The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration.

Tissue engineering for spinal cord injury (SCI) remains a complex and challenging task. Biomaterial scaffolds have been suggested as a potential solution for supporting cell survival and differentiation at the injury site. However, different biomaterials display multiple properties that significantly impact neural tissue at a cellular level. Here, we evaluated the behavior of different cell lines seeded on chitosan (CHI), poly (ε-caprolactone) (PCL), and poly (L-lactic acid) (PLLA) scaffolds. We demonstrated that the surface properties of a material play a crucial role in cell morphology and differentiation. While the direct contact of a polymer with the cells did not cause cytotoxicity or inhibit the spread of neural progenitor cells derived from neurospheres (NPCdn), neonatal rat spinal cord cells (SCC) and NPCdn only attached and matured on PCL and PLLA surfaces. Scanning electron microscopy and computational analysis suggested that cells attached to the material's surface emerged into distinct morphological populations. Flow cytometry revealed a higher differentiation of neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) into glial cells on all biomaterials. Immunofluorescence assays demonstrated that PCL and PLLA guided neuronal differentiation and network development in SCC. Our data emphasize the importance of selecting appropriate biomaterials for tissue engineering in SCI treatment.

3D Bioprinting Pluripotent Stem Cell Derived Neural Tissues Using a Novel Fibrin Bioink Containing Drug Releasing Microspheres.

3D bioprinting combines cells with a supportive bioink to fabricate multiscale, multi-cellular structures that imitate native tissues. Here, we demonstrate how our novel fibrin-based bioink formulation combined with drug releasing microspheres can serve as a tool for bioprinting tissues using human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs). Microspheres, small spherical particles that generate controlled drug release, promote hiPSC differentiation into dopaminergic neurons when used to deliver small molecules like guggulsterone. We used the microfluidics based RX1 bioprinter to generate domes with a 1 cm diameter consisting of our novel fibrin-based bioink containing guggulsterone microspheres and hiPSC-derived NPCs. The resulting tissues exhibited over 90% cellular viability 1 day post printing that then increased to 95% 7 days post printing. The bioprinted tissues expressed the early neuronal marker, TUJ1 and the early midbrain marker, Forkhead Box A2 (FOXA2) after 15 days of culture. These bioprinted neural tissues expressed TUJ1 (15 ± 1.3%), the dopamine marker, tyrosine hydroxylase (TH) (8 ± 1%) and other glial markers such as glial fibrillary acidic protein (GFAP) (15 ± 4%) and oligodendrocyte progenitor marker (O4) (4 ± 1%) after 30 days. Also, quantitative polymerase chain reaction (qPCR) analysis showed these bioprinted tissues expressed TUJ1, NURR1 (gene expressed in midbrain dopaminergic neurons), LMX1B, TH, and PAX6 after 30 days. In conclusion, we have demonstrated that using a microsphere-laden bioink to bioprint hiPSC-derived NPCs can promote the differentiation of neural tissue.

3D bioprinting models of neural tissues: The current state of the field and future directions.

3D bioprinting can potentially revolutionize the field of neural tissue engineering by increasing its throughput and reproducibility. However, many obstacles must be overcome to realize this immense potential. This review first discusses how 3D hydrogels can serve as powerful tools for engineering neural tissue, especially when combined with different types of cells. These tools enable us to gain a better understanding of neural tissue development and its associated disease states. Next, we define 3D bioprinting and detail the necessary tools for using this technique to produce neural tissue, along with reviewing relevant recent work in the area. We also compare with other approaches to generating 3D neural tissues while identifying key areas for future developments in the field of bioprinting.

3D Printing of Neural Tissues Derived from Human Induced Pluripotent Stem Cells Using a Fibrin-Based Bioink.

3D bioprinting offers the opportunity to automate the process of tissue engineering, which combines biomaterial scaffolds and cells to generate substitutes for diseased or damaged tissues. These bioprinting methods construct tissue replacements by positioning cells encapsulated in bioinks into specific locations in the resulting constructs. Human induced pluripotent stem cells (hiPSCs) serve as an important tool when engineering neural tissues. These cells can be expanded indefinitely and differentiated into the cell types found in the central nervous systems, including neurons. One common method for differentiating hiPSCs into neural tissue requires the formation of aggregates inside of defined diameter microwells cultured in chemically defined media. However, 3D bioprinting of such hiPSC-derived aggregates has not been previously reported in the literature, as it requires the development of specialized bioinks for supporting cell survival and differentiation into mature neural phenotypes. Here we detail methods including preparing base material components of the bioink, producing the bioink, and the steps involved in printing 3D neural tissues derived from hiPSC-derived neural aggregates using Aspect Biosystems' novel RX1 printer and their lab-on-a-printer (LOP) technology.

Guggulsterone-releasing microspheres direct the differentiation of human induced pluripotent stem cells into neural phenotypes.

Parkinson's disease (PD), a common neurodegenerative disorder, results from the loss of motor function when dopaminergic neurons (DNs) in the brain selectively degenerate. While pluripotent stem cells (PSCs) show promise for generating replacement neurons, current protocols for generating terminally differentiated DNs require a complicated cocktail of factors. Recent work demonstrated that a naturally occurring steroid called guggulsterone effectively differentiated PSCs into DNs, simplifying this process. In this study, we encapsulated guggulsterone into novel poly-ε-caprolactone-based microspheres and characterized its release profile over 44 d in vitro, demonstrating we can control its release over time. These guggulsterone-releasing microspheres were also successfully incorporated in human induced pluripotent stem cell-derived cellular aggregates under feeder-free and xeno-free conditions and cultured for 20 d to determine their effect on differentiation. All cultures stained positive for the early neuronal marker TUJ1 and guggulsterone microsphere-incorporated aggregates did not adversely affect neurite length and branching. Guggulsterone microsphere incorporated aggregates exhibited the highest levels of TUJ1 expression as well as high Olig 2 expression, an inhibitor of the STAT3 astrogenesis pathway previously known as a target for guggulsterone in cancer treatment. Together, this study represents an important first step towards engineered neural tissues consisting of guggulsterone microspheres and PSCs for generating DNs that could eventually be evaluated in a pre-clinical model of PD.

3D Bioprinting Stem Cell Derived Tissues.

Stem cells offer tremendous promise for regenerative medicine as they can become a variety of cell types. They also continuously proliferate, providing a renewable source of cells. Recently, it has been found that 3D printing constructs using stem cells, can generate models representing healthy or diseased tissues, as well as substitutes for diseased and damaged tissues. Here, we review the current state of the field of 3D printing stem cell derived tissues. First, we cover 3D printing technologies and discuss the different types of stem cells used for tissue engineering applications. We then detail the properties required for the bioinks used when printing viable tissues from stem cells. We give relevant examples of such bioprinted tissues, including adipose tissue, blood vessels, bone, cardiac tissue, cartilage, heart valves, liver, muscle, neural tissue, and pancreas. Finally, we provide future directions for improving the current technologies, along with areas of focus for future work to translate these exciting technologies into clinical applications.