Microfluidic Fabrication of Natural Polymer-Based Scaffolds for Tissue Engineering Applications: A Review

Natural polymers, thanks to their intrinsic biocompatibility and biomimicry, have been largely investigated as scaffold materials for tissue engineering applications. Traditional scaffold fabrication methods present several limitations, such as the use of organic solvents, the obtainment of a non-homogeneous structure, the variability in pore size and the lack of pore interconnectivity. These drawbacks can be overcome using innovative and more advanced production techniques based on the use of microfluidic platforms. Droplet microfluidics and microfluidic spinning techniques have recently found applications in the field of tissue engineering to produce microparticles and microfibers that can be used as scaffolds or as building blocks for three-dimensional structures. Compared to standard fabrication technologies, microfluidics-based ones offer several advantages, such as the possibility of obtaining particles and fibers with uniform dimensions. Thus, scaffolds with extremely precise geometry, pore distribution, pore interconnectivity and a uniform pores size can be obtained. Microfluidics can also represent a cheaper manufacturing technique. In this review, the microfluidic fabrication of microparticles, microfibers and three-dimensional scaffolds based on natural polymers will be illustrated. An overview of their applications in different tissue engineering fields will also be provided

Fiber-based tissue engineering: Progress, challenges, and opportunities.

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the abovementioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice

Development and characterization of a biopolymer direct-write process for 3D microvascular structures formation.

Engineering of bulk tissues has been limited by the lack of nutrient and waste exchange in these tissues without an adjacent capillary network. To produce microvasculature, a scaffold must be produced that provides temporary mechanical support and stimulate endothelial cell adhesion, growth, and morphogenesis into a vessel. However, current well-established techniques for producing microvasculature, such as electrospinning, are limited since they lack both the precision to control fiber placement in three-dimensional space and the ability to create fiber networks with predefined diameters to replicate the physiological microvascular progression from arteriole to capillary to venule. Our group has developed a “Direct-write” technique using a 3-Axis robotic dispensing system to process polymers into precisely positioned, three-dimensional, suspended fibers with controlled diameters. Within this dissertation, a conceptual scaffold-covering strategy is presented for the formation of the precisely positioned, three-dimensional microvascular structure with a controlled diameter in vitro. This study considers ways to extend the 3-Axis robotic dispensing system by incorporating new biodegradable materials into micro-fibers. First, a number of different biopolymers (natural, synthetic, composites, and copolymers) were used for demonstrating the capability of direct-writing micro-fibers and branched structures with microvascular-scale diameter through the 3-Axial robotic dispensing system. Then, the fabrication process was characterized by a design of experiments and a generalized mathematical model was developed through dimensional analysis. The empirical model determined the correlation between polymer fiber diameter and intrinsic properties of the polymer solution together with the processing parameters of the robotic dispensing system and allows future users the ability to employ the 3-Axis robotic dispensing system to direct-write micro-fibers without trial-and-error work. This study also considers ways to broaden the pre-vascularization methods by covering Human Dermal Microvascular Endothelial Cells (HDMECs) on the fabricated scaffold to generate the microvascular structure. HDMECs cultured on the produced micro-fiber scaffolds were observed to form a confluent monolayer spread along the axis and around the circumference of the fibers within two days of seeding. Once confluency was reached, the cell-covered scaffold was embedded into a collagen gel and a hybrid structure was formed. Through these experiments, we demonstrate the ability to obtain a cell-viable, flexible, and free-standing “modular tissue”, which could be potentially assembled to a three-dimensional microvascular network through angiogenesis mechanism

Application of microfabrication techniques for tissue engineering a cardiac conductive device

Impairment of the atrioventricular electrical conduction (AV-block) is a major cause for the implantation of an electronic pacemaker device. Even though this is the standard treatment today, it has its disadvantages. One major problem is the implementation of long-term pacing therapy in pediatric patients owing to the restrictions imposed by a child’s small size and their inevitable growth. Thus there is a genuine need for innovative therapies especially for children with cardiac rhythm disorders. The aim of the Biopacer project is to develop an autologous conductive tissue device that will serve as an electrical conduit between the upper and lower chambers of the heart. The idea is to use pediatric cardiomyocytes together with a fibrin-based scaffold to produce a tissue construct that is completely autologous and has the ability to grow with the patient. Mimicking the complexity and highly organized structure of native cardiac tissue sets significant challenges for the engineering process. To address this challenge, bottom-up tissue engineering, in which structural components of different cell types with high-degree organization are fabricated separately and later assembled together, was applied. The aim of this thesis was to utilize different microfabrication techniques for the realization of the fibrin scaffold with different cell types to fabricate building blocks for the assembly of a conductive tissue device with a controllable, spatially organized tissue microarchitecture. Two methods, micromolding in capillaries (MIMIC) and microfabrication of fibrin fibers by extrusion, were established and exploited in the fabrication of cell-seeded 3D fibrin tissue modules. Using the MIMIC technique, cells were spatially confined into a 3D fibrin gel structure and they were shown to align longitudinally in small diameter fibrin gels. In extruded fibrin fibers, endothelial cells were demonstrated to coalesce and bundle the surrounding fibrin into a core around which they arranged themselves resembling the phenomenon of tubulogenesis. The organization of these microfabricated tissue modules together to create a conductive tissue device consisting of separate functional units (e.g. aligned cardiomyocytes, vasculature, extracellular matrix) was additionally displayed. This way, all the structural components of native cardiac tissue are accounted for and highly organized in the engineered construct

Three-dimensional scaffolds as platforms for development of cell-based strategies for central nervous system rescue and repair

The mammalian central nervous system (CNS) has limited intrinsic repair mechanisms. Damage or trauma to the CNS causes a loss of cells, causing motor and cognitive impairments. Cell transplantation strategies have made great advances in the last few decades and have been essential in the field of regenerative medicine. Recently, biomaterials in conjunction with stem cells have been used to supplement therapeutic strategies. The objective of this project was using an interdisciplinary approach combining polymers, three-dimensional (3D) scaffolds and stem cells to provide a platform for enhancing cell proliferation and differentiation for future cell transplantation strategies. Poly (ε-caprolactone) (PCL) is a synthetic polymer that can be used to fabricate microfibers using a microfluidic technique. Adult rat hippocampal progenitor cells (AHPCs) were used to examine the ability of 3D microfibrous scaffolds to support the growth, proliferation and differentiation of cells in vitro. In contrast to conventional two-dimensional (2D) surfaces, our group revealed that PCL microfibers significantly increased proliferation and glial differentiation of AHPCs, demonstrating the importance of topographic cues on cellular behavior. Alternatively, we demonstrated the ability to encapsulate AHPCs within a hydrogel opposed to seeding cells onto a polymer surface. Encapsulation and recovery of AHPCs from alginate hydrogels demonstrated an increase in proliferation and neuronal differentiation, suggesting that fibrous hydrogels may mimic the natural microenvironment present in vivo and modulate cell behavior. To implement cell-based biomaterial strategies, we chose zebrafish (Danio rerio) as our model because they are optically transparent as larvae, enabling transplantation studies to be followed in vivo. First, we developed an efficient method to dissociate neural tissue from embryonic zebrafish brains in order to isolate neurons, with the ultimate goal of using these cells in conjunction with biomaterials to investigate novel therapeutic strategies. Second, we provided preliminary evidence on using zebrafish as a system to investigate biomaterial- based strategies. This work has provided evidence using a combinatorial approach of biomaterials and stem cells to promote cell proliferation and differentiation without the use of chemicals. Biomaterials provide a permissive environment leading to enhanced cell proliferation and differentiation, which may lead to the development of efficacious cell-based therapies

Bioengineering Tissue Constructs Using Elastic Alginate Hydrogels

Bioengineered 3-D tissue constructs have great potential for understanding tissue development and tissue repair in patients lacking functional organs. One of the major challenges faced in the field, however, is to build functional tissue constructs that resemble tissue found in vivo. Cells and tissues in the body are organized into three-dimensional architectures, which interact with fibrillar extracellular matrix (ECM) proteins at a nanoscale. Both the topology and elasticity of the ECM play critical roles in regulating tissue formation. Alginate, a naturally occurring polysaccharide, is a good candidate to use as a biomaterial to mimic the topography and elasticity of the ECM. In this study, the feasibility of synthesizing 3-D alginate microtubes, nanofibers and microbeads that simulate the elasticity and topography of the ECM has been investigated. Using a series of techniques, we fabricated tissue constructs with varying shapes, sizes, and elasticities. 3-D alginate microtubes, nanofibers, and microbeads were synthesized through the processes of microfluidics, electrospinning, and electrodroplet, respectively. The experiments conducted throughout this project provide a fundamental platform for bioengineering artificial salivary glands in future studies for patients who suffer from xerostomia (dry mouth) and salivary gland hypofunction

Advancement, applications, and future directions of 3D models in breast cancer research: a comprehensive review

3D models have popped up as indispensable tools for breast cancer study, they provide a closersemblance of the multiplex cellular and cancer tissue microenvironment as compared toancient 2D cultures. Their utilization in BC research permits a better interpretation ofhemostasis, cell-to-cell, and cell-to-extracellular matrix interactions, differentiation of cells,and tissue organization. 3D models qualify the exploration of numerous aspects regardingcancer progression, it also includes invasion of the tumor, cancer metastasis, and drugresistance, in a way that more precisely contemplates in vivo conditions. Hence, they provideda precise environment for research as compared to a complex in vivo host cell environment.This review highlights the importance of different 3D models in BC research, focusing on theircapability to enumerate complex disease physio-pathological features. This review explainsthe variety of 3D models utilized in BC research, encompassing Multicellular TumorSpheroids (MCTS), Three-Dimensional (3D) bioprinting, Organoid Models, Microfluidictechnologies, Organ on chip models, 3D hydrogel models and in silico approaches for BC,challenges and future of 3D models

Hollow Fiber and Nanofiber Membranes in Bioartificial Liver and Neuronal Tissue Engineering.

To date, the creation of biomimetic devices for the regeneration and repair of injured or diseased tissues and organs remains a crucial challenge in tissue engineering. Membrane technology offers advanced approaches to realize multifunctional tools with permissive environments well-controlled at molecular level for the development of functional tissues and organs. Membranes in fiber configuration with precisely controlled, tunable topography, and physical, biochemical, and mechanical cues, can direct and control the function of different kinds of cells toward the recovery from disorders and injuries. At the same time, fiber tools also provide the potential to model diseases in vitro for investigating specific biological phenomena as well as for drug testing. The purpose of this review is to present an overview of the literature concerning the development of hollow fibers and electrospun fiber membranes used in bioartificial organs, tissue engineered constructs, and in vitro bioreactors. With the aim to highlight the main biomedical applications of fiber-based systems, the first part reviews the fibers for bioartificial liver and liver tissue engineering with special attention to their multifunctional role in the long-term maintenance of specific liver functions and in driving hepatocyte differentiation. The second part reports the fiber-based systems used for neuronal tissue applications including advanced approaches for the creation of novel nerve conduits and in vitro models of brain tissue. Besides presenting recent advances and achievements, this work also delineates existing limitations and highlights emerging possibilities and future prospects in this field

Scalable production of 3D microtissues using novel microfluidic technologies

Tissue engineering approaches are widely studied with the goal to replace or repair human tissues. However, while studies are often promising in a laboratory environment, there remain difficulties in the translation of laboratory-based studies towards clinical applications due to low in vivo efficiency and/or complex impractical procedures.An interesting strategy for improving therapy effectiveness is by evolving from conventional 2D cell culture to more biomimetic 3D cell culture approaches. While therapy efficiency can be greatly improved using 3D cell culture, current 3D microtissue production techniques are often non-scalable batch processes, limiting clinical and industrial translation. A continuous production method is needed in order to improve the microtissue production rate and improve the feasibility of clinical application.Microfluidics offers the possibility to evolve microtissue production towards a continuous process. Using conventional on-chip microfluidics, microtissues can be produced in a controlled and continuous manner by cell encapsulation in hollow microcapsules. However, conventional on-chip microfluidics offers challenges such as complex multistep processes, the use of potentially harmful oils and surfactants and often low throughputs, which are currently hampering widespread clinical and industrial translation of microfluidically produced microtissues. There is therefore a need to evolve microfluidics towards a clean, fast and single step scalable approach to fulfill the clinical requirements for tissue engineering approaches that take advantage of 3D microtissues.This thesis describes multiple microfluidic solutions that focus on overcoming these challenges hampering the widespread clinical and industrial use of microtissues. A reusable, cleanroom-free, multifunctional microfluidic device is developed using standard cutting and abrasion technology, which allows the production of microtissue-laden microcapsules in a single step-manner. This on-chip process is then evolved towards an off-chip jetting approach which allows for the production of microtissue-laden microcapsules in an ultra-high throughput manner (&gt;10 ml/min) without the need of potentially harmful oils and surfactants. This in-air microfluidic approach is also utilized for mass production of microtissues in larger compartmentalized hydrogels, which are used for the production of large clinical-sized tissues. A multitude of microtissues are formed using these described microfluidic technologies such as human mesenchymal stem cell spheroids, chondrocyte spheroids, fibroblast spheroids, cholangiocyte and cholangiocarcinoma organoids, lumen-forming embryoid bodies, contracting cardiospheres, and clinical sized cartilage tissues.To summarize, this thesis introduces multiple microfluidic systems for scalable microcapsule and microtissue production with the aim to remove the hurdles towards clinical and industrial translation of 3D microtissues.<br/