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Under the Microscope: Callus Organoids derived from Human Periosteal Cells

Under the Microscope: Callus Organoids derived from Human Periosteal Cells

Gabriella Nilsson Hall, PhD
Postdoctoral Researcher

Niki Loverdou, MSc
PhD Student

Ioannis Papantoniou, PhD
Associate Professor

Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Belgium​
Prometheus the division of Skeletal Tissue Engineering, KU Leuven, Belgium​

Declarations of interest: none.

Callus organoids under the microscope.
A) Histological section stained with Alcian blue (extracellular matrix, ECM) and Fast Red (nuclei), imaged with an inverted widefield microscope. B) Whole mount immunostaining of the prehypertrophic marker osterix and DAPI (nuclei), imaged with confocal microscopy. Scale bars represent 50 µm.


Organoids are three-dimensional (3D) cell-based structures that can be grown in the laboratory using different types of stem/progenitor cells. Organoids recapitulate key features of both the development and functions of organs [1,2]. To do this, cells creating organoids must differentiate into tissue-specific lineages mimicking the cell and tissue patterns observed in their in vivo counterparts. Despite the extensive use of organoids in basic research to model human organ development and disease, their translation to clinical application is mostly restricted to drug testing [3–5] and preclinical proof-of-concept for cell replacement strategies [6,7].


What? Callus organoids are cartilaginous microtissues derived from adult progenitor cells (e.g. human periosteum-derived cells [8]) that form bone upon implantation. These organoids consist of 200-300 cells and exhibit semi-autonomous properties resulting robustly into “mini-bone structures” via endochondral ossification, upon implantation [9,10]. These can be assembled bottom-up into larger implants and regenerate critically-sized defects in small animal models.


Why? Periosteal cells are the main contributors to the formation of the fracture callus during native fracture healing [11]. Hence, for long-bone defects, they are an excellent source of progenitor cells for therapeutic applications. The recapitulation of robust developmental processes into engineering strategies for tissue-engineered implants has been termed “developmental engineering” [12,13].


Who? No product exists in the market with this cell type or tissue type yet. Therefore, academic researchers and clinicians are currently dedicating most efforts to the preclinical product development stage for this new therapeutic approach.


When? The demonstration that organoid-based implants possess the capacity to regenerate critical-size segmental tibial defects has been a key milestone [9]. The scale-up endeavour towards large animal models and clinical application is currently underway and will require the support of enabling technologies such as bioreactors and bioprinters for whole-tissue implant biomanufacturing. This has been supported by European Union funding such as the JOINTPROMISE H2020 program (, grant agreement number 874837). We forecast that we could reach the patient within the next 5 years.


Where? Currently, there are no Advanced Therapy Medicinal Products (ATMP) based on this cell type and Prometheus, at KU Leuven is exploring the use of this cell type in a translational setting. Clinical trials using other cell types and strategies for healing bone defects are also ongoing but commercially available products are limited. The use of organoids is predicted to increase the success rate of ATMP by improving biological robustness and scalability. Commercialization of organoids has increased in recent years [14] and public initiatives are currently ongoing for different types of organoids, such as pancreas organoids and liver organoids. In addition, initiatives to create organoid platforms and biobanks with collections of genetically and histologically characterized organoid models of disease states from many individuals can have a significant impact on personalized medicine [15,16].


How? The periosteal cells can be isolated from the periosteum of patients during surgery. Since the starting number of cells is low, efficient and scalable cell expansion is needed.  The expanded cells are aggregated into clusters for chondrogenic differentiation into bone-forming callus organoids. Subsequently, the callus organoids are assembled to fuse into larger constructs with shapes corresponding to the defect to be treated. Further research to more precisely characterize the quality attributes of these organoids is underway through multi-omics technologies.


Did you know that… human periosteum-derived progenitor cells exhibit trilineage differentiation potential such as bone marrow MSCs? And that callus organoids can self-assemble into scaffold-free implants?



[1]       H. Clevers, Modeling Development and Disease with Organoids, Cell. (2016). doi:10.1016/j.cell.2016.05.082.

[2]       T. Takebe, J.M. Wells, Organoids by design, Science (80-. ). 364 (2019) 956–959. doi:10.1126/science.aaw7567.

[3]       T. Takahashi, Organoids for drug discovery and personalized medicine, Annu. Rev. Pharmacol. Toxicol. 59 (2019) 447–462. doi:10.1146/annurev-pharmtox-010818-021108.

[4]       J.F. Dekkers, G. Berkers, E. Kruisselbrink, A. Vonk, H.R. de Jonge, H.M. Janssens, I. Bronsveld, E.A. van de Graaf, E.E.S. Nieuwenhuis, R.H.J. Houwen, F.P. Vleggaar, J.C. Escher, Y.B. de Rijke, C.J. Majoor, H.G.M. Heijerman, K.M. de Winter-de Groot, H. Clevers, C.K. van der Ent, J.M. Beekman, Characterizing responses to CFTR-modulating drugs using rectal organoids derived  from subjects with cystic fibrosis., Sci. Transl. Med. 8 (2016) 344ra84. doi:10.1126/scitranslmed.aad8278.

[5]       G. Rossi, A. Manfrin, M.P. Lutolf, Progress and potential in organoid research, Nat. Rev. Genet. 19 (2018) 671–687. doi:10.1038/s41576-018-0051-9.

[6]       T. Takebe, K. Sekine, M. Enomura, H. Koike, M. Kimura, T. Ogaeri, R.R. Zhang, Y. Ueno, Y.W. Zheng, N. Koike, S. Aoyama, Y. Adachi, H. Taniguchi, Vascularized and functional human liver from an iPSC-derived organ bud transplant, Nature. 499 (2013) 481–484. doi:10.1038/nature12271.

[7]       S. Sugimoto, Y. Ohta, M. Fujii, M. Matano, M. Shimokawa, K. Nanki, S. Date, S. Nishikori, Y. Nakazato, T. Nakamura, T. Kanai, T. Sato, Reconstruction of the Human Colon Epithelium In Vivo, Cell Stem Cell. 22 (2018) 171-176.e5. doi:10.1016/j.stem.2017.11.012.

[8]       C. De Bari, F. Dell’Accio, J. Vanlauwe, J. Eyckmans, I.M. Khan, C.W. Archer, E. a Jones, D. McGonagle, T. a Mitsiadis, C. Pitzalis, F.P. Luyten, Mesenchymal multipotency of adult human periosteal cells demonstrated by single-cell lineage analysis., Arthritis Rheum. 54 (2006) 1209–21. doi:10.1002/art.21753.

[9]       G. Nilsson Hall, L.F. Mendes, C. Gklava, L. Geris, F.P. Luyten, I. Papantoniou, Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing, Adv. Sci. 7 (2020) 1–16. doi:10.1002/advs.201902295.

[10]     G. Nilsson Hall, I. Rutten, J. Lammertyn, J. Eberhardt, L. Geris, F.P. Luyten, I. Papantoniou, Cartilaginous spheroid-assembly design considerations for endochondral ossification: towards robotic-driven biomanufacturing, Biofabrication. 13 (2021) 045025. doi:10.1088/1758-5090/ac2208.

[11]     O. Duchamp de Lageneste, A. Julien, R. Abou-Khalil, G. Frangi, C. Carvalho, N. Cagnard, C. Cordier, S.J. Conway, C. Colnot, Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin, Nat. Commun. 9 (2018) 773. doi:10.1038/s41467-018-03124-z.

[12]     P. Lenas, M. Moos, F.P. Luyten, Developmental engineering: a new paradigm for the design and manufacturing of cell-based products. Part I: from three-dimensional cell growth to biomimetics of in vivo development., Tissue Eng. Part B. Rev. 15 (2009) 381–94. doi:10.1089/ten.TEB.2008.0575.

[13]     I. Papantoniou, G. Nilsson Hall, N. Loverdou, R. Lesage, T. Herpelinck, L. Mendes, L. Geris, Turning Nature’s own processes into design strategies for living bone implant biomanufacturing: a decade of Developmental Engineering, Adv. Drug Deliv. Rev. 169 (2021) 22–39. doi:10.1016/j.addr.2020.11.012.

[14]     D. Choudhury, A. Ashok, M.W. Naing, Commercialization of Organoids, Trends Mol. Med. 26 (2020) 245–249. doi:10.1016/j.molmed.2019.12.002.

[15]     C. Bock, M. Boutros, J.G. Camp, L. Clarke, H. Clevers, J.A. Knoblich, P. Liberali, A. Regev, A.C. Rios, O. Stegle, H.G. Stunnenberg, S.A. Teichmann, B. Treutlein, R.G.J. Vries, The Organoid Cell Atlas, Nat. Biotechnol. 39 (2021) 13–17. doi:10.1038/s41587-020-00762-x.

[16]     F. Schutgens, H. Clevers, Human Organoids: Tools for Understanding Biology and Treating Diseases, Annu. Rev. Pathol. Mech. Dis. 15 (2020) 211–234. doi:10.1146/annurev-pathmechdis-012419-032611.



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