Dimensional Bioprinting Research Articles

3-Dimensional Bioprinting of a Tendon Stem Cell–Derived Exosomes Loaded Scaffold to Bridge the Unrepairable Massive Rotator Cuff Tear

Background: Unrepairable massive rotator cuff tears (UMRCTs) are challenging to surgeons owing to the severely retracted rotator cuff musculotendinous tissues and extreme defects in the rotator cuff tendinous tissues. Purpose: To fabricate a tendon stem cell–derived exosomes loaded scaffold (TSC-Exos-S) and investigate its effects on cellular bioactivity in vitro and repair in a rabbit UMRCT model in vivo. Study Design: Controlled laboratory study. Methods: TSC-Exos-S was fabricated by loading TSC-Exos and type 1 collagen (COL-I) into a 3-dimensional bioprinted and polycaprolactone (PCL)–based scaffold. The proliferation, migration, and tenogenic differentiation activities of rabbit bone marrow stem cells (BMSCs) were evaluated in vitro by culturing them in saline, PCL-based scaffold (S), COL-I loaded scaffold (COL-I-S), and TSC-Exos-S. In vivo studies were conducted on a rabbit UMRCT model, where bridging was repaired with S, COL-I-S, TSC-Exos-S, and autologous fascia lata (FL). Histological and biomechanical analyses were performed at 8 and 16 weeks postoperatively. Results: TSC-Exos-S exhibited reliable mechanical strength and subcutaneous degradation, which did not occur before tissue regeneration. TSC-Exos-S significantly promoted the proliferation, migration, and tenogenic differentiation of rabbit BMSCs in vitro. In vivo studies showed that UMRCT repaired with TSC-Exos-S exhibited significant signs of tendinous tissue regeneration at the bridging site with regard to specific collagen staining. Moreover, no significant differences were observed in the histological and biomechanical properties compared with those repaired with autologous FL. Conclusion: TSC-Exos-S achieved tendinous tissue regeneration in UMRCT by providing mechanical support and promoting the trend toward tenogenic differentiation. Clinical Relevance: The present study proposes a potential strategy for repairing UMRCT with severely retracted musculotendinous tissues and large tendinous tissue defects.

Bioengineering Cell Therapy for Treatment of Peripheral Artery Disease.

Peripheral artery disease is an atherosclerotic disease associated with limb ischemia that necessitates limb amputation in severe cases. Cell therapies comprised of adult mononuclear or stromal cells have been clinically tested and show moderate benefits. Bioengineering strategies can be applied to modify cell behavior and function in a controllable fashion. Using mechanically tunable or spatially controllable biomaterials, we highlight examples in which biomaterials can increase the survival and function of the transplanted cells to improve their revascularization efficacy in preclinical models. Biomaterials can be used in conjunction with soluble factors or genetic approaches to further modulate the behavior of transplanted cells and the locally implanted tissue environment in vivo. We critically assess the advances in bioengineering strategies such as 3-dimensional bioprinting and immunomodulatory biomaterials that can be applied to the treatment of peripheral artery disease and then discuss the current challenges and future directions in the implementation of bioengineering strategies.

Current Status of Auricular Reconstruction Strategy Development.

Microtia has severe physical and psychological impacts on patients, and auricular reconstruction offers improved esthetics and function, alleviating psychological issues. Microtia is a congenital disease caused by a multifactorial interaction of environmental and genetic factors, with complex clinical manifestations. Classification assessment aids in determining treatment strategies. Auricular reconstruction is the primary treatment for severe microtia, focusing on the selection of auricular scaffold materials, the construction of auricular morphology, and skin and soft tissue scaffold coverage. Autologous rib cartilage and synthetic materials are both used as scaffold materials for auricular reconstruction, each with advantages and disadvantages. Methods for achieving skin and soft tissue scaffold coverage have been developed to include nonexpansion and expansion techniques. In recent years, the application of digital auxiliary technology such as finite element analysis has helped optimize surgical outcomes and reduce complications. Tissue-engineered cartilage scaffolds and 3-dimensional bioprinting technology have rapidly advanced in the field of ear reconstruction. This article discusses the prevalence and classification of microtia, the selection of auricular scaffolds, the evolution of surgical methods, and the current applications of digital auxiliary technology in ear reconstruction, with the aim of providing clinical physicians with a reference for individualized ear reconstruction surgery. The focus of this work is on the current applications and challenges of tissue engineering and 3-dimensional bioprinting technology in the field of ear reconstruction, as well as future prospects.

Three Dimensional Bioprinting for Hepatic Tissue Engineering: From In Vitro Models to Clinical Applications.

Fabrication of functional organs is the holy grail of tissue engineering and the possibilities of repairing a partial or complete liver to treat chronic liver disorders are discussed in this review. Liver is the largest gland in the human body and plays a responsible role in majority of metabolic function and processes. Chronic liver disease is one of the leading causes of death globally and the current treatment strategy of organ transplantation holds its own demerits. Hence there is a need to develop an in vitro liver model that mimics the native microenvironment. The developed model should be a reliable to understand the pathogenesis, screen drugs and assist to repair and replace the damaged liver. The three-dimensional bioprinting is a promising technology that recreates in vivo alike in vitro model for transplantation, which is the goal of tissue engineers. The technology has great potential due to its precise control and its ability to homogeneously distribute cells on all layers in a complex structure. This review gives an overview of liver tissue engineering with a special focus on 3D bioprinting and bioinks for liver disease modelling and drug screening.

Advances in Three Dimensional Bioprinting for Wound Healing: A Comprehensive Review

The vulnerability of skin wounds has made efficient wound dressing a challenging issue for decades, seeking to mimic the natural microenvironment of cells to facilitate cell binding, augmentation, and metamorphosis. Many three-dimensional (3D) bioprinted hydrogel-based configurations have been developed using high-tech devices to overcome the limitations of traditional dressing materials. Based on a material perspective, this review examines current state-of-the-art 3D bioprinting for hydrogel-based dressings, including both their advantages and limitations. Accordingly, their potential applications in terms of their performance in vitro and in vivo, as well as their adaptability to clinical settings, were investigated. Moreover, different configurations of 3D bioprinters are discussed. Finally, a roadmap for advancing wound dressings fabricated with 3D bioprinting is presented.

Kidney Bioengineering for Transplantation.

The kidney is an important organ for maintenance of homeostasis in the human body. As renal failure progresses, renal replacement therapy becomes necessary. However, there is a chronic shortage of kidney donors, creating a major problem for transplantation. To solve this problem, many strategies for the generation of transplantable kidneys are under investigation. Since the first reports describing that nephron progenitors could be induced from human induced pluripotent stem cells, kidney organoids have been attracting attention as tools for studying human kidney development and diseases. Because the kidney is formed through the interactions of multiple renal progenitors, current studies are investigating ways to combine these progenitors derived from human induced pluripotent stem cells for the generation of transplantable kidney organoids. Other bioengineering strategies, such as decellularization and recellularization of scaffolds, 3-dimensional bioprinting, interspecies blastocyst complementation and progenitor replacement, and xenotransplantation, also have the potential to generate whole kidneys, although each of these strategies has its own challenges. Combinations of these approaches will lead to the generation of bioengineered kidneys that are transplantable into humans.

Development of Photosensitive Hydrogel-based 3-dimensional Bioprinting Using Locally Extracted Pectin From Durian Rind Waste and Cellulose for Pharmaceutical Application

Introduction: Hydrogels have gained prominence in a variety of fourth industrial revolution applications, including three-dimensional (3D) printing. However, there are limitations to 3D manufacturing, such as deformities in the final product. This is a significant obstacle to adopting this technology in the pharmaceutical industry, as printed products may have insufficient mechanical properties and a high brittleness, making further processing of these dosage forms problematic. The objective of this study is to produce a new 3D bioink from a mixture of locally produced pectin-based material from durian rind waste and cellulose-based material (pectin/cellulose hydrogel) and to partially characterize the bioink hydrogel. Methods: Four formulations of pectin/cellulose-based hydrogel (3:1, 3:2, 4:1, and 5:3 ratio of pectin/cellulose) from durian rind waste and carboxymethyl cellulose (CMC) powder, as well as cross-linking agents, were developed and evaluated using a rheometer to evaluate viscoelastic properties, FTIR Spectroscopy to identify compounds, and thermogravimetric analysis (TGA) to evaluate thermal stability. Results: All bioink formulations exhibit outstanding shear-thinning behavior suitable for 3D printing. The viscosity of edible ink increases as the pectin/cellulose concentration increases. The formulation of 3:1 pectin/cellulose has greater heat resistance than others (highest thermal stability with 21.69% of residual weight) and the lowest percentage of weight loss (76.18%). Conclusion: The study of a pectin/cellulose hydrogel mixture provides an attractive outcome for the creation of bioink due to the effective synthesis of 3D printing shapes that are both smooth and uniform.

Printability assessment of modified filament deposition modelling three dimensional bioprinter printer using polymeric formulations

Three dimensional (3D) bioprinting is a rapid prototyping technology that can be used to accurately position living cells and biomaterials called bioink, to fabricate functional living tissue constructs or organs. The bioink are deposited in 3D in a layer-by-layer manner using a bioprinter. However, commercially available 3D bioprinters are expensive, limiting the widespread adoption of this technology in low-resource laboratories. To overcome this limitation the conventional Filament Deposition Modelling (FDM) 3D printer can be modified to a 3D bioprinter by replacing the print head unit. During the makeover, The device has to perform in its most efficient capacity in synergy with the bioink used. Hence it is essential to check the bio-printabililty of bioink in the modified FDM printer. In this study we created certain specific G-codes for the evaluation of hybrid bioinks and authenticated the printability. This study focuses on quantifying the printability of a hybrid hydrogel composed of sodium salt carboxymethyl cellulose (CMC) and gelatin using a 3D bioprinter based on the RepRap prototyper. The 3D design and slicing parameters were generated using opensource software and manually edited for printability evaluation. The results of these experiments indicate the importance of printability evaluation of custom bioprinters and provide some key aspects of how to modify CAD design parameters for printability evaluation. This approach can also be adopted to evaluate the printability of other hydrogels for bio-printing. The codes are created to evaluate the printability of bioink using FDM modified bioprinters. The printability evaluation is limited to high viscous bioink for extrusion bioprinting. A user friendly, simple G-Codes and methodology for evaluation of printability of bioink using FDM modified bioprinters. To authors knowledge, this is the first report on 3D printability evaluation of bioink using FDM modified bioprinters. The study also fulfills an identified need to study printability of bioink in biofabrication by additive manufacturing.

A Century-long Journey From the Discovery of Insulin to the Implantation of Stem Cell-derived Islets.

For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing β cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing β cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature β-like cells in vitro that produce insulin but do not yet fully mimic healthy mature β cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell-derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.

SAT202 - Microfluidic 3-dimensional bioprinting and 2-dimensional micropatterns as tools to bioengineer biliary ducts

SAT202 - Microfluidic 3-dimensional bioprinting and 2-dimensional micropatterns as tools to bioengineer biliary ducts

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.

SKIN TISSUE ENGINEERING: A REVIEW

Tissue engineering (TE) for skin grafting, also known as skin tissue engineering (STE) is a strategy involving the generation of artificial skin by using widely available natural or synthetic materials as substitutes that resemble the native skin i.e., it involves in-vitro fabrication of the biocompatible scaffolds. Earlier the skin grafting needed a healthy donor making the therapy limited due to the chances of immune rejection. Besides this, skin grafting may often result in poor healing in diabetic patients and bleeding problems in the individuals suffering from hemophilia. It may often result in infection of either the donor or the recipient at transplantation site. The emergence of novel methods of TE has overcome the limitations associated with the conventional methods. Various tissues and organs like the heart, skin, lung, liver, cartilage, etc, can be regenerated using TE. TE can be facilitated with the aid of nanotechnology for the generation of scaffolds due to various properties it possess, of which, the major advantageous property involves a large surface area to volume ratio to serve wider range function as well as antimicrobial properties to prevent infections near the damaged area. Often, the different types of stem cells can be used for tissue repairing, due to their self-renewable properties. The skin mimics are often prepared using 3-dimensional bioprinting. This review deals with the applications of TE in skin grafting, typically by manipulation of naturally available materials.

The Effects of 3-Dimensional Bioprinting Calcium Silicate Cement/Methacrylated Gelatin Scaffold on the Proliferation and Differentiation of Human Dental Pulp Stem Cells.

A calcium silicate cement/methacrylated gelatin (GelMa) scaffold has been applied in tissue engineering; however, the research on its applications in dental tissue regeneration remains lacking. We investigate the effect of this scaffold on human dental pulp stem cells (hDPSCs). hDPSCs were cultured in 3D-printed GelMa and MTA-GelMa scaffolds. Cell adhesion was evaluated using scanning electron microscopy images. Cells were cultured in an osteogenic differentiation medium, which contained a complete medium or α-MEM containing aqueous extracts of the 3D-printd GelMa or MTA-GelMa scaffold with 2% FBS, 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 10 nM dexamethasone; cell viability and differentiation were shown by WST-1 assay, Alizarin Red S staining, and alkaline phosphatase staining. Quantitative real-time PCR was used to measure the mRNA expression of DSPP and DMP-1. One-way analysis of variance followed by Tukey’s post hoc test was used to determine statistically significant differences, identified at p < 0.05. hDPSCs adhered to both the 3D-printed GelMa and MTA-GelMa scaffolds. There was no statistically significant difference between the GelMa and MTA-GelMa groups and the control group in the cell viability test. Compared with the control group, the 3D-printed MTA-GelMa scaffold promoted the odontogenic differentiation of hDPSCs. The 3D-printed MTA-GelMa scaffold is suitable for the growth of hDPSCs, and the scaffold extracts can better promote odontoblastic differentiation.

Biomimetic &lt;i&gt;in vitro&lt;/i&gt; heart platforms for drug development

New drug development is currently very expensive and time-consuming. In addition, some drugs that are approved after animal and clinical trials have their approval revoked because of adverse effects. About 30% of such drugs have heart side effects. Conventional cell-based drug toxicity tests are performed under conditions entirely different from the in vivo environment, and animal testing for drug evaluation has limitations because of differences between species. Therefore, researchers are increasingly focusing on developing models that can overcome these limitations to enable accurate drug toxicity tests. This review outlines biomimetic in vitro heart platforms, such as heart organoids, 3-dimensional bioprinting, and heart-on-a-chip devices, and describes their advantages, limitations, future perspectives. The development and use of effective cardiac biomimetic models could contribute to the development of alternatives to animal testing by providing more specific information on drug metabolism and reducing the rate of failure in later stages of drug development.

Harnessing shear stress preconditioning to improve cell viability in 3D post-printed biostructures using extrusion bioprinting

Extrusion-based three dimensional bioprinting has emerged as a promising fabrication technique in the field of tissue engineering. However, it still suffers from reduced cell survival post-printing due to process-induced shear stress. Great research efforts have been made to reduce such an effect, yet none have investigated the ability of preconditioned cells to tolerate stress induced by printing. In this study, we investigated the effect of shear stress preconditioning of C2C12 murine myoblasts on post-printing cell viability using a custom-built device to induce shear stress. Here, we present the first study investigating the link between shear stress preconditioning, cellular protective mechanisms and cell survival post-printing. We hypothesized that moderate shear stress preconditioning increases the ability of cells to tolerate bioprinting induced stress, thus improving their viability after printing. We used HSP70 expression as an indicator of cellular stress and of the activation of cell adaptation/repair mechanisms. Our results showed an increase in level and a translocation of HSP70 in shear stress preconditioned cells compared to non-conditioned cells. In addition, our results demonstrated that exposure to shear stress prior to printing enhances viability of cells after printing. In particular, viability of preconditioned cells printed via a nozzle was 7.8% higher than that of non-conditioned cells while viability of preconditioned cells printed via a needle was 6.6%, higher than that of non-conditioned cells. Taken together, these findings indicate that shear stress cell preconditioning is a promising method to enhance cell viability post-printing, which is of vital importance in tissue engineering applications.

Progress and Challenges in Microengineering the Dental Pulp Vascular Microenvironment

Introduction: The dental pulp is highly vascularized and innervated tissue that is uniquely designed, being highly biologically active, while being enclosed within the calcified structure of the tooth. It is well-established that the dental pulp vasculature is a key requirement for the functional performance of the tooth. Therefore, controlled regeneration of the dental pulp vasculature is a challenge that must be met for future regenerative endeavors in endodontics. Methods: In this perspective review, we address recent progress and challenges on the use of microengineering methods and biomaterials scaffolds to fabricate the dental pulp vascular microenvironment. Results: The conditions required to control the growth and differentiation of vascular capillaries are discussed, together with the conditions required for the formation of mature and stable pericyte-supported microvascular networks in 3-dimensional hydrogels and fabricated microchannels. Recent biofabrication methods, such as 3-dimensional bioprinting and micromolding are also discussed. Moreover, recent advances in the field of organs-on-a-chip are discussed regarding their applicability to dental research and endodontic regeneration. Conclusion: Collectively, this short review offers future directions in the field that are presented with the objective of pointing toward successful pathways for successful clinical and translational strategies in regenerative endodontics, with especial emphasis on the dental pulp vasculature.

Bioprinting Organs-Progress Toward a Moonshot Idea.

Bioprinting Organs-Progress Toward a Moonshot Idea.

Current challenges in three-dimensional bioprinting heart tissues for cardiac surgery.

SummaryPrevious attempts in cardiac bioengineering have failed to provide tissues for cardiac regeneration. Recent advances in 3-dimensional bioprinting technology using prevascularized myocardial microtissues as ‘bioink’ have provided a promising way forward. This review guides the reader to understand why myocardial tissue engineering is difficult to achieve and how revascularization and contractile function could be restored in 3-dimensional bioprinted heart tissue using patient-derived stem cells.

The application and prospects of three dimensional bioprinting in urinary system reconstruction

Three dimensional (3D) bioprinting is a new biological tissue engineering technology in recent years. The development of 3D bioprinting is conducive to solving the current problems of clinical tissue and organ repairing. This article provides a review about the clinical and research status of 3D bioprinting and urinary system reconstruction. Furthermore, the feasibility and clinical value of 3D bioprinting in urinary system reconstruction will be also discussed.

In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid.

One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.