Extrusion bioprinting of hydrogel scaffolds: printability and mechanical behavior

Extrusion bioprinting (known as dispensing-based bioprinting as well) has been widely used to extrude or dispense continuous strands or fibers of biomaterials (e.g. hydrogel) and cells (such a mixture is referred to as "bioink"), layer-by-layer, to form three-dimensional (3D) scaffolds for tissue engineering. For extrusion bioprinting, one key issue is printability or the capability to print and maintain reproducible 3D scaffolds from bioink, which is typically measured by the difference in structure between the designed scaffold and the printed one. Due to the structural difference (or the difference caused by printability), the printed scaffold's mechanical properties are also different from those of the designed scaffold, notably affecting the scaffold performance as applied subsequently to tissue engineering. This dissertation aims to perform a comprehensive study on the printability and mechanical behavior of hydrogel scaffolds fabricated by extrusion bioprinting. The specific objectives are (1) to investigate the influence of design-, bioink-, and printing-related factors on the printability of hydrogel scaffolds, (2) develop an indirect printing technique to improve the printability of low-concentration hydrogels, (3) develop a numerical model representative of the elastic modulus of hydrogel scaffolds by considering the influence of printability, and (4) investigate the effect of crosslinkers on the scaffold's mechanical properties through experimental and numerical approaches. While studies on printing scaffolds from hydrogel(s) have been conducted, limited knowledge has been documented on hydrogels' printability. Current studies often consider one aspect of studying hydrogel printability (for example, bioink properties solely). The first part of this dissertation studies the multiple dimensions of printability for hydrogel scaffolds, including identifying the influence of hydrogel composition and printing parameters/conditions. Specifically, by using the hydrogels synthesized from alginate, gelatin, and methylcellulose (MC), flow behavior and mechanical properties, as well as their influence on the printability of hydrogels, were investigated. Pore size, strand diameter, and other dimensions of the printed scaffolds were examined; then, pore/ strand/ angular/ printability and irregularity were studied to characterize the printability. The results revealed that the printability could be affected by many factors; among them, the most important are those related to the hydrogel composition and printing parameters. This chapter also presents a framework to evaluate alginate hydrogel printability systematically, which can be adopted and used in the studies of other hydrogels for bioprinting. Low-concentration hydrogels have favorable properties for many cell functions in tissue engineering, but they are considerably limited from a scaffold fabrication point of view due to poor 3D printability. The second part of this dissertation is developing an indirect printing method to fabricate scaffolds made from a low-concentration of hydrogels as the second objective. This chapter briefly presents an indirect bioprinting technique to biofabricate scaffolds with low (0.5%w/v) to moderate (3%w/v) concentrations of alginate hydrogel using gelatin as a sacrificial bioink. Indirect-fabricated scaffolds were evaluated using compression, swelling, degradation, biological (primary rat Schwann cells), and morphological assessments. Results indicated that 0.5% alginate scaffolds have steep swelling changes, while 3.0% alginate scaffolds had gradual changes. 0.5% alginate demonstrated better cell viability throughout the study than 3.0% counterparts, though. It was concluded that this indirect bioprinting approach could be extended to other types of hydrogels to improve the printability of low-concentration hydrogels along with the biological performance of cells and avoid high shear stress during direct 3D bioplotting causing cell damage. One issue involved in 3D bioplotting is achieving the scaffold structure with the desired mechanical properties. To overcome this issue, various numerical methods have been developed to predict scaffolds' mechanical properties, but they are limited by the imperfect representation of scaffolds as fabricated. The third part of this dissertation is developing a numerical model to predict the elastic modulus (one important index of mechanical properties) of scaffolds, considering the penetration or fusion of strands in one layer into the previous layer as the third objective. For this purpose, the finite element method was used for the model development, while medium-viscosity alginate was selected for scaffold fabrication by the 3D bioplotting technique. The elastic modulus of the bioplotted scaffolds was characterized using mechanical testing; the results were compared with those predicted from the developed model, demonstrating a strong congruity amongst them. Our results showed that the penetration, pore size, and the number of printed layers have significant effects on the elastic modulus of bioplotted scaffolds and suggest that the developed model can be used as a powerful tool to modulate the mechanical behavior of bioplotted scaffolds. For improvement, the fourth part of the dissertation (or the fourth objective) is improving the developed model by considering the crosslinker's effect on the modeling. The use of a cation solution (a crosslinker agent such as CaCl2) is important for regulating the mechanical properties, but this use has not been well documented in the literature. Here, the effect of varied crosslinking agent volume and crosslinking time on 3D extrusion-based alginate scaffolds' mechanical behavior were evaluated using both experimental and numerical methods. Compression tests were used to measure each scaffold's elastic modulus; then, a finite element model was developed, and a power model was used to predict scaffold mechanical behavior. Results showed that crosslinking time and crosslinker volume both play a decisive role in modulating 3D bioplotted scaffolds' mechanical properties. Because scaffolds' mechanical properties can affect cell response, this study's findings can be implemented to modulate the elastic modulus of scaffolds according to the intended application. In conclusion, this dissertation presents the development of methods/models to study/represent the printability and mechanical properties of hydrogel scaffolds by using extrusion bioprinting, along with meaningful experimental and model-simulation results. The developed methods/models/results would represent an advance in bioprinting scaffolds for tissue engineering

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

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. Keywords: 3D bioprinting, Tissue engineering, Vascularization, Extrusion, Laser-based printing, Co-axial printin

Printability of 3D Printed Hydrogel Scaffolds: Influence of Hydrogel Composition and Printing Parameters

Extrusion-based bioprinting of hydrogel scaffolds is challenging due to printing-related issues, such as the lack of capability to precisely print or deposit hydrogels onto three-dimensional (3D) scaffolds as designed. Printability is an index to measure the difference between the designed and fabricated scaffold in the printing process, which, however, is still under-explored. While studies have been reported on printing hydrogel scaffolds from one or more hydrogels, there is limited knowledge on the printability of hydrogels and their printing processes. This paper presented our study on the printability of 3D printed hydrogel scaffolds, with a focus on identifying the influence of hydrogel composition and printing parameters/conditions on printability. Using the hydrogels synthesized from pure alginate or alginate with gelatin and methyl-cellulose, we examined their flow behavior and mechanical properties, as well as their influence on printability. To characterize the printability, we examined the pore size, strand diameter, and other dimensions of the printed scaffolds. We then evaluated the printability in terms of pore/strand/angular/printability and irregularity. Our results revealed that the printability could be affected by a number of factors and among them, the most important were those related to the hydrogel composition and printing parameters. This study also presented a framework to evaluate alginate hydrogel printability in a systematic manner, which can be adopted and used in the studies of other hydrogels for bioprinting

Fused Deposition Modeling and Fabrication of a Three-dimensional Model in Maxillofacial Reconstruction

The utilization of customized three-dimentional (3D) models based on patient's computed tomography (CT) scan data and by assistance of additive manufacturing/rapid prototyping (AM/RP) techniques for 3D reconstruction is one of the applicable trends for reducing the errors and time saving during surgeries. In the present study, the methodology of the fabrication of a custom-made 3D model based on converting CT scan data to standard triangle language (STL) format for a 33-years old male patient who was suffering from an accident trauma was described. The 3D model of the patient’s skull was fabricated and applied in preoperative planning. It was used for designing a comprehensive plan for rehabilitation of the damaged orbit to restore the appearance and bone reconstruction of the patient. Before fabricating the model, the accuracy of protocols used in converting CT scan data into STL file was evaluated. Then, the model was fabricated by a fused deposition modeling (FDM) machine. Using this procedure led to a maximum of 1.4% difference between the virtual model in the software and the fabricated 3D model in the fracture site. The present technique reduced operation time significantly. In addition, following eight months from the operation, the treatment approach ensured the patient's fractures healing process

Modeling of the Mechanical Behavior of 3D Bioplotted Scaffolds Considering the Penetration in Interlocked Strands

Three-dimensional (3D) bioplotting has been widely used to print hydrogel scaffolds for tissue engineering applications. One issue involved in 3D bioplotting is to achieve the scaffold structure with the desired mechanical properties. To overcome this issue, various numerical methods have been developed to predict the mechanical properties of scaffolds, but limited by the imperfect representation of one key feature of scaffolds fabricated by 3D bioplotting, i.e., the penetration or fusion of strands in one layer into the previous layer. This paper presents our study on the development of a novel numerical model to predict the elastic modulus (one important index of mechanical properties) of 3D bioplotted scaffolds considering the aforementioned strand penetration. For this, the finite element method was used for the model development, while medium-viscosity alginate was selected for scaffold fabrication by the 3D bioplotting technique. The elastic modulus of the bioplotted scaffolds was characterized using mechanical testing and results were compared with those predicted from the developed model, demonstrating a strong congruity between them. Once validated, the developed model was also used to investigate the effect of other geometrical features on the mechanical behavior of bioplotted scaffolds. Our results show that the penetration, pore size, and number of printed layers have significant effects on the elastic modulus of bioplotted scaffolds; and also suggest that the developed model can be used as a powerful tool to modulate the mechanical behavior of bioplotted scaffolds