Tissue-engineered heart chambers as a platform technology for drug discovery and disease modeling.

Current drug screening approaches are incapable of fully detecting and characterizing drug effectiveness and toxicity of human cardiomyocytes. The pharmaceutical industry uses mathematical models, cell lines, and in vivo models. Many promising drugs are abandoned early in development, and some cardiotoxic drugs reach humans leading to drug recalls. Therefore, there is an unmet need to have more reliable and predictive tools for drug discovery and screening applications. Biofabrication of functional cardiac tissues holds great promise for developing a faithful 3D in vitro disease model, optimizing drug screening efficiencies enabling precision medicine. Different fabrication techniques including molding, pull spinning and 3D bioprinting were used to develop tissue-engineered heart chambers. The big challenge is to effectively organize cells into tissue with structural and physiological features resembling native tissues. Some advancements have been made in engineering miniaturized heart chambers that resemble a living pump for drug screening and disease modeling applications. Here, we review the currently developed tissue-engineered heart chambers and discuss challenges and prospects.

Fabrication of chitosan/alginate/hydroxyapatite hybrid scaffolds using 3D printing and impregnating techniques for potential cartilage regeneration.

Three-dimensional (3D) printed hydrogel scaffolds enhanced with ceramics have shown potential applications for cartilage regeneration, but leaving biological and mechanical properties to be desired. This paper presents our study on the development of chitosan /alginate scaffolds with nano hydroxyapatite (nHA) by combining 3D printing and impregnating techniques, forming a hybrid, yet novel, structure of scaffolds for potential cartilage regeneration. First, we incorporated nHA into chitosan scaffold printing and studied the printability by examining the difference between the printed scaffolds and their designs. Then, we impregnated alginate with nHA into the printed chitosan scaffolds to forming a hybrid structure of scaffolds; and then characterized the scaffolds mechanically and biologically, with a focus on identifying the influence of nHA and alginate for potential cartilage regeneration. The results of compression tests on the scaffolds showed that the inclusion of nHA increased the elastic moduli of scaffolds; while the live/dead assay illustrated that nHA had a great effect on improving attachment and viability of ATCD5 cells on the scaffolds. Also, our results illustrated scaffolds with nHA impregnated in alginate hydrogel enhanced the cell viability and attachment. Furthermore, antibacterial activity of hybrid scaffolds was characterized with results indicating that the chitosan scaffolds had favourable antibacterial ability, which was further enhanced with the impregnated nHA. Taken together, our study has illustrated that chitosan/HA/alginate hybrid scaffolds are promising for cartilage regeneration and the methods developed to create hybrid scaffolds based on 3D printing and impregnating techniques, which can also be extended to fabricating scaffolds for other tissue engineering applications.

Latest Advances in 3D Bioprinting of Cardiac Tissues.

Cardiovascular diseases (CVDs) are known as the major cause of death worldwide. In spite of tremendous advancements in medical therapy, the gold standard for CVD treatment is still transplantation. Tissue engineering, on the other hand, has emerged as a pioneering field of study with promising results in tissue regeneration using cells, biological cues, and scaffolds. Three-dimensional (3D) bioprinting is a rapidly growing technique in tissue engineering because of its ability to create complex scaffold structures, encapsulate cells, and perform these tasks with precision. More recently, 3D bioprinting has made its debut in cardiac tissue engineering, and scientists are investigating this technique for development of new strategies for cardiac tissue regeneration. In this review, the fundamentals of cardiac tissue biology, available 3D bioprinting techniques and bioinks, and cells implemented for cardiac regeneration are briefly summarized and presented. Afterwards, the pioneering and state-of-the-art works that have utilized 3D bioprinting for cardiac tissue engineering are thoroughly reviewed. Finally, regulatory pathways and their contemporary limitations and challenges for clinical translation are discussed.

Printability-A key issue in extrusion-based bioprinting.

Three-dimensional (3D) extrusion-based bioprinting is widely used in tissue engineering and regenerative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique. One critical issue in 3D extrusion-based bioprinting is printability or the capability to form and maintain reproducible 3D scaffolds from bioink (a mixture of biomaterials and cells). Research shows that printability can be affected by many factors or parameters, including those associated with the bioink, printing process, and scaffold design, but these are far from certain. This review highlights recent developments in the printability assessment of extrusion-based bioprinting with a focus on the definition of printability, printability measurements and characterization, and printability-affecting factors. Key issues and challenges related to printability are also identified and discussed, along with approaches or strategies for improving printability in extrusion-based bioprinting.

Biofabrication Strategies for Musculoskeletal Disorders: Evolution towards Clinical Applications.

Biofabrication has emerged as an attractive strategy to personalise medical care and provide new treatments for common organ damage or diseases. While it has made impactful headway in e.g., skin grafting, drug testing and cancer research purposes, its application to treat musculoskeletal tissue disorders in a clinical setting remains scarce. Albeit with several in vitro breakthroughs over the past decade, standard musculoskeletal treatments are still limited to palliative care or surgical interventions with limited long-term effects and biological functionality. To better understand this lack of translation, it is important to study connections between basic science challenges and developments with translational hurdles and evolving frameworks for this fully disruptive technology that is biofabrication. This review paper thus looks closely at the processing stage of biofabrication, specifically at the bioinks suitable for musculoskeletal tissue fabrication and their trends of usage. This includes underlying composite bioink strategies to address the shortfalls of sole biomaterials. We also review recent advances made to overcome long-standing challenges in the field of biofabrication, namely bioprinting of low-viscosity bioinks, controlled delivery of growth factors, and the fabrication of spatially graded biological and structural scaffolds to help biofabricate more clinically relevant constructs. We further explore the clinical application of biofabricated musculoskeletal structures, regulatory pathways, and challenges for clinical translation, while identifying the opportunities that currently lie closest to clinical translation. In this article, we consider the next era of biofabrication and the overarching challenges that need to be addressed to reach clinical relevance.

Printability in extrusion bioprinting.

Extrusion bioprinting has been widely used to extrude continuous filaments of bioink (or the mixture of biomaterial and living cells), layer-by-layer, to build three-dimensional constructs for biomedical applications. In extrusion bioprinting, printability is an important parameter used to measure the difference between the designed construct and the one actually printed. This difference could be caused by the extrudability of printed bioink and/or the structural formability and stability of printed constructs. Although studies have reported in characterizing printability based on the bioink properties and printing process, the concept of printability is often confusingly and, sometimes, conflictingly used in the literature. The objective of this perspective is to define the printability for extrusion bioprinting in terms of extrudability, filament fidelity, and structural integrity, as well as to review the effect of bioink properties, bioprinting process, and construct design on the printability. Challenges related to the printability of extrusion bioprinting are also discussed, along with recommendations for improvements.

Printability-A key issue in extrusion-based bioprinting / 药物分析学报

Three-dimensional(3D)extrusion-based bioprinting is widely used in tissue engineering and regener-ative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique.One critical issue in 3D extrusion-based bioprinting is printability or the capability to form and maintain reproducible 3D scaffolds from bioink(a mixture of biomaterials and cells).Research shows that printability can be affected by many factors or parameters,including those associated with the bioink,printing process,and scaffold design,but these are far from certain.This review highlights recent de-velopments in the printability assessment of extrusion-based bioprinting with a focus on the definition of printability,printability measurements and characterization,and printability-affecting factors.Key issues and challenges related to printability are also identified and discussed,along with approaches or strategies for improving printability in extrusion-based bioprinting.

Extrusion-based printing of chitosan scaffolds and their in vitro characterization for cartilage tissue engineering.

Researchers have looked to cartilage tissue engineering to address the lack of cartilage regenerative capability related to cartilage disease/trauma. For this, a promising approach is extrusion-based three-dimensional (3D) printing technique to deliver cells, biomaterials, and growth factors within a scaffold to the injured site. This paper evaluates the printability of chitosan scaffolds for a cartilage tissue engineering, with a focus on identifying the influence of drying technique implemented before crosslinking on the improvement of chitosan printability. First, the printability of chitosan with concentrations of 8%, 10%, and 12% (w/v) was evaluated and 10% chitosan was selected for further studies. Then, different drying methods, including air drying, warm drying, and vacuum drying followed by crosslinking, were used to study their effect on the mechanical properties of the 10% chitosan scaffolds. Our compression testing results showed the highest elastic modulus for the scaffolds crosslinked with the air-drying technique; as a major part of experiemtn, pore sizes were studies and scaffolds with smaller pore sizes showed higher elastic modulus. Additionally, the geometrical features of scaffolds were examined using a scanning electron microscopy (SEM) technique. The morphology of scaffolds, dried with the aformentioned methods, was assess using SEM images to evaluate the dimensional stability of scaffolds. Chondrocyte cells cultured on the 3D-printed chitosan scaffolds dried using the air-drying technique showed high cell attachment while retaining round cellular morphology. Also, the results of the cytotoxicity test indicated that there was proper biocompatibility of the chitosan for the ATDC5 cells. Results showed that the drying method plays a decisive role in the mechanical and biological behavior of chitosan scaffolds. Considering biological and mechanical properties, the proposed 3D-printed chitosan scaffold can be of a potential structure for cartilage tissue engineering applications.

Electrospinning of polyurethane/graphene oxide for skin wound dressing and its in vitro characterization.

Electrospinning polyurethane has been utilized as skin wound dressing for protecting skin wounds from infection and thus facilitating their healings, but also limited by its imperfect biocompatibility, mechanical and antibacterial properties. This paper presents our study on the addition of graphene oxide to electrospinning polyurethane for improved properties, as well as its in vitro characterization. Polyurethane/graphene oxide wound dressing was electrospun with varying amount of graphene oxide (from 0.0% to 2.0%); and in vitro tests was carried out to characterize the wound dressing properties and performance from the structural, mechanical, and biological perspectives. Scanning electron microscopy and Fourier-transform infrared spectroscopy were used to confirm the interaction between graphene oxide particles and polyurethane fibers, while the scanning electron microscopy images further illustrated that the wound dressing was of a porous structure with fibre diameters depending on the amount of graphene oxide added; specifically, 20 to 180 nm were for composite polyurethane/graphene oxide fibers and 600 to 900 nm for pure polyurethane. Our results also revealed that the hydrophilicity and swelling properties of the wound dressing could be regulated by the amount of graphene oxide added to the polyurethane/graphene oxide composites. Mechanical, antibacterial, and cytotoxicity properties of the composite polyurethane/graphene oxide wound dressing were examined with the results illustrating that the addition of graphene oxide could improve the properties of the electrospun wound dressing. Combined together, our study illustrates that electrospinning polyurethane/graphene oxide composite is promising as skin wound dressing.

Micromechanisms of Cortical Bone Failure Under Different Loading Conditions.

Bone being a hierarchical composite material has a structure varying from macro- to nanoscale. The arrangement of the components of bone material and the bonding between fibers and matrix gives rise to its unique material properties. In this study, the micromechanisms of cortical bone failure were examined under different loading conditions using scanning electron microscopy. The experimental tests were conducted in longitudinal and transverse directions of bone diaphysis under tensile as well as compressive loading. The results show that bone material has maximum stiffness under longitudinal tensile loading, while the strength is higher under transverse compressive loading. A reverse trend of compressive mechanical properties of bone is observed for longitudinal and transverse loading as compared to trends reported in the previous studies. Therefore, micromechanisms of cortical bone failure were analyzed for different loading conditions to reveal such type of behavior of cortical bone and to correlate bone microstructure with mechanical response of bone.

Assessment of Elastic-Plastic Fracture Behavior of Cortical Bone Using a Small Punch Testing Technique.

The fracture properties of cortical bone are directly coupled to its complex hierarchical structure. The limited availability of bone material from many anatomic locations creates challenges for assessing the effect of bone heterogeneity and anisotropy on fracture properties. The small punch technique was employed to examine the fracture behavior of cortical bone in terms of area under the curve values obtained from load-load point displacement behavior. Fracture toughness of cortical bone was also determined in terms of J-toughness values obtained using a compact tension (CT) test. Area under the curve values obtained from the small punch test were correlated with the J-toughness values of cortical bone. The effects of bone density and compositional parameters on area under the curve and Jtoughness values were also analyzed using linear and multiple regression analysis. Area under the curve and J-toughness values are strongly and positively correlated. Bone density and %mineral content are positively correlated with both area under the curve and J-toughness values. The multiple regression analysis outcomes support these results. Overall, the findings support the hypothesis that area under the curve values obtained from small punch tests can be used to assess the fracture behavior of cortical bone.

Electrospinning of Scaffolds from the Polycaprolactone/Polyurethane Composite with Graphene Oxide for Skin Tissue Engineering.

Creating scaffolds for skin tissue engineering remain challenging in terms of their mechanical and biological properties. In this paper, we present a study on the nanocomposite polyurethane (PU)/polycaprolactone (PCL) scaffolds with graphene oxide (GO), which were fabricated by using electrospinning method, for potential skin tissue engineering. For this, homogenous and soft PU nanofibers containing varying percent of polycaprolactone (12% and 15%) and nano GO (0.5-4%) were electrospun, respectively, and then characterized by different techniques/assays in vitro. For the scaffold characterization, scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were used. The SEM results show the spun scaffolds have 3D porous structure (90%) with the fiber diameter increased with the GO concentration, while the FTIR results confirmed the presence of PU, PCL, and Go in the scaffolds. Also, the biocompatibility, via the cytotoxicity, of the scaffolds was examined by MTT assay with the human skin fibroblast cells, along with their wettability in terms of contact angle. Our results show that the scaffolds are biocompatible to the skin fibroblast cell, illustrating their potential use in skin tissue engineering. Also, our results illustrate that the addition of GO to the PU/PCL composite can increase the wettability (or hydrophilicity) and biocompatibility of scaffolds. Combined together, the nanocomposite PU/PCL scaffolds with GO are promising as biocompatible constructs for skin tissue engineering.

Bioprinting of Vascularized Tissue Scaffolds: Influence of Biopolymer, Cells, Growth Factors, and Gene Delivery.

Over the past decades, tissue regeneration with scaffolds has achieved significant progress that would eventually be able to solve the worldwide crisis of tissue and organ regeneration. While the recent advancement in additive manufacturing technique has facilitated the biofabrication of scaffolds mimicking the host tissue, thick tissue regeneration remains challenging to date due to the growing complexity of interconnected, stable, and functional vascular network within the scaffold. Since the biological performance of scaffolds affects the blood vessel regeneration process, perfect selection and manipulation of biological factors (i.e., biopolymers, cells, growth factors, and gene delivery) are required to grow capillary and macro blood vessels. Therefore, in this study, a brief review has been presented regarding the recent progress in vasculature formation using single, dual, or multiple biological factors. Besides, a number of ways have been presented to incorporate these factors into scaffolds. The merits and shortcomings associated with the application of each factor have been highlighted, and future research direction has been suggested.

Indirect 3D bioprinting and characterization of alginate scaffolds for potential nerve tissue engineering applications.

Low-concentration hydrogels have favorable properties for many cell functions in tissue engineering but are considerably limited from a scaffold fabrication point of view due to poor three-dimensional (3D) printability. Here, we developed an indirect-bioprinting process for alginate scaffolds and characterized the potential of these scaffolds for nerve tissue engineering applications. The indirect-bioprinting process involves (1) printing a sacrificial framework from gelatin, (2) impregnating the framework with low-concentration alginate, and (3) removing the gelatin framework by an incubation process, thus forming low-concentration alginate scaffolds. The scaffolds were characterized by compression testing, swelling, degradation, and morphological and biological assessment of incorporated or seeded Schwann cells. For comparison, varying concentrations of alginate scaffolds (from 0.5% to 3%) were fabricated and sterilized using either ultraviolet light or ethanol. Results indicated that scaffolds can be fabricated using the indirect-bioprinting process, wherein the scaffold properties are affected by the concentration of alginate and sterilization technique used. These factors provide effective means of regulating the properties of scaffolds fabricated using the indirect-bioprinting process. Cell-incorporated scaffolds demonstrated better cell viability than bulk gels. In addition, scaffolds showed better cell functionality when fabricated with a lower concentration of alginate compared to a higher concentration. The indirect-bioprinting process that we implemented could be extended to other types of low-concentration hydrogels to address the tradeoffs between printability and properties for favorable cell functions.

Studies on the Stress-Strain Relationship Bovine Cortical Bone Based on Ramberg-Osgood Equation.

Bone is a complex material that exhibits an amount of plasticity before bone fracture takes place, where the nonlinear relationship between stress and strain is of importance to understand the mechanism behind the fracture. This brief presents our study on the examination of the stress-strain relationship of bovine femoral cortical bone and the relationship representation by employing the Ramberg-Osgood (R-O) equation. Samples were taken and prepared from different locations (upper, middle, and lower) of bone diaphysis and were then subjected to the uniaxial tensile tests under longitudinal and transverse loading conditions, respectively. The stress-strain curves obtained from tests were analyzed via linear regression analysis based on the R-O equation. Our results illustrated that the R-O equation is appropriate to describe the nonlinear stress-strain behavior of cortical bone, while the values of equation parameters vary with the sample locations (upper, middle, and lower) and loading conditions (longitudinal and transverse).

3D biofabrication of vascular networks for tissue regeneration: A report on recent advances.

Rapid progress in tissue engineering research in past decades has opened up vast possibilities to tackle the challenges of generating tissues or organs that mimic native structures. The success of tissue engineered constructs largely depends on the incorporation of a stable vascular network that eventually anastomoses with the host vasculature to support the various biological functions of embedded cells. In recent years, significant progress has been achieved with respect to extrusion, laser, micro-molding, and electrospinning-based techniques that allow the fabrication of any geometry in a layer-by-layer fashion. Moreover, decellularized matrix, self-assembled structures, and cell sheets have been explored to replace the biopolymers needed for scaffold fabrication. While the techniques have evolved to create specific tissues or organs with outstanding geometric precision, formation of interconnected, functional, and perfused vascular networks remains a challenge. This article briefly reviews recent progress in 3D fabrication approaches used to fabricate vascular networks with incorporated cells, angiogenic factors, proteins, and/or peptides. The influence of the fabricated network on blood vessel formation, and the various features, merits, and shortcomings of the various fabrication techniques are discussed and summarized.

Regeneration of peripheral nerves by nerve guidance conduits: Influence of design, biopolymers, cells, growth factors, and physical stimuli.

Injuries to the peripheral nervous system (PNS) cause neuropathies that lead to weakness and paralysis, poor or absent sensation, unpleasant and painful neuropathies, and impaired autonomic function. In this regard, implanted artificial nerve guidance conduits (NGCs) used to bridge an injured site may provide appropriate biochemical and biophysical guidance cues required to stimulate regeneration across a nerve gap and restore the function of PNS. Advanced conduit design and fabrication techniques have made it possible to fabricate autograft-like structures in the NGCs with incredible precision. To this end, strategies involving the use of biopolymers, cells, growth factors, and physical stimuli have been developed over the past decades and have led to the development of varying NGCs, from simple hollow tubes to complex conduits that incorporate one or more guidance cues. This paper briefly reviews the recent progress in the development of these NGCs for nerve regeneration, focusing on the design and fabrication of NGCs, as well as the influence of biopolymers, cells, growth factors, and physical stimuli. The advanced techniques used to fabricate NGCs that incorporate cells/growth factors are also discussed, along with their merits and flaws. Key issues and challenges with regard to the development of NGCs have been identified and discussed, and recommendations for future research have been included.

Strategic Design and Fabrication of Nerve Guidance Conduits for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) have been drawing considerable attention as an aid to promote regeneration of injured axons across damaged peripheral nerves. Ideally, NGCs should include physical and topographic axon guidance cues embedded as part of their composition. Over the past decades, much progress has been made in the development of NGCs that promote directional axonal regrowth so as to repair severed nerves. This paper briefly reviews the recent designs and fabrication techniques of NGCs for peripheral nerve regeneration. Studies associated with versatile design and preparation of NGCs fabricated with either conventional or rapid prototyping (RP) techniques have been examined and reviewed. The effect of topographic features of the filler material as well as porous structure of NGCs on axonal regeneration has also been examined from the previous studies. While such strategies as macroscale channels, lumen size, groove geometry, use of hydrogel/matrix, and unidirectional freeze-dried surface are seen to promote nerve regeneration, shortcomings such as axonal dispersion and wrong target reinnervation still remain unsolved. On this basis, future research directions are identified and discussed.

Influence of crosslinking on the mechanical behavior of 3D printed alginate scaffolds: Experimental and numerical approaches.

Tissue scaffolds fabricated by three-dimensional (3D) bioprinting are attracting considerable attention for tissue engineering applications. Because the mechanical properties of hydrogel scaffolds should match the damaged tissue, changing various parameters during 3D bioprinting has been studied to manipulate the mechanical behavior of the resulting scaffolds. Crosslinking scaffolds using a cation solution (such as CaCl2) is also important for regulating the mechanical properties, but has not been well documented in the literature. Here, the effect of varied crosslinking agent volume and crosslinking time on the mechanical behavior of 3D bioplotted alginate scaffolds was evaluated using both experimental and numerical methods. Compression tests were used to measure the elastic modulus of each scaffold, then a finite element model was developed and a power model used to predict scaffold mechanical behavior. Results showed that crosslinking time and volume of crosslinker both play a decisive role in modulating the mechanical properties of 3D bioplotted scaffolds. Because mechanical properties of scaffolds can affect cell response, the findings of this study can be implemented to modulate the elastic modulus of scaffolds according to the intended application.

Dispensing-based bioprinting of mechanically-functional hybrid scaffolds with vessel-like channels for tissue engineering applications - A brief review.

Over the past decades, significant progress has been achieved in the field of tissue engineering (TE) to restore/repair damaged tissues or organs and, in this regard, scaffolds made from biomaterials have played a critical role. Notably, recent advances in biomaterials and three-dimensional (3D) printing have enabled the manipulation of two or more biomaterials of distinct, yet complementary, mechanical and/or biological properties to form so-called hybrid scaffolds mimicking native tissues. Among various biomaterials, hydrogels synthesized to incorporate living cells and/or biological molecules have dominated due to their hydrated tissue-like environment. Moreover, dispensing-based bioprinting has evolved to the point that it can now be used to create hybrid scaffolds with complex structures. However, the complexities associated with multi-material bioprinting and synthesis of hydrogels used for hybrid scaffolds pose many challenges for their fabrication. This paper presents a brief review of dispensing-based bioprinting of hybrid scaffolds for TE applications. The focus is on the design and fabrication of hybrid scaffolds, including imaging techniques, potential biomaterials, physical architecture, mechanical properties, cell viability, and the importance of vessel-like channels. The key issues and challenges for dispensing-based bioprinting of hybrid scaffolds are also identified and discussed along with recommendations for future research directions. Addressing these issues will significantly enhance the design and fabrication of hybrid scaffolds to and pave the way for translating them into clinical applications.