HYALURONIC ACID BASED NEXT-GENERATION BIOINK FOR 3D BIOPRINTING OF A HUMAN STEM CELL DERIVED CORNEAL STROMA EQUIVALENT AND A 3D CORNEA TISSUE MODEL WITH INNERVATION

Jun 29, 2022, 11:20 AM
10m
Room: S4 A

Room: S4 A

Speaker

Mörö, Anni (Eye group, Faculty of Medicine and Health Technology, Tampere University )

Description

Introduction
There is a dire short of donor corneas for cornea transplantation, leaving millions of visually impaired patients without treatment1. 3D bioprinting holds tremendous potential for fabrication of cornea mimicking structures. One of the key technological challenges in 3D bioprinting is the establishment of bioink compositions that allow both ideal printability as well as biocompatibility. To address these needs, we developed a hyaluronic acid (HA)-based bioink for 3D bioprinting of cornea tissue engineering (TE).

Methodology
HA-based bioink was prepared using hydrazone crosslinking chemistry2. Crosslinking components were combined with rheological modifiers to obtain a printable bioink. The shear thinning property and viscosity of the bioink as well as the mechanical stability of the printed structures were determined with a rheometer. Extrusion-based 3D bioprinting was used. The shape fidelity and self-healing properties of the bioink were explored. Human stem cells, such as human adipose stem cells (hASCs) and hASC-derived cells were chosen for printing cornea stroma equivalents. The printed constructs were evaluated for their cell viability, proliferation and microstructure with LIVE/DEAD® and PrestoBlue™ viability assays, immunofluorescence (IF) and hematoxylin and eosin stainings. Key protein expression was determined with IF and quantitative PCR. Moreover, 3D printed stromal equivalents were implanted into ex vivo porcine corneal organ cultures to explore integration to host tissue. Finally, human pluripotent stem cell derived neurons (hPSC-neurons) were 3D bioprinted to the periphery of the cornea stroma equivalents, and the integration of neuronal extensions to the printed structures was explored.

Results
The developed HA-based bioink showed excellent shear thinning property, viscosity as well as printability. Quality prints with high-resolution and good shape fidelity were achieved. Moreover, HA-DA bioink discs showed self-healing after 24 hours of healing. Importantly, HA-based bioink demonstrated excellent biocompatibility with all explored human stem cells and human stem cell derived cells. Cells in printed structures showed good tissue formation seen with positive expression for connexin 43 and formation of cellular networks. Corneal stroma equivalents with appropriate cell organization and positive expression of lumican were successfully manufactured. Moreover, 3D bioprinted cornea stromal equivalents demonstrated excellent integration to host tissue in ex vivo organotypic cultures after 21 days. While inspecting the innervation of cornea stromal equivalents in 3D bioprinted cornea tissue model, the printed structures with HA-based bioink allowed the ingrowth of long neuronal extensions. Target cells in the cornea stromal equivalents accelerated the neuronal extension growth compared to printed structures without cells.

Conclusions
We have developed a HA-based bioink using click chemistry that fills the demands of next-generation bioinks with excellent printability, stability, biocompatibility as well as tissue formation. Here, we demonstrated that the developed bioink is feasible for 3D bioprinting of cornea stroma equivalents. Moreover, we manufactured the first 3D bioprinted cornea tissue model with innervation. The developed bioink and the printed human stem cell derived cornea stromal equivalents hold great potential for future cornea TE applications.

References:
1. Gain, P. et al., JAMA Ophthalmol. 134(2):167-73 (2016).
2. Koivusalo, L. et al. Biomaterials. 225, 119516 (2019).

20941860968

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