in vitro bone constructs. Statement of significanceIn this study, we evaluated for the first time how the hydrogel structure dampens the effect of flow-induced mechanical stimulation during the culture of 3D bioprinted bone tissue constructs. By combining computational and experimental techniques we demonstrated that those shear stress thresholds generally considered for culturing cells seeded on scaffold surface, are no longer applicable when cells are embedded in 3D bioprinted constructs. Significantly, more bone volume was formed in constructs exposed to shear stress values generally considered as detrimental than in constructs exposed shear stress values generally considered as beneficial after 3 weeks and 6 weeks of dynamic culture using a perfusion bioreactor.

Application of mesenchymal stem cells combined with nano-polypeptide hydrogel in tissue engineering blood vessel

At present, the vascular grafts used in clinic are mainly autologous blood vessels, but they often face the dilemma of no blood vessels available due to limited sources. However, synthetic blood vessels made of polytetrafluoroethylene (ePTFE), which is commonly used in clinic, are prone to thrombosis and intimal hyperplasia, and the long-term patency rate is poor, so its effectiveness is severely limited, which is far from meeting the clinical needs. With the development of nano-materials, stem cells and 3D bio-printing technology, people began to explore the preparation of new endothelialized vascular grafts through this technology. Nano-peptide materials have excellent biocompatibility, can be compounded with different bioactive molecules, and have unique advantages in cultivating stem cells. It has been reported that self-assembled nano-polypeptide hydrogel was successfully constructed, mesenchymal stem cells were correctly isolated and cultured, and their transformation into blood vessels was studied. It was confirmed that the 3D bio-printed nano-polypeptide hydrogel tissue ADMSCs still had strong vascular endothelial differentiation ability. The application of mesenchymal stem cells and nano-polypeptide hydrogel in tissue engineering blood vessels has gradually become a research hotspot, and it is expected to develop a new type of transplanted blood vessel that meets the physiological functions of human body in terms of vascular endothelialization, cell compatibility and histocompatibility, so as to realize the customized and personalized printing of the endothelialized transplanted blood vessel according to the shape of the target blood vessel, which has attractive prospects and far-reaching social and economic benefits.

Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications.

Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of -80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.

Hybrid extracellular vesicles-liposome incorporated advanced bioink to deliver microRNA

In additive manufacturing, bioink formulations govern strategies to engineer 3D living tissues that mimic the complex architectures and functions of native tissues for successful tissue regeneration. Conventional 3D-printed tissues are limited in their ability to alter the fate of laden cells. Specifically, the efficient delivery of gene expression regulators (i.e. microRNAs (miRNAs)) to cells in bioprinted tissues has remained largely elusive. In this study, we explored the inclusion of extracellular vesicles (EVs), naturally occurring nanovesicles (NVs), into bioinks to resolve this challenge. EVs show excellent biocompatibility, rapid endocytosis, and low immunogenicity, which lead to the efficient delivery of miRNAs without measurable cytotoxicity. EVs were fused with liposomes to prolong and control their release by altering their physical interaction with the bioink. Hybrid EVs-liposome (hEL) NVs were embedded in gelatin-based hydrogels to create bioinks that could efficiently encapsulate and deliver miRNAs at the target site in a controlled and sustained manner. The regulation of cells’ gene expression in a 3D bioprinted matrix was achieved using the hELs-laden bioink as a precursor for excellent shape fidelity and high cell viability constructs. Novel regulatory factors-loaded bioinks will expedite the translation of new bioprinting applications in the tissue engineering field.

Bioink based on the dECM for 3D bioprinting of bionic tissue, the first results obtained on murine model

Tissue engineering is an intensively developing field of modern medicine. However, to transfer the research from the laboratory scale to the clinic, it is necessary to produce biocompatible bioinks, that will be safe for both patients and cells used in the bioprinting process itself.The aim of this researches was to produce a bioink that would enable bioprinting of structures with biological material, improving their functionality by recreating their natural environment.The results showed the appropriate physicochemical properties of the biomaterial, which is stable in vitro and in vivo. In vitro studies showed the highest functionality of islets (7 days) in the bioprinted cell-printed constructs. The cytotoxicity tests showed the cell viability at the level of 120%. Moreover, the paper presents the first in vivo results for biodegradation of bionic cell-printed constructs. During these studies, no inflammatory reaction to the implanted cell-printed construct was found for 12 months. The appearance of the first neovascular processes was shown in the 8th week of the experiment. At that time, there were no deviations in the level of blood parameters: AST,ALT,KC, IL-6 and TNF-α.Summing up, the created bioink composition is safe for living organisms and contributes to the improvement of the functionality of pancreatic islets.

CRIMINAL AND LEGAL PROTECTION OF RELATIONS IN THE FIELD OF 3D-BIOPRINTING

The presented publication is devoted to identifying criminal risks and threats arising from the active use of 3D bioprint technologies in medicine and developing proposals for improving criminal legislation in this area. The choice of this topic is updated by the fact that at the moment there is no comprehensive study of issues related to 3D bioprinting, including the consideration of legal issues. The active introduction of biotechnologies can contribute to the emergence of biocrime. Due to the fact that the use of bioprinting is a type of medical intervention and a type of high-tech medical care, there is a risk of harm to life and health to patients who plan to transplant a bioprinted organ or tissue, or to donors who donate cells for the subsequent bioprinting procedure, as well as personnel involved in the bioprinting process (bioengineers, doctors). The methodological basis of the study is general scientific (synthesis, generalization, analysis, deduction, induction) and special (formal legal, comparative legal) methods of scientific knowledge. As a result of the study, measures were proposed to improve criminal legislation in the field of bioprinting, as well as rules were developed for qualifying illegal actions of persons in the field of bioprinting technologies. The results obtained can be used in the development of proposals for improving the current criminal legislation, as well as in educational and scientific processes.

Gellan Gum Is a Suitable Biomaterial for Manual and Bioprinted Setup of Long-Term Stable, Functional 3D-Adipose Tissue Models.

Due to its wide-ranging endocrine functions, adipose tissue influences the whole body’s metabolism. Engineering long-term stable and functional human adipose tissue is still challenging due to the limited availability of suitable biomaterials and adequate cell maturation. We used gellan gum (GG) to create manual and bioprinted adipose tissue models because of its similarities to the native extracellular matrix and its easily tunable properties. Gellan gum itself was neither toxic nor monocyte activating. The resulting hydrogels exhibited suitable viscoelastic properties for soft tissues and were stable for 98 days in vitro. Encapsulated human primary adipose-derived stem cells (ASCs) were adipogenically differentiated for 14 days and matured for an additional 84 days. Live-dead staining showed that encapsulated cells stayed viable until day 98, while intracellular lipid staining showed an increase over time and a differentiation rate of 76% between days 28 and 56. After 4 weeks of culture, adipocytes had a univacuolar morphology, expressed perilipin A, and secreted up to 73% more leptin. After bioprinting establishment, we demonstrated that the cells in printed hydrogels had high cell viability and exhibited an adipogenic phenotype and function. In summary, GG-based adipose tissue models show long-term stability and allow ASCs maturation into functional, univacuolar adipocytes.

Tissue bioprinting for biology and medicine

Bioprinting merges additive manufacturing and tissue engineering to generate functional tissues and organs. The field has experienced tremendous growth over the past few years. Here, we highlight recent breakthroughs in bioprinting and discuss the challenges that are yet to be addressed before this technology can be widely utilized in biology and medicine.

Development and Application of Three-Dimensional Bioprinting Scaffold in the Repair of Spinal Cord Injury.

Spinal cord injury (SCI) is a disabling and destructive central nervous system injury that has not yet been successfully treated at this stage. Three-dimensional (3D) bioprinting has become a promising method to produce more biologically complex microstructures, which fabricate living neural constructs with anatomically accurate complex geometries and spatial distributions of neural stem cells, and this is critical in the treatment of SCI. With the development of 3D printing technology and the deepening of research, neural tissue engineering research using different printing methods, bio-inks, and cells to repair SCI has achieved certain results. Although satisfactory results have not yet been achieved, they have provided novel ideas for the clinical treatment of SCI. Considering the potential impact of 3D bioprinting technology on neural studies, this review focuses on 3D bioprinting methods widely used in SCI neural tissue engineering, and the latest technological applications of bioprinting of nerve tissues for the repair of SCI are discussed. In addition to introducing the recent progress, this work also describes the existing limitations and highlights emerging possibilities and future prospects in this field.

Release Kinetics and In Vitro Characterization of Sodium Percarbonate and Calcium Peroxide to Oxygenate Bioprinted Tissue Models.

Oxygen-generating materials have been used in several tissue engineering applications; however, their application as in situ oxygen supply within bioprinted constructs has not been deeply studied. In this study, two oxygen-generating materials, sodium percarbonate (SPO) and calcium peroxide (CPO), were studied for their oxygen release kinetics under a 0.1% O2 condition. In addition, a novel cell-culture-insert setup was used to evaluate the effects of SPO and CPO on the viability of skeletal muscle cells under the same hypoxic condition. Results showed that SPO had a burst oxygen release, while CPO had a more stable oxygen release than SPO. Both SPO and CPO reduced cell viability when used alone. The addition of catalase in SPO and CPO increased the oxygen release rate, as well as improving the viability of skeletal muscle cells; however, CPO still showed cytotoxicity with catalase. Additionally, the utilization of 1 mg/mL SPO and 20 U catalase in a hydrogel for bioprinting significantly enhanced the cell viability under the hypoxic condition. Moreover, bioprinted muscle constructs could further differentiate into elongated myotubes when transferring back to the normoxic condition. This work provides an excellent in vitro model to test oxygen-generating materials and further discover their applications in bioprinting, where they represent promising avenues to overcome the challenge of oxygen shortage in bioprinted constructs before their complete vascularization.

3D Bioprinted Silk-Based In Vitro Osteochondral Model for Osteoarthritis Therapeutics.

3D bioprinting of osteochondral tissue offers unique opportunities for enabling precise pharmacological interventions in osteoarthritis (OA). The current study investigates the screening potential of anti-inflammatory drugs using bioprinted inflamed human osteochondral units. The biomimetic hierarchical geometry is bioprinted using silk-based bioinks encapsulating pre-differentiated stem cells, creating an in vitro model. Inflammation is stimulated in the model, using tumor necrosis factor-alpha and Interleukin-1 beta pro-inflammatory cytokines. The resultant degeneration, akin to OA, is flagged by key markers like sulfated glycosaminoglycan, collagen, alkaline phosphatase, and downregulation of osteochondral transcript levels. In the next step, the screening of anti-inflammatory drugs is validated using celecoxib and rhein. Consequently, in the inflamed constructs, the initial upregulation of the key inflammatory mediators (nitric oxide, Prostaglandin E2), is subsequently downregulated, post-drug treatment. In addition, catabolic markers (matrix metalloproteinases and aggrecanase-1), indicative of hypertrophic and apoptosing chondrocytes, are significantly downregulated in the treatment groups; while the transcript and protein levels required for osteochondral health are attenuated. Therefore, the in vitro model mimicks the inflammation in the early stages of OA, and corroborates a potential high-throughput platform for screening novel anti-inflammatory drugs in OA therapeutics.

Bioprinting of a Hepatic Tissue Model Using Human-Induced Pluripotent Stem Cell-derived Hepatocytes for Drug-Induced Hepatotoxicity Evaluation.

Three-dimensional (3D) bioprinting technology is an effective method for exploring the biological functions of hepatocytes by building biomimetic 3D microenvironments. Various hepatic tissue models have been developed for disease modeling, drug screening, and tissue regeneration using 3D bioprinting technology. Human-induced pluripotent stem cells (hiPSCs) are a promising cell source for the generation of functional hepatocytes for bioprinting. In this study, we introduced hiPSC-derived hepatocytes (hiPSC-Heps) as mature hepatocytes for the bioprinting of a 3D hepatic tissue model. The 3D-printed (3DP) model facilitated the formation of hiPSC-Hep spheroids with higher viability and proliferation than the commonly used non-printed sandwich-cultured model. hiPSC-Heps in the 3DP model exhibited higher mRNA expression of liver-specific functions than those in the two-dimensional-cultured model. Moreover, enhanced secretion of liver function-related proteins, including α-1-antitrypsin, albumin, and blood urea nitrogen, was observed in the 3DP model. For the evaluation of acetaminophen-induced hepatotoxicity, the 3DP model exhibited a favorable drug response with upregulation of the drug metabolism-related gene cytochrome P450-1A2 (CYP1A2). Overall, the bioprinted hepatic tissue model showed great biofunctional and drug-responsive performance, which could be potentially applied in in vitro toxicological studies.

Molecularly cleavable bioinks facilitate high-performance digital light processing-based bioprinting of functional volumetric soft tissues

Digital light processing bioprinting favors biofabrication of tissues with improved structural complexity. However, soft-tissue fabrication with this method remains a challenge to balance the physical performances of the bioinks for high-fidelity bioprinting and suitable microenvironments for the encapsulated cells to thrive. Here, we propose a molecular cleavage approach, where hyaluronic acid methacrylate (HAMA) is mixed with gelatin methacryloyl to achieve high-performance bioprinting, followed by selectively enzymatic digestion of HAMA, resulting in tissue-matching mechanical properties without losing the structural complexity and fidelity. Our method allows cellular morphological and functional improvements across multiple bioprinted tissue types featuring a wide range of mechanical stiffness, from the muscles to the brain, the softest organ of the human body. This platform endows us to biofabricate mechanically precisely tunable constructs to meet the biological function requirements of target tissues, potentially paving the way for broad applications in tissue and tissue model engineering.

816-P: Bioprinted Allogeneic Islet–Containing Implants Normalize Blood Glucose Control in Diabetic Rat Models without Immune Suppression

Transplantation of cadaveric islets can reverse type 1 diabetes (T1D) but requires life long immunosuppression. Cell encapsulation has potential to overcome this challenge by blocking immune cell access to islet grafts while allowing nutrient exchange, glucose sensing and insulin release. In this study, 3D tissues consisting of patterned, multi-shell fibres with an islet-containing core and immunoprotective shell were manufactured using Aspect Biosystems’ microfluidic 3D bioprinting technology for implantation in diabetic rats. Blood glucose and animal body weight were monitored for several months following surgery. Upon retrieval, bioprinted tissues were assayed for islet cell viability and function by glucose-stimulated insulin secretion (GSIS) , followed by histology and immunohistochemistry (IHC) to analyze foreign body response (FBR) and immune cell infiltration. 3D bioprinted implants containing reaggregated allogeneic rat islets re-established normoglycemia for > 90 days in streptozotocin-treated diabetic immunodeficient rats, and > 30 days in diabetic immunocompetent rats. Post-retrieval, tissue implants demonstrate high viability and functionality, with absence of immune cell penetration through the shell, confirming its immune protective properties. Modification of the outer shell biomaterial to reduce FBR was shown to extend islet tissue implant functionality. This is the first study describing a 3D bioprinted implant composed of a core/shell micro-fibre used to deliver therapeutic islets into diabetic animals. The promising results obtained in rodent models open the door to further testing in larger animal models and, eventually, T1D patients. Disclosure V.Russo: None. S.Getsios: None. T.J.Kieffer: Consultant; Fractyl Health, Inc., Sigilon Therapeutics, Inc., Employee; ViaCyte, Inc., Research Support; Aspect Biosystems. S.Wadsworth: None. R.Jalili: None. Y.Yu: None. R.Agarwal: None. S.Pan: None. N.Hakimi: None. K.Jahan: None. E.M.Wilts: None. S.Ida: None. Funding National Research Council Canada - Industrial Research Assistance Program (#935249) , Canadian International Innovation Program (#938664) , Genome British Columbia (#GEN009) , Stem Cell Network (#FY20/FBP-7 Kieffer) , Mitacs (#IT19764) ,

Programmable multicellular and spatially patterned organoids: A one-pot strategy

The simultaneous differentiation of human-induced pluripotent stem cells (hiPSCs) into different cell types is the basis for obtaining organoids and tissues with complex cell components, structures, and functions. Lewis and colleagues' latest work published in Nature Biomedical Engineering presented a new one-pot orthogonally differentiation strategy combined with a multi-material 3D bioprinting method to generate programmatic patterned organoids and bioprinted tissues. This approach will provide a new roadmap to developing engineered tissues for extensive biomedical applications.

Air-loaded Gas Vesicle Nanoparticles Promote Cell Growth in Three-dimensional Bioprinted Tissue Constructs.

Three-dimensional (3D) bioprinting has emerged as a promising method for the engineering of tissues and organs. Still, it faces challenges in its widespread use due to issues with the development of bioink materials and the nutrient diffusion barrier inherent to these scaffold materials. Herein, we introduce a method to promote oxygen diffusion throughout the printed constructs using genetically encoded gas vesicles derived from haloarchaea. These hollow nanostructures are composed of a protein shell that allows gases to permeate freely while excluding the water flow. After printing cells with gas vesicles of various concentrations, the cells were observed to have increased activity and proliferation. These results suggest that air-filled gas vesicles can help overcome the diffusion barrier throughout the 3D bioprinted constructs by increasing oxygen availability to cells within the center of the construct. The biodegradable nature of the gas vesicle proteins combined with our promising results encourage their potential use as oxygen-promoting materials in biological samples.

Radical scavenging gelatin methacrylamide based bioink formulation for three dimensional bioprinting of parenchymal liver construct

Methacrylated gelatin (GelMA) in the form of methacryloyl, methacrylate, and methacrylamide is an established and widely accepted photocrosslinkable bioink, for three dimensional bioprinting of various tissues. One of the limitations of photocrosslinkable bioinks is the inability to control the free radicals generated by photoinitiators and ultraviolet (UV) rays. The presence of excess free radicals compromises the viability and functionality of cells during crosslinking. In this study, ascorbic acid, a known free radical scavenger (FRS) molecule, was introduced into the GelMA bioink formulation to protect the cell viability, proliferation, and tissue functions of 3D bioprinted parenchymal liver constructs. The concentration of FRS in the bioink was optimized and used for 3D bioprinting of HepG2 cells. The results confirmed that the inclusion of 3.4 mM FRS in the GelMA bioink formulation nullified the excess ROS formed inside the cells. Furthermore, the optimized GelMA formulation containing FRS preserved and improved the cell activity, albumin, and urea synthesis in the 3D construct over 7 days in culture. In the future, this concept could be implemented in the biofabrication of large liver constructs that require multiple or longer durations of UV irradiation.