Generation of a 3D gradient inducing a precise control over phenotype and pre-vasculature for osteochondral tissue modelling

Introduction

Gradients of 3-D hierarchical tissues are common in nature and present specific architectures, as this is the case of the anisotropic subchondral bone interfaced with articular cartilage [1]. While diverse fabrication techniques based on 3D printing, microfabrication, and microfluidics have been used to recreate tailored biomimetic tissues and their respective microenvironment [2,3], an alternative solution is still need for improved biomimetic gradient tissues under dynamic conditions with control over pre-vasculature formation.

Goal

Here, we engineered a gradient osteochondral human-based tissue with precise control over both cell/tissue phenotype and pre-vasculature formation, which opens-up possibilities for the study of complex tissues interfaces, with broader applications in drug testing and regenerative medicine.

Method

The fabrication of three-dimensional gradient of microparticles was performed combining methacrylated gelatin (GelMA) and gellan gum (GG) with hydroxyapatite microparticles (HAp). The mixing of the interface was controlled by the temperature of two polymeric layers, being the second added at 10 ºC higher than the first one. This subsequent addition of polymeric solutions at different temperatures promoted convection, which drove the microparticles through the interface of the two gels forming the gradient. After ionic and photo-crosslinking, the freezing step was programmed using an external cover of styrofoam forcing the ice crystals to grow linearly, generating an anisotropic gradient scaffold. A dual-chamber microreactor device was designed (figure 1A) to culture fat pad adipose-derived stem cells and microvascular endothelial cells under two biochemical microenvironments.

Results

Using control over temperature and crosslinking, hydrogel-like structures were built in 3-D isotropic and anisotropic HAp gradients. Then, an in vitro osteochondral tissue model was obtained using a dual-chamber platform (figure 1Bi). Results showed a significant difference of SOX9 (p < 0.05) and RUNX2 (p < 0.05) from the top to the bottom regions of the 3-D gradient structures under dynamic conditions (figure 1Bii). Finally, a pre-vasculature was controlled over 7 days, stimulating the subchondral bone-like region 35% more (p < 0.05) when compared to the cartilage-like region (figure 1C).

Conclusions and recommendations

In this work, microparticle and biochemical gradients were fabricated into iso- and anisotropic architectures. The obtained outcomes enable the precise control of 3-D gradients in different architectures, such as anisotropic or isotropic structures, with broad applications in interfaced tissue engineering, regenerative medicine and drug testing.

ACKNOWLEDGEMENTS: The authors are grateful for the FLAD and FCT funding (SFRH/BD/92565/2013).

REFERENCES

[1] Gurdon J B et al. Nature. 2001; 413:797-803

[2] Canadas R F et al. Biomat. 2018; 181:402-414

[3] Polacheck W J et al. PNAS. 2014; 111:2447-2452