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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Decoene, Isaak (KU Leuven/Prometheus Division of Skeletal Tissue Engineering)


"Introduction: Bottom-up developmental engineering is showing promising results in skeletal tissue engineering1. In this approach, microtissues are formed from periosteal cells, the main contributors in the formation of the cartilaginous fracture callus. These microtissues can be chondrogenically differentiated towards hypertrophy resulting in a cartilaginous template in vitro which can be remodeled after implantation resulting in bone ossicles mimicking closely fracture healing. However, further clinical translation of these promising implants is hampered by (1) a lack of information on donor variation, and (2) a lack of non-invasive potency monitoring techniques. In this study, the protein secretome of bone-forming microtissues was monitored over time for 6 individual donors to extract secreted end-point efficacy biomarkers.

Methods: Human periosteum-derived cells from 6 donors were seeded in non-adherent microwells (Aggrewell TM 800, STEMCELL Technologies) to form microtissues of 1000 cells each. These microtissues were cultured for 21 days in a low-protein chemically defined chondrogenic medium. After 5 hours, 7, 14, and 21 days microtissues were collected for histological staining and DNA quantification, and their culture supernatant was analyzed by mass spectrometry for identification of the protein secretome. Then, 600 microtissues were fused and implanted ectopically for 4 weeks in nude mice to assess bone-forming potency.

Results: Histological analysis of microtissues showed a clear distinction between 3 donors with a high production versus 3 donors with low production of extracellular matrix, generating large and smaller microtissues. These differences were related to the gender of the donors. Similar differences were seen in the total amount of soluble protein in their culture supernatant (38.8±6.1 µg/mL v.s. 22.1±1.4 µg/mL, p = 2.5 x 10-5). A total of 172 proteins were detected in the culture supernatant by LC-MS/MS. Interestingly, most proteins were already detected after 7 days and showed a decrease towards day 21. However, when implanted, cartilaginous implants resulted in the formation of bone ossicles for all donors, showing high protocol robustness. Therefore, we identified 10 secreted proteins that were commonly found for all donors that could be promising candidates for bone-forming potency biomarkers.

Discussion: We carried out a series of orthogonal quality characterization studies for human donor-derived cartilaginous microtissues evaluating also bone-forming capacity in vivo. Furthermore, we used high-sensitivity proteomics for mapping the secretome during chondrogenic differentiation. This is promising for the identification of biomarkers that will enable the implementation of quality-by-design strategies by predictively controlling the microtissue differentiation processes in vitro to ensure skeletal defect regeneration upon implantation.

References: Nilsson Hall, G. et al. Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing. Adv. Sci. 1902295, 1–16 (2019)."

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