Bioprinting Vessel-like Constructs Using Hyaluronan Hydrogels Crosslinked With

Tetrahedral Polyethylene Glycol Tetracrylates

Aleksander Skardal, Jianxing Zhang, Glenn D. Prestwich

by, Syed Baseeruddin AlviBM16RESCH11005Sub: Bio fabrication

Content

• Introduction• Literature• Materials & Method• Results• Importance • Drawbacks • Conclusion

Introduction

• Tissue engineering is an emerging field which allows us to generate tissues mimicking that of a natural system.

• The ultimate goal of tissue engineering is to fabricate a fully functional organ, that can perform all the complex functions.

• Mimicking the vascular architecture of a tissue is a mile stone in tissue engineering.

• An engineered blood vessel that posses compliance, lack of thrombogenicity, ability to heal, contract, remodel, secret normal blood vessel products can be of great importance.

• Problem statement: fabrication of a miniaturized blood vessel is difficult with the existing modalities.

Literature review • Electrospinning of collagen, elastin, and synthetic polymer nano- fibers into

lumenized scaffolds and centrifugal casting of hyaluronic acid (HA) hydrogels into cellularized hydrogel tubes have yielded tubular structures

• In a related approach, smooth muscle cells and fibroblasts were cultured in the presence of ascorbic acid to create cohesive cellular sheets, which were then wrapped around a mandrel and seeded with endothelial cells to form three-layered tubes (Cytograft’s Lifeline)

Materials & Method

• Chemical synthesis of TetraPEG intermediates and TetraPAc crosslinkers– four-armed PEG derivatives, TetraPEG8 with four PEG 2000

chains and TetraPEG13 with four PEG 3400 chains – TetraPEG tetra-acrylate deriva- tives (TetraPAcs) to co-crosslink

thiolated hyaluronic acid

• Rheology: Rheometer • Biocompatibility:

– Murine fibroblasts (NIH 3T3), human hepatoma cells (HepG2 C3A), and human intestinal epithelial cells (Int 407).

– cell viability was determined using MTS assays at day 3 and day 7.

• Bioprinting of hydrogel macrofilaments– 1 million (M), 5 M, 10 M, 25 M, 100 M, 250, M, and 500 M cells

of each type were added to hydrogel and gelation was observed after 1h incubation.

Materials & Method

• 3D Printer– Fab@Home printing assembly

Results

• Rheology: – The TetraPAc crosslinked hydrogels were significantly stiffer than the

corresponding PEGDA-crosslinked hydrogels at weight/volume percentages of 1.0%, 1.5%, and 2% for both TetraPAc8 or TetraPac13 gels.

Biocompatibility

Cell growth and proliferation were assessed on

day 3 and day 7 after encapsulation in the

PEGDA, TetraPAc8, and TetraPAc13 hydrogels

using an MTS assay. Three cell types were

evaluated in each hydrogel, and increased

proliferation was observed at day 7 compared to

day 3 for each experimental group.

Cell viabilityCross-sectional views of the bioprinted construct taken (A) immediately after

printing with encapsulated a fluorescent HA-BODIPY tracer for increased visualization, (B) at 14 days, and (C) at 28 days of culture using LIVE/DEAD

staining to highlight viable and dead cells. Green fluorescence indicates calcein AM-stained live cells and red fluorescence indicates ethidium homodimer-1-

stained dead cells

Importance of the work

• Engineering of tissue-like constructs on a human-relevant size scale is limited by diffusion of nutrients and oxygen to the cells within the construct. By incorporating functional vasculature into engineered tissue constructs, the size and complexity of engineered constructs can be increased and viability can be maintained

• HA-based sECM exhibits necessary physical and mechanical properties to be easily printable

• Fab@Home system can be customizable and comparatively economical alternative to conventional bio printers.

Drawbacks

• Medium-resolution printing of millimeter to centimeter scale geometries.

• Time-consuming • lacks efficiency

Conclusion HA sECM hydrogels are easy to prepare and fully

biocompatible and allows higher cell entrapment, extruded cellularized sECM macrofilaments held their shape both during and after bioprinting. These structures maintained viability in

culture for up to 4 weeks. Thus providing an alternative to existing modalities.