"Despite immense interests, growing organoids resembling the human musculoskeletal system (including bone, cartilage, muscle, tendon, ligament) in a petri dish remains a major challenge in front of the tissue engineering and regenerative medicine (TERM) community. Bone is a vital organ that contains billions of bone cells as well as a sophisticated internal architecture across several length scales. Recapitulating the structural and functional complexity in bone requires the development of high resolution biofabrication techniques that faithfully recreate tissue architecture down to the micrometer-scale accuracy. One promising approach is to combine computer models derived from biomedical imaging data with light-based tissue manufacturing.
Here, we present an image-based subtractive biomanufacturing process to create microengineered 3D bone cell models in biocompatible hydrogels. To this end, new computer models that mimic the topology of lacuno-canalicular network (LCN) in bone are developed by sequential immunostaining and confocal microscopic imaging of osteocytes in bone specimen. These models are converted into stereolithography (.STL) files through image processing. Using two-photon subtractive lithography, we demonstrate the fabrication of LCN-mimicking microstructures inside a photodegradable polyethylene glycol hydrogel at high spatial resolution. The structural fidelity is highly dependent on the laser processing parameters such as laser power, writing speed and photosensitivity of the hydrogel matrices. The inclusion of a soluble two-photon photoinitiator can greatly decrease the laser threshold. Lastly, preliminary success on biomimetic subtractive 3D microprinting in the presence of living bone cells and guided 3D cell growth will be presented towards a living bone organoid."