No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Bio-inks for 3D extrusion-based bio-printed scaffolds: Printability assessment
Abstract
Three-dimensional bioprinting is a new technology that should be integrated into several areas, including medical technology. However, before designing and applying it on a large scale, several biophysical parameters and particularly printability need to be established. In the present work, general characteristics of the extrusion method, bioinks, and scaffolds are reviewed. Printability analysis of 3D bioprinting is also included.
Cartilage damage is one of the main causes of disability, and 3D bioprinting technology can produce complex structures that are particularly suitable for constructing a customized and irregular tissue engineering scaffold for cartilage repair. Alginate is an attractive biomaterial for bioinks because of its good biological safety profile and fast ionic gelation. However, ionically crosslinked alginate hydrogels are recognized as lacking enough mechanical property and long-term stability due to ion exchange. Here, we developed a double crosslinked alginate (DC-Alg) hydrogel for 3D bioprinting, and human umbilical cord mesenchymal stem cells (huMSCs) could differentiate into chondrocytes on its printed 3D scaffold after 4 weeks ' culture. We performed sequential modification of alginate with L-cysteine and 5-norbornene-2-methylamine, and the DC-Alg hydrogels were obtained in the presence of CaCl2 and ultraviolet light with stronger mechanical properties than those of the single ionic crosslinked alginate hydrogels, which was similar to natural cartilage. They also had better stability and could be maintained in DMEM medium for over one month, as well good viability for huMSCs. Moreover, the DC-Alg as 3D printing inks demonsrated a better printing accuracy(~200 μm). After 4 weeks culture of huMSCs in the 3D printed DC-Alg scaffolds, the expressions of chondrogenic genes such as aggrecan (agg), collagen Ⅱ (col II), and SRY-box transcription factor 9 (sox- 9) were obviously observed, indicating the differentiation of huMSCs into cartilage. Immumohistochemical staining analysis further exhibited cartilage tissue developed well in the 3D printed scaffolds. Our study is the first demonstration of DC-Alg in 3D printing for MSC differentiation into cartilage, which shows a potential application in cartilage defect repair.
Amorphous solid dispersions (ASDs) are popular for enhancing the solubility and bioavailability of poorly water-soluble drugs. Various approaches have been employed to produce ASDs and novel techniques are emerging. This review provides an updated overview of manufacturing techniques for preparing ASDs. As physical stability is a critical quality attribute for ASD, the impact of formulation, equipment, and process variables, together with the downstream processing on physical stability of ASDs have been discussed. Selection strategies are proposed to identify suitable manufacturing methods, which may aid in the development of ASDs with satisfactory physical stability.
Understanding how cancer cells migrate, and how this migration is affected by the mechanical and chemical composition of the extracellular matrix (ECM) is critical to investigate and possibly interfere with the metastatic process, which is responsible for most cancer-related deaths. In this article we review the state of the art about the use of hydrogel-based three-dimensional (3D) scaffolds as artificial platforms to model the mechanobiology of cancer cell migration. We start by briefly reviewing the concept and composition of the extracellular matrix (ECM) and the materials commonly used to recreate the cancerous ECM. Then we summarize the most relevant knowledge about the mechanobiology of cancer cell migration that has been obtained using 3D hydrogel scaffolds, and relate those discoveries to what has been observed in the clinical management of solid tumors. Finally, we review some recent methodological developments, specifically the use of novel bioprinting techniques and microfluidics to create realistic hydrogel-based models of the cancer ECM, and some of their applications in the context of the study of cancer cell migration.
Owing to their tunable properties, controllable degradation, and ability to protect labile drugs, hydrogels are increasingly investigated as local drug delivery systems. However, a lack of standardized methodologies used to characterize and evaluate drug release poses significant difficulties when comparing findings from different investigations, preventing an accurate assessment of systems. Here, we review the commonly used analytical techniques for drug detection and quantification from hydrogel delivery systems. The experimental conditions of drug release in saline solutions and their impact are discussed, along with the main mathematical and statistical approaches to characterize drug release profiles. We also review methods to determine drug diffusion coefficients and in vitro and in vivo models used to assess drug release and efficacy with the goal to provide guidelines and harmonized practices when investigating novel hydrogel drug delivery systems.
3D bioprinting has seen a tremendous growth in recent years in a variety of fields such as tissue engineering, drug testing and regenerative medicine, which has led researchers and manufacturers to continuously advance and develop novel bioprinting techniques and materials. Although new bioprinting methods are emerging (e.g. contactless and volumetric bioprinting), micro-extrusion bioprinting remains the most widely used method. Micro-extrusion bioprinting, however, is still largely dependent on the conventional pneumatic extrusion process, which relies heavily on homogenous biomaterial inks and bioinks to maintain a constant material flow rate. Augmenting the functionality of the bioink with the addition of nanoparticles, cells or biopolymers can induce inhomogeneities resulting in uneven material flow during printing and/or clogging of the nozzle, leading to defects in the printed construct. In this work, we evaluated a novel extrusion technique based on a miniaturized progressive cavity pump which allows precise control over the volumetric flow rate by positive displacement. We compared the accuracy and precision of this system to the pneumatic extrusion system and tested both systems for their effect on cell viability after extrusion. The progressive cavity pump achieved a significantly higher accuracy and precision compared to the pneumatic system, while maintaining good viability. These improvements were independent of the bioink composition, printing speed or nozzle size. This study demonstrates the merit of precise extrusion-process control in bioprinting by progressive cavity pumps and investigates their influence on process-induced cell damage. Progressive cavity pumps are a promising tool for bioprinting and could help provide standardized and validated bioprinted constructs while leaving the researcher more freedom in the design of the bioinks.
Bioprinting technologies, which have the ability to combine various human cell phenotypes, signaling proteins, extracellular matrix components, and other scaffold-like biomaterials, are currently being exploited for the fabrication of human skin in regenerative medicine. We performed a systematic review to appraise the latest advances in 3D bioprinting for skin applications, describing the main cell phenotypes, signaling proteins, and bioinks used in extrusion platforms. To understand the current limitations of this technology for skin bioprinting, we briefly address the relevant aspects of skin biology. This field is in the early stage of development, and reported research on extrusion bioprinting for skin applications has shown moderate progress. We have identified two major trends. First, the biomimetic approach uses cell-laden natural polymers, including fibrinogen, decellularized extracellular matrix, and collagen. Second, the material engineering line of research, which is focused on the optimization of printable biomaterials that expedite the manufacturing process, mainly involves chemically functionalized polymers and reinforcement strategies through molecular blending and postprinting interventions, i.e., ionic, covalent, or light entanglement, to enhance the mechanical properties of the construct and facilitate layer-by-layer deposition. Skin constructs manufactured using the biomimetic approach have reached a higher level of complexity in biological terms, including up to five different cell phenotypes and mirroring the epidermis, dermis and hypodermis. The confluence of the two perspectives, representing interdisciplinary inputs, is required for further advancement toward the future translation of extrusion bioprinting and to meet the urgent clinical demand for skin equivalents.
Three-dimensional (3D) bioprinting as a technology is being researched and applied since 2003. It is actually several technologies (inkjet, extrusion, laser, magnetic bioprinting, etc.) under an umbrella term “3D bioprinting.” The versatility of this technology allows widespread applications in several; however, after almost 20 years of research, there is still a limited number of cases of commercialized applications. This article discusses the potential for 3D bioprinting in regenerative medicine, drug discovery, and food industry, as well as the existing cases of companies that create commercialized products and services in the aforementioned areas and even in fashion, including their go-to-market route and financing received. We also address the main barriers to creating practical applications of 3D bioprinting within each sphere the technology that is being studied for.
Three‐dimensional (3D) bioprinting has recently advanced as an important tool to produce viable constructs that can be used for regenerative purposes or as tissue models. To develop biomimetic and sustainable 3D constructs, several important processing aspects need to be considered, among which crosslinking is most important for achieving desirable biomechanical stability of printed structures, which is reflected in subsequent behavior and use of these constructs. In this work, crosslinking methods used in 3D bioprinting studies are reviewed, parameters that affect bioink chemistry are discussed, and the potential toward improving crosslinking outcomes and construct performance is highlighted. Furthermore, current challenges and future prospects are discussed. Due to the direct connection between crosslinking methods and properties of 3D bioprinted structures, this Review can provide a basis for developing necessary modifications to the design and manufacturing process of advanced tissue‐like constructs in future.
Next generation engineered tissue constructs with complex and ordered architectures aim to better mimic the native tissue structures, largely due to advances in 3D bioprinting techniques. Extrusion bioprinting has drawn tremendous attention due to its widespread availability, cost‐effectiveness, simplicity, and its facile and rapid processing. However, poor printing resolution and low speed have limited its fidelity and clinical implementation. To circumvent the downsides associated with extrusion printing, microfluidic technologies are increasingly being implemented in 3D bioprinting for engineering living constructs. These technologies enable biofabrication of heterogeneous biomimetic structures made of different types of cells, biomaterials, and biomolecules. Microfluiding bioprinting technology enables highly controlled fabrication of 3D constructs in high resolutions and it has been shown to be useful for building tubular structures and vascularized constructs, which may promote the survival and integration of implanted engineered tissues. Although this field is currently in its early development and the number of bioprinted implants is limited, it is envisioned that it will have a major impact on the production of customized clinical‐grade tissue constructs. Further studies are, however, needed to fully demonstrate the effectiveness of the technology in the lab and its translation to the clinic.
It is difficult to fabricate tubular-shaped tissues and organs (e.g., trachea, blood vessel, and esophagus tissue) with traditional biofabrication techniques (e.g., electrospinning, cell-sheet engineering, and mold-casting) because these have complicated multiple processes. In addition, the tubular-shaped tissues and organs have their own design with target-specific mechanical and biological properties. Therefore, the customized geometrical and physiological environment is required as one of the most critical factors for functional tissue regeneration. 3D bioprinting technology has been receiving attention for the fabrication of patient-tailored and complex-shaped free-form architecture with high reproducibility and versatility. Printable biocomposite inks that can facilitate to build tissue constructs with polymeric frameworks and biochemical microenvironmental cues are also being actively developed for the reconstruction of functional tissue. In this review, we delineated the state-of-the-art of 3D bioprinting techniques specifically for tubular tissue and organ regeneration. In addition, this review described biocomposite inks, such as natural and synthetic polymers. Several described engineering approaches using 3D bioprinting techniques and biocomposite inks may offer beneficial characteristics for the physiological mimicry of human tubular tissues and organs.
Bioprinting is an emerging and promising technique for fabricating 3D cell-laden constructs for various biomedical applications. In this paper, we employed 3D bioprinted GelMA-based models to investigate the trophoblast cell invasion phenomenon, enabling studies of key placental functions. Initially, a set of optimized material and process parameters including GelMA concentration, UV crosslinking time and printing configuration were identified by systematic, parametric study. Following this, a multiple-ring model (2D multi-ring model) was tested with the HTR-8/SVneo trophoblast cell line to measure cell movement under the influence of EGF (chemoattractant) gradients. In the multi-ring model, the cell front used as a cell invasion indicator moves at a rate of 85 ± 33 µm/day with an EGF gradient of 16 µM. However, the rate was dramatically reduced to 13 ± 5 µm/day, when the multi-ring model was covered with a GelMA layer to constrain cells within the 3D environment (3D multi-ring model). Due to the geometric and the functional limitations of multi-ring model, a multi-strip model (2D multi-strip model) was developed to investigate cell movement in the presence and absence of the EGF chemoattractant. The results show that in the absence of an overlying cell-free layer of GelMA, movement of the cell front shows no significant differences between control and EGF-stimulated rates, due to the combination of migration and proliferation at high cell density (6 × 106 cells/ml) near the GelMA surface. When the model was covered by a layer of GelMA (3D multi-strip model) and migration was excluded, EGF-stimulated cells showed an invasion rate of 21 ± 3 µm/day compared to the rate for unstimulated cells, of 5 ± 4 µm/day. The novel features described in this report advance the use of the 3D bioprinted placental model as a practical tool for not only measurement of trophoblast invasion but also the interaction of invading cells with other tissue elements.
During the bioprinting processes that employ either pneumatic or screw-driven mechanisms, living cells are subject to process-induced forces, which may cause cell injury or damage. However, the similarities and differences between these two mechanisms have not been discovered and documented in terms of process-induced forces and cell damage. In this paper, we examined the process-induced forces, including hydrostatic pressure, shear stress, extensional stress, and tensile/compressive forces that the cells experienced during the bioprinting processes by means of these two mechanisms; we also experimentally investigated the process-induced cell damage (featured by the rupture of the cell membrane) under various printing conditions or factors, including the volumetric flow rates, cell types, bioink solutions, needle types and sizes, and printing head-movement speeds. On this basis, we correlated the percent of cell damage to the process-induced forces, which were considered mainly responsible for the rupture of the cell membrane. Our results illustrate that compared to the pneumatic bioprinting process, the screw-driven bioprinting process generally induces more cell damage, varying with the printing conditions. This study, for the first time, discovers the similarities and differences between the pneumatic and screw-driven bioprinting processes and further demonstrates their merits and demerits for bioprinting in terms of printing-process control, process-induced forces, and cell damage.
Nowadays, biopolymers as intelligent and active biopolymer systems in the food and pharmaceutical industry are of considerable interest in their use. With this association in view, biopolymers such as chitosan, alginate, pectin, cellulose, agarose, guar gum, agar, carrageenan, gelatin, dextran, xanthan, and other polymers have received significant attention in recent years due to their abundance and natural availability. Furthermore, their versatile properties such as non-toxicity, biocompatibility, biodegradability, and flexibility offer significant functionalities with multifunctional applications. The purpose of this review is to summarize the most compatible biopolymers such as chitosan, alginate, and pectin, which are used for application in food, biotechnological processes, and biomedical applications. Therefore, chitosan, alginate, and pectin are biopolymers (used in the food industry as a stabilizing, thickening, capsular agent, and packaging) with great potential for future developments. Moreover, this review highlights their characteristics, with a particular focus on their potential for biocompatibility, biodegradability, bioadhesiveness, and their limitations on certain factors in the human gastrointestinal tract.
Hyaluronic acid (HA)-based hydrogels are widely used in biomedical applications due to their excellent biocompatibility. HA can be Ultraviolet (UV)-crosslinked by modification with methacrylic anhydride (HA-MA) and crosslinked by modification with 3,3′-dithiobis(propionylhydrazide) (DTP) (HA-SH) via click reaction. In the study presented in this paper, a 3D-bioprinted, double-crosslinked, hyaluronic-acid-based hydrogel for wound dressing was proposed. The hydrogel was produced by mixing HA-MA and HA-SH at different weight ratios. The rheological test showed that the storage modulus (G’) of the HA-SH/HA-MA hydrogel increased with the increase in the HA-MA content. The hydrogel had a high swelling ratio and a high controlled degradation rate. The in vitro degradation test showed that the hydrogel at the HA-SH/HA-MA ratio of 9:1 (S9M1) degraded by 89.91% ± 2.26% at 11 days. The rheological performance, drug release profile and the cytocompatibility of HA-SH/HA-MA hydrogels with loaded Nafcillin, which is an antibacterial drug, were evaluated. The wound dressing function of this hydrogel was evaluated by Live/Dead staining and CCK-8 assays. The foregoing results imply that the proposed HA-SH/HA-MA hydrogel has promise in wound repair applications.
Over the last decades, the fabrication of 3D tissues has become commonplace in tissue engineering and regenerative medicine. However, conventional 3D biofabrication techniques such as scaffolding, microengineering, and fiber and cell sheet engineering are limited in their capacity to fabricate complex tissue constructs with the required precision and controllability that is needed to replicate biologically relevant tissues. To this end, 3D bioprinting offers great versatility to fabricate biomimetic, volumetric tissues that are structurally and functionally relevant. It enables precise control of the composition, spatial distribution, and architecture of resulting constructs facilitating the recapitulation of the delicate shapes and structures of targeted organs and tissues. This Review systematically covers the history of bioprinting and the most recent advances in instrumentation and methods. It then focuses on the requirements for bioinks and cells to achieve optimal fabrication of biomimetic constructs. Next, emerging evolutions and future directions of bioprinting are discussed, such as freeform, high‐resolution, multimaterial, and 4D bioprinting. Finally, the translational potential of bioprinting and bioprinted tissues of various categories are presented and the Review is concluded by exemplifying commercially available bioprinting platforms.
Cardiovascular disease is the leading cause of mortality worldwide. Atherosclerosis, one of the most common forms of the disease, is characterized by a gradual formation of atherosclerotic plaque, hardening, and narrowing of the arteries. Nanomaterials can serve as powerful delivery platforms for atherosclerosis treatment. However, their therapeutic efficacy is substantially limited in vivo due to nonspecific clearance by the mononuclear phagocytic system. In order to address this limitation, rapamycin (RAP)‐loaded poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles are cloaked with the cell membrane of red blood cells (RBCs), creating superior nanocomplexes with a highly complex functionalized bio‐interface. The resulting biomimetic nanocomplexes exhibit a well‐defined “core–shell” structure with favorable hydrodynamic size and negative surface charge. More importantly, the biomimetic nature of the RBC interface results in less macrophage‐mediated phagocytosis in the blood and enhanced accumulation of nanoparticles in the established atherosclerotic plaques, thereby achieving targeted drug release. The biomimetic nanocomplexes significantly attenuate the progression of atherosclerosis. Additionally, the biomimetic nanotherapy approach also displays favorable safety properties. Overall, this study demonstrates the therapeutic advantages of biomimetic nanotherapy for atherosclerosis treatment, which holds considerable promise as a new generation of drug delivery system for safe and efficient management of atherosclerosis.
Generation of thick vascularized tissues that fully match the patient still remains an unmet challenge in cardiac tissue engineering. Here, a simple approach to 3D‐print thick, vascularized, and perfusable cardiac patches that completely match the immunological, cellular, biochemical, and anatomical properties of the patient is reported. To this end, a biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to become pluripotent stem cells, and differentiated to cardiomyocytes and endothelial cells, the extracellular matrix is processed into a personalized hydrogel. Following, the two cell types are separately combined with hydrogels to form bioinks for the parenchymal cardiac tissue and blood vessels. The ability to print functional vascularized patches according to the patient's anatomy is demonstrated. Blood vessel architecture is further improved by mathematical modeling of oxygen transfer. The structure and function of the patches are studied in vitro, and cardiac cell morphology is assessed after transplantation, revealing elongated cardiomyocytes with massive actinin striation. Finally, as a proof of concept, cellularized human hearts with a natural architecture are printed. These results demonstrate the potential of the approach for engineering personalized tissues and organs, or for drug screening in an appropriate anatomical structure and patient‐specific biochemical microenvironment. A small biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to induce pluripotent stem cells and differentiate to cardiac and endothelial cells, the extra‐cellular matrix is processed into a thermoresponsive, hydrogel‐based bioink. These components are used to 3D‐print functional vascularized cardiac patches and even small scale, whole cellularized human hearts.
Recent advances in regenerative medicine have confirmed the potential to manufacture viable and effective tissue engineering 3D constructs comprising living cells for tissue repair and augmentation. Cell printing has shown promising potential in cell patterning in a number of studies enabling stem cells to be precisely deposited as a blueprint for tissue regeneration guidance. Such manufacturing techniques, however, face a number of challenges including; (i)post-printing cell damage, (ii)proliferation impairment and, (iii)poor or excessive final cell density deposition. The use of hydrogels offers one approach to address these issues given the ability to tune these biomaterials and subsequent application as vectors capable of delivering cell populations and as extrusion pastes. While stem cell-laden hydrogel 3D constructs have been widely established in vitro, clinical relevance, evidenced by in vivo long-term efficacy and clinical application, remains to be demonstrated. This review explores the central features of cell printing, cell-hydrogel properties and cell-biomaterial interactions together with the current advances and challenges in stem cell printing. A key focus is the translational hurdles to clinical application and how in vivo research can reshape and inform cell printing applications for an ageing population.
Recently, 3D bioprinting is developed as an emerging approach, increasingly applied to materials for healthcare; while, the precise placement of cells and materials, and the shape fidelity of forming constructs is of great importance for successful application of 3D bioprinting. Research efforts have been made to develop new bioinks as “raw materials” with better biocompatibility and biofunctionality, but the printability of bioinks is largely ignored and still needs to be carefully examined to enable robotic bioprinting. This article aims to introduce a recent published review (Appl. Phys. Rev. 2018, 5, 041304) on the evaluation of bioink printability by Huang’s research group from University of Florida. Huang et al. comprehensively reviewed the bioink printability based on the physical point of view during inkjet printing, laser printing, and microextrusion, and a series of self-consistent time scales and dimensionless quantities were utilized to physically understand and evaluate bioink printability. This article would be helpful to know the trends on physical understanding of bioink printability.
Purpose:
Mesenchymal stem cells (MSCs) have demonstrated great promises for the treatment of ischemic stroke. Previously, we identified a new source of MSCs located in the inferior turbinate. We investigated therapeutic potentials of human turbinate- derived mesenchymal stem cells (hTMSCs) in ischemic stroke.
Methods:
Ischemic stroke was induced by the intraluminal occlusion of middle cerebral artery (MCAo) for 50 minutes in rats. At one day after MCAo, hTMSCs, adipose tissue-derived MSCs (AdMSCs), or phosphate buffered saline (PBS) were transplanted into the striatum. Functional recovery was assessed by repeating behavioral tests including modified neurologic severity score and corner test. At 14 days after MCAo, brains were stained with hematoxylin and eosin (H&E) for measuring infarct volume. The survival of grafted MSCs was evaluated by immunohistochemistry to human nuclei (hNU). Immunohistochemistry with anti-doublecortin (anti-DCX) was performed to assess hippocampal neurogenesis.
Results:
Transplantation of hTMSCs following MCAo showed improvements of neurologic function, which was comparable with that of AdMSCs. H&E staining showed no difference in infarct volume among 3 groups. Regarding the survival of grafted MSCs, the number of hNU-expressing cells was not different between hTMSCs- and AdMSCs-treated groups. Finally, hTMSCs increased the number of subgranular DCX-positive cells compared to PBS-treated controls, without affecting hilar ectopic migration of newborn neurons.
Conclusion:
hTMSCs could improve functional recovery following ischemic stroke, of which efficacy was similar to AdMSCs. Although hTMSCs showed comparable infarct size and survival of grafted MSCs, transplantation of hTMSCs could upregulate subgranular neurogenesis with no impact on ectopically migrating newborn neurons.
Stem cell transplantation has been recognized as a promising strategy to induce the regeneration of injured and diseased tissues and sustain therapeutic molecules for prolonged periods in vivo. However, stem cell-based therapy is often ineffective due to low survival, poor engraftment, and a lack of site-specificity. Hydrogels can offer several advantages as cell delivery vehicles, including cell stabilization and the provision of tissue-like environments with specific cellular signals; however, the administration of bulk hydrogels is still not appropriate to obtain safe and effective outcomes. Hence, stem cell encapsulation in uniform micro-sized hydrogels and their transplantation in vivo have recently garnered great attention for minimally invasive administration and the enhancement of therapeutic activities of the transplanted stem cells. Several important methods for stem cell microencapsulation are described in this review. In addition, various natural and synthetic polymers, which have been employed for the microencapsulation of stem cells, are reviewed in this article.
Residing under the umbrella term of ‘material extrusion’, semi-solid extrusion (SSE) 3D printing deposits gels or pastes in sequential layers to create a solid object. The physical nature of the feedstock allows SSE to print quickly at low temperatures with little compromise in accuracy. As a result, the technology has been extensively adopted in the field of bioprinting in which SSE can print living cells able to create large, complex structures of human tissue. In terms of pharmaceutical formulation, SSE is relatively unused. By building on the advancements made in other fields, the unique attributes of SSE printing are beginning to come to the fore. In particular, the ability of SSE to create complex tablets at a low heat means the technology has the potential to make on demand, personalised dosages within a clinical setting. As a result, the infancy of SSE in pharmaceutics should not be seen as a sign of inferiority but rather a real opportunity to bring 3D printing into the clinic.
3D printing, an additive manufacturing based technology for precise 3D construction, is currently widely employed to enhance applicability and function of cell laden scaffolds. Research on novel compatible biomaterials for bioprinting exhibiting fast crosslinking properties is an essential prerequisite toward advancing 3D printing applications in tissue engineering. Printability to improve fabrication process and cell encapsulation are two of the main factors to be considered in development of 3D bioprinting. Other important factors include but are not limited to printing fidelity, stability, crosslinking time, biocompatibility, cell encapsulation and proliferation, shear-thinning properties, and mechanical properties such as mechanical strength and elasticity. In this review, we recite recent promising advances in bioink development as well as bioprinting methods. Also, an effort has been made to include studies with diverse types of crosslinking methods such as photo, chemical and ultraviolet (UV). We also propose the challenges and future outlook of 3D bioprinting application in medical sciences and discuss the high performance bioinks.
Background
The worldwide demand for the organ replacement or tissue regeneration is increasing steadily. The advancements in tissue engineering and regenerative medicine have made it possible to regenerate such damaged organs or tissues into functional organ or tissue with the help of 3D bioprinting. The main component of the 3D bioprinting is the bioink, which is crucial for the development of functional organs or tissue structures. The bioinks used in 3D printing technology require so many properties which are vital and need to be considered during the selection. Combination of different methods and enhancements in properties are required to develop more successful bioinks for the 3D printing of organs or tissue structures.
Main body
This review consists of the recent state-of-art of polymer-based bioinks used in 3D printing for applications in tissue engineering and regenerative medicine. The subsection projects the basic requirements for the selection of successful bioinks for 3D printing and developing 3D tissues or organ structures using combinations of bioinks such as cells, biomedical polymers and biosignals. Different bioink materials and their properties related to the biocompatibility, printability, mechanical properties, which are recently reported for 3D printing are discussed in detail.
Conclusion
Many bioinks formulations have been reported from cell-biomaterials based bioinks to cell-based bioinks such as cell aggregates and tissue spheroids for tissue engineering and regenerative medicine applications. Interestingly, more tunable bioinks, which are biocompatible for live cells, printable and mechanically stable after printing are emerging with the help of functional polymeric biomaterials, their modifications and blending of cells and hydrogels. These approaches show the immense potential of these bioinks to produce more complex tissue/organ structures using 3D bioprinting in the future.
Featured Application
Bioprinting of complex cell-laden tissue constructs that mimic the human vascular tissue structure.
Abstract
Microextrusion-based bioprinting within a support bath material is an emerging additive manufacturing paradigm for complex three-dimensional (3D) tissue construct fabrication. Although a support bath medium enables arbitrary in-process geometries to be printed, a significant challenge lies in preserving the shape fidelity upon the extraction of the support bath material. Based on the bioprinting in a support bath paradigm, this paper advances quantitative analyses to systematically determine the printability of cell-laden liquid hydrogel precursors towards filament-based tissue constructs. First, a yield stress nanoclay material is judiciously selected as the support bath medium owing to its insensitivity to temperature and ionic variations that are considered in the context of the current gelatin-alginate bio-ink material formulation. Furthermore, phenomenological observations for the rheology-mediated print outcomes enable the compositions for the bio-ink material (10% gelatin, 3% alginate), in tandem with the support bath medium (4% nanoclay, 0.5% CaCl2), to be identified. To systematically evaluate the performance outcomes for bioprinting within a support bath, this paper advances an experimental parametric study to optimize the 3D structural shape fidelity by varying parameters such as the layer height, extrusion flowrate, printing temperature, and printhead speed, towards fabricating complex 3D structures with the stabilization of the desired shape outcome. Specifically, it is found that the layer height and printhead speed are determinant parameters for the extent of successive layer fusion. Moreover, maintenance of an optimal bath temperature is identified as a key parameter for establishing the printability for the hydrogel bio-ink. Studying this effect is enabled by the custom design of a PID temperature control system with integration with the bioprinter for real-time precision control of the support bath temperature. In order to qualify the printed construct, a surface irregularity metric, defined as the average height difference between consecutive local maximum and minimum points of the binary image contour for the printed structure, is advanced to evaluate the quality of the printed constructs. Complex one-to-four bifurcating tubular structure prints demonstrate the applicability of the optimized bioprinting parameter space to create exemplar 3D human vessel-like structures. Finally, a cell viability assay and perfusion test for a printed cell-laden tubular element demonstrates high cell survival rates and leakage-free flow, respectively.
Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccpAx). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000–3000 μm and a diameter of 10–20 μm, which were remarkably larger than the fibres of BC typically produced by G. hansenii.
Electronic supplementary material
The online version of this article (10.1007/s00449-017-1864-1) contains supplementary material, which is available to authorized users.
Stem cells are recognized by their self-renewal ability and can give rise to
specialized progeny. Hydrogels are an established class of biomaterials with
the ability to control stem cell fate via mechanotransduction. They can mimic
various physiological conditions to influence the fate of stem cells and are an
ideal platform to support stem cell regulation. This review article provides a
summary of recent advances in the application of different classes of hydrogels
based on their source (e.g., natural, synthetic, or hybrid). This classification is
important because the chemistry of substrate affects stem cell differentiation
and proliferation. Natural and synthetic hydrogels have been widely used in
stem cell regulation. Nevertheless, they have limitations that necessitate a
new class of material. Hybrid hydrogels obtained by manipulation of the
natural and synthetic ones can potentially overcome these limitations and
shape the future of research in application of hydrogels in stem cell regulation.
The awareness of global warming, fossil scarcity and pollution are some current eminent burdens which are necessary to be acknowledged in order to find alternative solutions to mitigate its detrimental impacts. Taking this into account, this article focuses on displaying a historical overview, critical trends, recent challenges and employment of vegetal biomass as feedstock for the sustainable and environmentally friendly production of promising microbial-derived molecules through fermentative strategies. Among, bioethanol, xylitol, biopolymers, biosurfactants, organic acids and others such as butanol, butanediol, single cell protein and biopigments will be elucidated. Nevertheless, each one of these biomolecules presents specific applications.
3D printing technology has grown exponentially since its introduction due to its ability to print complex structures quickly and simply. The ink used in 3D printers is one of the most discussed areas and a variety of hydrogel-based inks were developed. Carboxymethyl cellulose (CMC) is derived from cellulose, which is a natural, biocompatible, biodegradable, and wildly abounded biopolymer. CMC is a very qualified candidate in the preparation of hydrogels because it has good solubility in water with multiple carboxyl groups. Various physical and chemical cross-linking methods and mechanisms have been used by researchers to prepare CMC-based hydrogels. Bioprinting is a powerful technology for tissue engineering applications that have been able to design and simulate different tissue and organs with digital control. Among many advantages, which were reported for bioprinting, its high throughput, as well as precise control of scaffolding and cells, is very valuable. Considering all these tips and capabilities, in this study, the methods of preparation and improvement of CMC-based hydrogels, applied 3D printer, and the latest inks designed using this biopolymer in terms of combination, features, and performance in tissue engineering are reported.
Cancer research depends on the challenging task of producing representative and reliable models of human disease; these have largely been limited to mouse models or human cancer cell lines cultured in monolayers. Three-dimensional (3D) cell culture offers more realistic options, but conventional 3D models still fail to recreate the human tumor microenvironment. One biofabrication technique that has emerged as a powerful tool is 3D bioprinting, which can generate tumor constructs with increasing complexity. By incorporating factors like stromal cells, vasculature, hydrogels, and functional molecules into the bioprinting process, researchers are now able to create human tumor models that quite realistically represent human glioblastoma, breast, cervical, ovarian, hepatoma, lung, colon, and oral cancers. The obtained structures range from coaxially extruded fibers and monolayered grids to cylinders, cubes, discs, beads, and even mini-organs. Here, we discuss recent advances in cancer research based on 3D bioprinting. Our aim is to provide a broad perspective of the possibilities provided by this biofabrication technique for the generation of complex tumor models. We also review the different structures and characterization techniques used with these models. The use of 3D bioprinted tumors is increasing in areas like tumor biology, migration, invasion, and metastasis, as well as in pharmaceutical testing and even personalized medicine. Future work will involve improvement of the mechanical properties and chemical cues provided to the cells within the 3D constructs. The inclusion of several cell types within a single construct will upgrade current recapitulations of real tumor tissues. Bioprinting of cells cultured from patients’ own biopsies will generate personalized models of the tumor niche.
People with weakened immune systems are at risk of developing candidiasis which is a fungal infection caused by several species of Candida genus. In this work, polymeric nanoparticles containing miconazole nitrate and the anesthetic lidocaine clorhydrate were developed. Miconazole was chosen as a typical drug to treat buccopharyngeal candidiasis whereas lidocaine may be useful in the management of the pain burning, and pruritus caused by the infection. Nanoparticles were synthesized using chitosan and gelatin at different ratios ranging from 10:90 to 90:10. The nano-systems presented nanometric size (between 80 and 300 nm in water; with polydispersion index ranging from 0.120 to 0.596), and positive Z potential (between 20.11 and 37.12 mV). The determined encapsulation efficiency ranges from 65 to 99% or 34 to 91% for miconazole nitrate and lidocaine clorhydrate, respectively. X-ray diffraction and DSC analysis suggested that both drugs were in amorphous state in the nanoparticles. Finally, the systems fitted best the Korsmeyer-Peppas model showing that the release from the nanoparticles was through diffusion allowing a sustained release of both drugs and prolonged the activity of miconazole nitrate over time against Candida albicans for at least 24 h.
Polyethylene glycols (PEGs) or macrogols are hydrophilic polymers found in everyday products such as foods, cosmetics, and medications. We present 5 cases of confirmed PEG allergy, which to our knowledge is the largest case series to date. Four of the 5 cases developed anaphylaxis to medications containing PEGs, with 1 near-fatal case resulting in cardiac arrest. Skin tests were undertaken to the index medications and to PEGs of different molecular weights. Three were confirmed with positive skin prick test result to PEG, 1 confirmed with a positive intradermal test result, and 1 confirmed after positive oral challenge. Two patients developed anaphylaxis following intradermal test to PEG and 1 a systemic allergic reaction (without hypotension or respiratory distress) following PEG skin prick tests. Before diagnosis, all 5 patients were mislabeled as allergic to multiple medications and their clinical management had become increasingly challenging. An algorithm is proposed to safely investigate suspected PEG allergy, with guidance on PEG molecular weights and skin test dilutions to minimize the risk of systemic allergic reaction. Investigation carries considerable risk without knowledge and informed planning so should only be conducted in a specialist drug allergy center.
The advancement of 3D printing techniques has given rise to tissue engineering in the field of biological sciences due to the availability of biocompatible natural and synthetic polymers. The ability to use various polymers of different properties has led to the establishment of several printing approaches capable of accommodating the desired properties of the selected polymer to print either a scaffold or a tissue in-vitro. A very common synthetic polymer used in 3D printing is poly-ε-caprolactone (PCL). PCL being a multifaceted thermoplastic polymer possesses exceptional mechanical properties that have made it a favourite among the biomedical researchers. It is a well-known fact that PCL can be printed by a wide array of 3D printing techniques, however the application of PCL in bioprinting is still a new concept. In this review, we have explored: any limiting properties of PCL which could hinder its use in bioprinting; the various techniques of modification of PCL that allow it be bioprinted; and the suitable bioprinter for bioprinting PCL. We further investigated the impact of using PCL in bioprinting and the properties it renders to the construct, observing that PCL is seldomly used to encapsulate cells, instead it is used as supporting polymer which is co-printed in extrusion based bioprinting. Moreover, these PCL reinforced grafts have shown promising results in terms of cell viability, adherence, proliferation and differentiation.
3D bioprinting technology has been an interest in constructing tissue engineering scaffolds due to its high resolution and repeatability, ability to fabricate complex construct, and mimic 3D microenvironment of the cells. Bioinks are the basic building blocks for the manufacture of 3D bioprinted constructs. Bioink plays a crucial role in building advanced 3D structures and as well as adhesion, proliferation, and differentiation of incorporated cells to achieve functional constructs. In this review, we introduce scaffold-free based bioinks, including living cell-only aggregations without any biomaterial carrier for functional tissue formation. Cell aggregations in the forms of spheroid and strand are popular as bioink to fabricate 3D printed constructs. The different methods of cell aggregations fabrication are discussed in detail. Then the current advantages and disadvantages of scaffold-free bioink, as well as the future prospects to overcome the limitations are presented.
Most available 3D biofabrication technologies rely on single-component deposition methods, such as inkjet, extrusion, or light-assisted printing. It is unlikely that any of these technologies used individually would be able to replicate the complexity and functionality of living tissues. Recently, new biofabrication approaches have emerged that integrate multiple manufacturing technologies into a single biofabrication platform. This has led to fabricated structures with improved functionality. In this review, we provide a comprehensive overview of recent advances in the integration of different manufacturing technologies with the aim to fabricate more functional tissue structures. We provide our vision on the future of additive manufacturing (AM) technology, digital design, and the use of artificial intelligence (AI) in the field of biofabrication.
Developing green and non-toxic biomaterials, derived from renewable sources and processable through 3D bioprinting technologies, is an emerging challenge of sustainable tissue engineering. Here, pectin from citrus peels was crosslinked for the first time with (3-Glycidyloxypropyl)trimethoxysilane (GPTMS) through a one-pot procedure. Freeze-dried porous pectin sponges, with tunable properties in terms of porosity, swelling degree and compressive modulus, were obtained by controlling GPTMS content. Cell experiments showed that GPTMS did not affect the cytocompatibility of pectin. The addition of GPTMS improved the printability of pectin due to an increase of viscosity and yield stress. Three-dimensional woodpile and complex anatomical shaped scaffolds with interconnected micro- and macro-pores were therefore bioprint-ed without the use of any additional support material. These results show the great potential of using pectin crosslinked with GPTMS as biomaterial ink to fabricate patient specific scaffolds, which could be used to promote tissue regeneration in vivo.
Hydrogel plays a vital role in cell-laden three dimensional (3D) bioprinting, whereas those hydrogels mimic the physical and biochemical characteristics of native extracellular matrix (ECM). The complex microenvironment of the ECM does not replicate from the traditional static microenvironment of the hydrogel, but the evolution of the 3D bioprinting facilitates to accommodate the dynamic modulation and spatial heterogeneity of the hydrogel system. Selection of hydrogel for 3D bioprinting depends on the printing techniques including microextrusion, inkjet, laser-assisted printing, and stereolithography. In this review, we specifically cover the 3D printable hydrogels where cells can be encapsulated without significant reduction in the cell viability. The recent research highlights of the most widely used hydrogel materials are elucidated in terms of stability of the hydrogel system, cross-linking method, support cell types and their post-printing cell viability. Also, the techniques used to improve the mechanical and biological properties of the hydrogels, such as adding various organic and inorganic materials and making microchannels, are discussed. Furthermore, the recent advances in vascularized tissue construct and scaffold-free bioprinting as a promising method for vascularization are covered in this review. The recent trends in four-dimensional (4D) bioprinting as a stimuli-responsive formation of new organs, and 3D bioprinting based organ-on-chip systems are also discussed.
In this study, we designed a polyvinyl alcohol (PVA)‐alginate based hydrogel and evaluated its cytocompatibility and printability. The samples were fabricated by 3D printing using a freeze–thaw process. The scanning electron microscope, material testing machine, rheometer, and cell counting kit‐8 assay were used to examine the morphology, mechanical properties, rheological properties, and cytocompatiblity of the scaffolds, respectively. The mechanical strength, cytocompatiblity, crosslinking time, and printability were remarkably improved with the use of PVA. To sum up, our data suggest that hybrid bio‐ink is more appropriate for precise 3D bioprinting due to its rapid prototyping capability and better cytocompatibility.
We present the first cell attachable and visible light crosslinkable hydrogels based on gelatin methacryloyl (GelMA) with eosin Y (EY) photoinitiation for stereolithography 3D bioprinting. In order to develop visible a light crosslinkable hydrogel, we systematically studied five combinations of the GelMA and EY photoinitiator with various concentrations. Their mechanical properties, microstructures, and cell viability and confluency after encapsulation were investigated rigorously to elucidate the effects of the EY and GelMA macromer concentration on the characteristics of the hydrogel. Experimental results show that the compressive Young's Modulus and pore size are positively affected by the concentration of EY, while the mass swelling ratio and cell viability are negatively affected. Increasing the concentration of GelMA helps to improve the compressive Young's Modulus and cell attachment. We further employed the developed visible light-based stereolithography bioprinting system to print the patterned cell-laden hydrogels to demonstrate the bioprinting applications of the developed hydrogel. We observed good cell proliferation and the formation of a 3D cellular network inside the printed pattern at day 5, which proves the great feasibility of using EY-GelMA as the bioinks for biofabrication and tissue engineering.
Large-scale additive manufacturing processes for construction utilise computer-controlled placement of extruded cement-based mortar to create physical objects layer-by-layer. Demonstrated applications include component manufacture and placement of in-situ walls for buildings. These applications vary the constraints on design parameters and present different technical issues for the production process. In this paper, published and new work are utilised to explore the relationship between fresh and hardened paste, mortar, and concrete material properties and how they influence the geometry of the created object. Findings are classified by construction application to create a matrix of issues that identifies the spectrum of future research exploration in this emerging field.
Three-dimensional printing (3DP) is gaining momentum in the field of pharmaceuticals, offering innovative opportunities for medicine manufacture. Selective laser sintering (SLS) is a novel, high resolution and single-step printing technology that we have recently introduced to the pharmaceutical sciences. The aim of this work was to use SLS 3DP to fabricate printlets (3D printed tablets) with cylindrical, gyroid lattice and bi-layer structures having customisable release characteristics. Paracetamol-loaded constructs from four different pharmaceutical grade polymers, including polyethylene oxide, Eudragit (L100-55 and RL) and ethyl cellulose, were created using SLS 3DP. The novel gyroid lattice structure was employed to modulate the drug release from all four polymers. This work is the first to demonstrate the feasibility of using SLS to achieve customised drug release properties of several polymers, in a swift, cost-effective process, avoiding the need to alter the formulation composition. As such, by creating these constructs, it is possible to modify drug release, which in practice could enable the tailoring of drug performance to the patient simply by changing the 3D design.
The manufacture of immediate release high drug loading paracetamol oral tablets was achieved using an extrusion based 3D printer from a premixed water based paste formulation. The 3D printed tablets demonstrate that a very high drug (paracetamol) loading formulation (80% w/w) can be printed as an acceptable tablet using a method suitable for personalisation and distributed manufacture. Paracetamol is an example of a drug whose physical form can present challenges to traditional powder compression tableting. Printing avoids these issues and facilitates the relatively high drug loading. The 3D printed tablets were evaluated for physical and mechanical properties including weight variation, friability, breaking force, disintegration time, and dimensions and were within acceptable range as defined by the international standards stated in the United States Pharmacopoeia (USP). X-Ray Powder Diffraction (XRPD) was used to identify the physical form of the active. Additionally, XRPD, Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC) were used to assess possible drug-excipient interactions. The 3D printed tablets were evaluated for drug release using a USP dissolution testing type I apparatus. The tablets showed a profile characteristic of the immediate release profile as intended based upon the active/excipient ratio used with disintegration in less than 60 seconds and release of most of the drug within 5 minutes. The results demonstrate the capability of 3D extrusion based printing to produce acceptable high-drug loading tablets from approved materials that comply with current USP standards.
Alginates are natural exopolysaccharides produced by seaweeds and bacteria belonging to the genera Pseudomonas and Azotobacter. Due to exhibiting unique physicochemical properties, they have been widely applied for various industrial purposes such as in food, agricultural, cosmetic, pharmaceutical, and biomedical industries. In the last two decades, they have found their way into the advanced pharmaceutical and biomedical applications, owing to their biocompatibility and non-toxicity as well as versatility in view of modifications. So far, algal alginates have been the sole commercialized products applied for various purposes, while the potential uses of bacterial alginates remain unharnessed. Importantly, algal and bacteria alginates differ substantially from each other with respect to their composition, modifications, molecular mass, viscoelastic properties, and polydispersity. Indeed, bacterial alginates may meet current needs in the field of advanced pharmaceutical and biomedical engineering. In this chapter, after a brief overview of alginate discovery, general properties, applications, and comparative assessment of algal and bacterial resources, current findings about the biosynthesis of alginates, mainly in bacteria, will be discussed. Furthermore, we will discuss the current understanding of alginate polymerizing and modifying enzymes and their structure-function relationship. Knowledge about alginate biosynthesis/modification enzymes provides foundation for rational design of cell factories for producing tailor-made alginates. As a conclusion, advanced understanding of alginate biosynthesis pathway and involved enzymes creates an opportunity for bioengineering and synthetic biology approaches toward the production of alginates exhibiting desired material properties suitable for pharmaceutical and biomedical applications.
Gelatin Methacrylate (GelMA) is an inexpensive, photocrosslinkable, cell-responsive hydrogel which has drawn attention for a wide range of tissue engineering applications. The potential of GelMA scaffolds was demonstrated to be tunable for different TE applications through modifying the polymer concentration, methacrylation degree or UV light intensity. Despite the promising results of GelMA hydrogels in tissue engineering, the influence of polymer concentration for bone tissue engineering scaffolds was not established yet. Thus, in this study, we have demonstrated the effect of polymer concentration in GelMA scaffolds on osteogenic differentiation. We prepared GelMA scaffolds with 5 and 10% polymer concentrations and characterized the scaffolds in terms of porosity, pore size, swelling characteristics and mechanical properties. Subsequent to the scaffolds characterization, the scaffolds were seeded with bone marrow drived rat mesenchymal stem cells and cultured in osteogenic media to evaluate the possible osteogenic differentiation effect exerted by the polymer concentration. After 7, 14, 21 and 28 days, DNA content, calcium deposition and ALP activity of scaffolds were evaluated quantitatively by colorimetric bioassays. Furthermore, the distribution of the calcium deposition within the scaffolds was attained qualitatively and quantitatively by micro computer tomography (µCT). Our data suggests, GelMA hydrogels prepared with 5% polymer concentration has promoted homogeneous extracellular matrix calcification and it is a great candidate for bone tissue engineering applications. This article is protected by copyright. All rights reserved.
In the last decade, interest in the field of three-dimensional (3D) bioprinting has increased enormously. 3D bioprinting combines the fields of developmental biology, stem cells, and computer and materials science to create complex bio-hybrid structures for various applications. It is able to precisely place different cell types, biomaterials and biomolecules together in a predefined position to generate printed composite architectures. In the field of tissue engineering, 3D bioprinting has allowed the study of tissues and organs on a new level. In clinical applications, new models have been generated to study disease pathogenesis. One of the most important aspects of 3D bio-printing is the bio-ink, which is a mixture of cells, biomaterials and bioactive molecules that creates the printed article. This review describes all the currently used bio-printing inks, including polymeric hydrogels, polymer bead microcarriers, cell aggregates and extracellular matrix proteins. Amongst the polymeric components in bio-inks are: natural polymers including gelatin, hyaluronic acid, silk proteins and elastin; and synthetic polymers including amphiphilic block copolymers, PEG, poly(PNIPAAM) and polyphosphazenes. Furthermore, photocrosslinkable and thermoresponsive materials have been described. To provide readers with an understanding of the context, the review also contains an overview of current bio-printing techniques and finishes with a summary of bio-printing applications.
Yield-stress support bath-enabled extrusion printing is emerging as a promising filament-based direct-write strategy for different applications in tissue engineering and regenerative medicine. Central to the printing quality of complex three-dimensional structures fabricated by the support bath-enabled fabrication approach is the formation of a continuous filament with well-defined geometry. The objective of this research is to study the printability of hydrogel precursor solutions in a Laponite nanoclay yield-stress bath during extrusion printing where the printed hydrogel precursor solutions remain liquid. The printability herein is mainly evaluated based on the morphology and dimensions of printed liquid filaments. Seven filament types are observed during extrusion in the nanoclay bath: three types of well-defined filaments (swelling, equivalent diameter, and stretched) and four types of irregular filaments (rough surface, over-deposited, compressed, and discontinuous). When the alginate concentration increases, the diameter of filaments made of alginate-gelatin blends decreases. The nanoclay concentration significantly affects the morphology of deposited filaments: low concentration Laponite bath (such as 0.5% (w/v)) may lead to the formation of irregular filaments such as rough surface and over-deposited filaments while high concentration bath (such as 8.0% (w/v)) may result in the formation of compressed filaments. Operating conditions affect the filament diameter and morphology similar to those as observed during conventional extrusion printing. The printability knowledge enables the successful fabrication of cellular vascular constructs in the Laponite nanoclay bath.