Brillouin micro-spectroscopy as an advanced tool for mechanical characterization of 3D-bioprinted scaffolds for tissue engineering
Supervisor: Dr Giedrė Astrauskaitė and Aleksandra Kozyrina
Industry Partner: Cell Link
School: Engineering
Description:
Development of biomimetic scaffolds with customisable mechanical properties is crucial for advancing regenerative medicine and tissue engineering. In vivo, tissues exhibit complex, heterogeneous mechanical environments that influence cell behaviour, differentiation, and function. To replicate these conditions in vitro, advanced 3D bio-printing technologies are needed to fabricate materials with physiologically relevant micro-architectures and mechanics.
Advanced digital light processing (DLP)-based 3D bio-printing allows the creation of complex tissue models with precise placement of a wide range of functional elements, including biomaterials (bioinks), live cells, and biomolecules. Bioinks are synthetic or natural polymers, which support cell adhesion, proliferation and differentiation; they allow rapid fabrication, with spatial stiffness control, by modulating the light exposure time and/or intensity. For example, using the commercial BIONOVA X light-based bioprinter (CELLINK) healthy, soft, and cirrhotic (stiff) liver 3D in vitro models have been demonstrated (Figure 1.1 C, D).
Accurate characterisation of stiffness gradients, bioink performance as well as photopatterning resolution and reproducibility is essential for optimisation and wider adoption of printed models. Nevertheless, conventional methods to probe material stiffness are invasive or low-resolution and do not allow 3D testing, uncovering only large-scale, bulk material properties.
Brillouin micro-spectroscopy is a technique capable of measuring material mechanical properties. It is non-invasive, contact- and label-free, and offers contrast based on the scattering of photons off acoustic vibrational modes. This allows micrometre-scale resolution visualisation of the longitudinal storage and loss moduli, describing the sample’s stiffness and viscosity, respectively. Brillouin microscopy is gaining interest in mechanical characterisation of biological samples, including hydrogel-based biomaterials. As a proof-of-concept, we have already successfully resolved the stiffness transition in a multi-hydrogel sample from CELLINK with Brillouin microscopy (Figure 1.2A, C).
The goal of the project is to characterise and optimise samples produced with the CELLINK’s BIONOVA X using a commercial LightMachinery Brillouin system. The work will focus on evaluating commercial bioinks and light-based bioprinting features in multi-material samples, and assessing the precision of crosslinking gradients, to achieve various stiffness levels that closely replicate the mechanical properties of native tissues. The student will optimise the data acquisition protocol, as well as the analysis pipeline with a programming language of choice (Python or MATLAB) and present her/his findings to the industry partner. The project is therefore an exciting opportunity to gain cross-disciplinary experience in advanced microscopy, data analysis and establish a contact with a leading bio-material company.
Figure 1.1: Example overview of the 3D bio-printing workflow with the BIONOVA X. (A) Digital grayscale mask to guide the printing geometry; the lighter colour represents longer exposure time to provide different stiffness in different regions on a single hexagonal scaffold; (B) Bioink solution is loaded into the multi-well plate in the bioprinter; (C, D) Brightfield images of 3D bio-printed acellular (C) and cell laden (D) scaffolds with regionally varied stiffness for liver mimetic models. 1.2 An example of Brillouin micro-spectroscopy method to probe the stiffness gradient between printed multi-material hydrogel structures. A) Diagram of sample set-up and brightfield image of the boundary between gels. Scale bar is 100 µm. B) Upon illumination, each region in the raster scan yields a scattered light spectrum with Brillouin Stokes and anti-Stokes frequency-shifted peaks. (C) 2D map of Brillouin shift across the boundary between the two gels.