Cartilaginous spheroid-assembly design considerations for endochondral ossification: towards robotic-driven biomanufacturing

Abstract

Spheroids have become essential building blocks for biofabrication of functional tissues. Spheroid formats allow high cell-densities to be efficiently engineered into tissue structures closely resembling the native tissues. In this work, we explore the assembly capacity of cartilaginous spheroids (d ∼ 150 µm) in the context of endochondral bone formation. The fusion capacity of spheroids at various degrees of differentiation was investigated and showed decreased kinetics as well as remodeling capacity with increased spheroid maturity. Subsequently, design considerations regarding the dimensions of engineered spheroid-based cartilaginous mesotissues were explored for the corresponding time points, defining critical dimensions for these type of tissues as they progressively mature. Next, mesotissue assemblies were implanted subcutaneously in order to investigate the influence of spheroid fusion parameters on endochondral ossification. Moreover, as a step towards industrialization, we demonstrated a novel automated image-guided robotics process, based on targeting and registering single-spheroids, covering the range of spheroid and mesotissue dimensions investigated in this work. This work highlights a robust and automated high-precision biomanufacturing roadmap for producing spheroid-based implants for bone regeneration.

Bioprinting of Stem Cell Spheroids Followed by Post‐Printing Chondrogenic Differentiation for Cartilage Tissue Engineering

“…[ 38,40–43,47,48 ] Despite the success of using these chondrogenic induced spheroids, fusion capacity and remodeling may be impaired after new tissue formation, preventing overall new tissue fusion in a macro scale. [ 43 ]…”

“…[38,[40][41][42][43]47,48] Despite the success of using these chondrogenic induced spheroids, fusion capacity and remodeling may be impaired after new tissue formation, preventing overall new tissue fusion in a macro scale. [43] On the other hand, excessive contact between spheroids can also lead to intense fusion which generally results in a big cell mass, resulting in a hypoxic aggregate with a necrotic core without organization. [23] Thus, instead of using chondrogenic induced spheroids, hMSC not differentiated spheroids formed during 5 days showed to be the ideal timepoint when compared to 7-day spheroids and these were bioprinted in combination with a polysaccharide-based hydrogel using XG and A.…”

Robotics-driven manufacturing of cartilaginous microtissues for the bio-assembly of skeletal implants

“…One of the challenges for culturing microtissues in non-adherent well plates, is that over time they are prone to move, leading to uncontrolled fusion and the development of large agglomerates (23). This can result in tissue heterogeneity and influence the homogeneity of differentiation cascades over time leading also to the development of non-differentiated tissue locations (17) By introducing robotic medium changes, we investigated the effect of dispension and aspiration speed in ranges close to and beyond manual speed and found no statistically significant effect. Therefore, robotic media changes can be time saving.…”

“…Monitoring of microtissue cultures and their morphometric quality profiles can be done non-invasively through imaging. Software is already available to segment and analyse microtissues in hydrogel microwells and floating cultures, even enabling selection of desirable microtissues and studying their fusion kinetics (13–17). Commercial microwell systems are typically produced from PCL or PDMS in different microwell shapes, which interact with light, creating complex backgrounds in brightfield images.…”

Close-to-native bone repair via tissue-engineered endochondral ossification approaches

“… Hall et al. 141 Human periosteum-derived cells LG-DMEM, 1% antibiotic-antimycotic, 1 × 10 −3 M AA, 100 × 10 −9 M DEX, 40 μg mL −1 proline, 20 × 10 −6 M of Rho-kinase inhibitor Y27632, ITS + Premix, 100 ng mL −1 BMP-2, 100 ng mL −1 GDF5, 10 ng mL −1 TGF-β1, 1 ng mL −1 BMP-6, and 0.2 ng mL −1 basic FGF-2 21 days — — 4 weeks This work proposes a proof-of-concept on the feasibility of image-guided robotic biomanufacturing of spheroid-based implants. After cartilaginous spheroids design and assembly, they were implanted subcutaneously in order to investigate the influence of spheroid fusion parameters on endochondral ossification.…”