Breast cancer often results in the removal of the breast, creating a need for replacement tissue. Tissue engineering offers the promise of generating such replacements by combining cells with biomaterial scaffolds and serves as an attractive potential alternative to current surgical repair methods. Such engineered tissues can also serve as important tools for drug screening and provide in vitro models for analysis. 3D bioprinting serves as an exciting technology with significant implications and applications in the field of tissue engineering. Here we review the work that has been undertaken in hopes of generating the recognized in-demand replacement breast tissue using different types of bioprinting. We then offer suggestions for future work needed to advance this field for both in vitro and in vivo applications.
Recent work demonstrated that 3D fibrin scaffolds function as an effective substrate for engineering tissues from pluripotent stem cells. However, the rapid degradation rate of fibrin remains a major limitation when differentiating human pluripotent stem cells for tissue engineering applications. The addition of crosslinking agents, such as genipin, during the polymerization process increases scaffold stability while decreasing the degradation rate of fibrin. Genipin crosslinking alters the physical characteristics of the fibrin scaffolds, which influences the behaviour of the differentiating cells seeded inside. It also possesses neuritogenic and neuroprotective properties, making it particularly attractive for engineering neural tissue from pluripotent stem cells. Here we show that genipin enhances neuronal differentiation of neural progenitors derived from human induced pluripotent stem cells (hiPSCs) in 2D culture and genipin concentration influences the morphological and mechanical properties of 3D fibrin scaffolds. These mechanically stable genipin-crosslinked fibrin scaffolds support hiPSC-derived neural aggregates and induce neurite outgrowth while remaining intact for 2 weeks as opposed to 5 days for unmodified fibrin scaffolds.
Parkinson's disease (PD) is a neurodegenerative disorder that results when the dopaminergic neurons (DNs) present in the substantia nigra necessary for voluntary motor control are depleted, making patients with this disorder ideal candidates for cell replacement therapy. Human induced pluripotent stem cells (hiPSCs), obtained by reprogramming adult cells, possess the properties of pluripotency and immortality while enabling the possibility of patient-specific therapies. An effective cell therapy for PD requires an efficient, defined method of DN generation, as well as protection from the neuroinflammatory environment upon engraftment. Although similar in pluripotency to human embryonic stem cells (hESCs), hiPSCs differentiate less efficiently into neuronal subtypes. Previous work has shown that treatment with guggulsterone can efficiently differentiate hESCs into DNs. Our work shows that guggulsterone is able to derive DNs from hiPSCs with comparable efficiency, and furthermore, this differentiation can be achieved inside three-dimensional fibrin scaffolds that could enhance cell survival upon engraftment.
We have designed and validated a set of robust and non-toxic protocols for directly evaluating the properties of engineered neural tissue. These protocols characterize the mechanical properties of engineered neural tissues and measure their electrophysical activity. The protocols obtain elastic moduli of very soft fibrin hydrogel scaffolds and voltage readings from motor neuron cultures. Neurons require soft substrates to differentiate and mature, however measuring the elastic moduli of soft substrates remains difficult to accurately measure using standard protocols such as atomic force microscopy or shear rheology. Here we validate a direct method for acquiring elastic modulus of fibrin using a modified Hertz model for thin films. In this method, spherical indenters are positioned on top of the fibrin samples, generating an indentation depth that is then correlated with elastic modulus. Neurons function by transmitting electrical signals to one another and being able to assess the development of electrical signaling serves is an important verification step when engineering neural tissues. We then validated a protocol wherein the electrical activity of motor neural cultures is measured directly by a voltage sensitive dye and a microplate reader without causing damage to the cells. These protocols provide a non-destructive method for characterizing the mechanical and electrical properties of living spinal cord tissues using novel biosensing methods.
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