The effect of toughness on differentiate in the bioprinted scaffold

Amirhossein Bakhtiari,1,* Hamidreza behboodi,2

1. University of tehran

Abstract

Introduction

3d-bioprinting as a novel method use multiple aspects to produce a precise deposition of strands that result in the construction of scaffolds with controllable porosity. one of the most critical problems on the way of using this technique is about bio-ink. bio-ink must be biocompatible, biodegradable and have an appropriate viscosity which can print precise structures without harmful effect on cells located in them. high viscosity needs high printing pressure that causes high shear stress and cell damage. otherwise, low viscosity cause collapsing the strands into each other. for printing scaffolds along with cells, it needs to print in room temperature, for this purpose it needs to adjust the gel point in which the printed strand immediately do the sol-gel transition in room temperature till can print an accurate structure. here, we use a pre-crosslinking step by cacl2 that result in printing an accurate cell-embedded structure by different pore size in two different sides. we use vitamin d to study differentiation possibility on the stiff and smooth sides to understand the effects of the porosity on the differentiation process.

Methods

For selecting the best composition of three components, rheology analysis was done on 10 different samples, further select the composition with gel-point near the room temperature (25±1 ̊c). after sterilizing the hydrogel, mesenchymal stem cells with density 5e+5 cells/ml was mixed with hydrogel and centrifuge in 3500rpm for 1.5 min. for resulting in the more accurate printed structure, we use a novel method of two-step crosslinking by cacl2. design of the scaffold was done by solidwork as a 4 layer structure by strands in 45, 135, 0, 90 degree position. the print was done by 3d-printed extrusion-base (the printer was design and manufactured by our team). cartilage and platform were located in 32 ̊c and 25 ̊c respectively till provide the appropriate temperature terms for living the cells. the degradation rate of the scaffolds in different crosslinking conditions was analyzed in pbs (ph=7.4 and temperature 37 ̊c), the weight of the samples till complete destruction was recorded. for evaluation the roughness of crosslinked scaffold we use 3d-laser measuring microscopy and use sem for indicating the pore diameters. vitamin d for 20day was used to produce a bone tissue on two different sides of the printed scaffold.

Results

First of all, we select the bio-ink composition of 7%gelatin, 3%alginat, and 1%collagen as a composition that has the possibility to turn solid gel in room temperature. we indicate the gel point of this composition is 25 ̊c. it is noticeable that in thermoreversible polymers, gel-point detects where the loss tangent is independent of frequency in the oscillatory experiment. after selecting the best composition of bio-ink, we find the concentration and the time of crosslinking in an independent test to find a condition with degradation time between 4-6 week that is satisfying for replacing of scaffold with ecm. as a result, we mixed 80mm cacl2 with bio-ink in the 1-1 ratio as a pre-crosslinking step and feed it into cartilage that according to laboratory observations let to having better control on the accuracy of printed structure. as a secondary step of crosslinking, 100mm cacl2 was sprayed on the printed scaffold, and after 1 hr, the samples were washed by deionized water. sem analyses of the printed scaffold show average width of 140 μm and 430 μm for small and large pore side, respectively. laser scanning indicate roughness of 1.23μm and 0.32μm for different surfaces that show a rigider surface for larger pore side. our observation indicated that differentiation of the mesenchymal stem cells to bone-tissue on the side with bigger pores is more significant compare to the smooth side.

Conclusion

Herein, we use alginate, collagen, and gelatin as a novel composition with compatible viscosity to print a precise scaffold for guided tissue regeneration aspects. for better control of the porosity of printed structure, we use a pre-crosslinking step by cacl2. further, use vitamin d to differentiate the printed scaffold into bone tissue.

Keywords

Cellular bio-printing, pre crosslinking step, differentiation