Plenary Poster 33rd ASM of the Australian & New Zealand Bone & Mineral Society 2023

Development of in vitro 3D osteocyte culture models in ECM-derived hydrogels (#104)

Thanuja Herath Mudiyanselage 1 2 , Agathe Bessot 1 2 , Flavia Medeiros Savi 2 3 , Marie-Luise Wille 2 3 , Dietmar W Hutmacher 1 2 3 , Nathalie Bock 1 2
  1. School of Biomedical Sciences, Queensland University of Technology, Woolloongabba, QLD, Australia
  2. Materials Science of Extracellular Matrices, Max Planck Queensland Centre, Brisbane, QLD, Australia
  3. School of Mechanical, Medical and Process Engineering, Queensland University of Technology , Brisbane, QLD, Australia

Currently, most bone knowledge derived from in vitro models use cells cultured in 2D, due to inherent technical difficulties in establishing osteocyte cultures in 3D. Unlike other bone cells, osteocytes cannot faithfully be studied in vitro in the absence of their surrounding extracellular components, including collagen proteins and minerals. We propose to use extracellular matrix (ECM)-derived 3D hydrogels composed of tuneable gelatin methacrylamide (GelMA)1, for the establishment and study of 3D osteocyte networks in vitro. Gelatin is relevant as it chemically resembles native collagenous bone matrix, contains integrin attachment sites that facilitate interaction of the cytoskeleton and ECM, and is conducive to mineralization, as shown by us and others2,3. We hypothesised that combining GelMA and biomimetic culture would provide new avenues to control the formation and mineralisation of osteocyte 3D cultures in vitro and identify how ECM and culture cues support network formation and functions. We used either human primary osteoprogenitor cells (derived from knee/hip arthroplasty from Prof. Ross Crawford, QUT) or murine DMP1-GFP OCY454 cell lines (gift from Prof Natalie Sims, St Vincent’s Institute) and encapsulated cells in 5% w/v GelMA (3D) and also cultured them in 2D. We assessed the use of growth medium (GM) or osteogenic medium (OM) up to 28 days with/without the use of a mineralisation medium (MM) boost for 3 days following cell encapsulation, known to enhance nanoscale biomineralisation4, and compared normoxic/hypoxic (20%/2% O2) culture conditions in 2D and 3D settings. Mineralisation was assessed by micro-computed tomography; osteocyte phenotype and branching by brightfield and confocal laser microscopy; and protein and gene expression by downstream analysis. Ongoing experiments preliminarily showed that supportive environmental cues such as MM and OM positively modulated mineralisation and osteocyte phenotype. Further optimisation of in vitro 3D osteocyte culture models will enable understanding the effects of ECM-cell interactions and addressing fundamental osteocyte biology questions.

  1. Loessner, D. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat. Protoc. 11, 727–746 (2016).
  2. Mestres, G. et al. Advantages of microfluidic systems for studying cell-biomaterial interactions-focus on bone regeneration applications Biomedical Materials. Biomed. Mater 17, 65027 (2022)
  3. Bessot, A. et al. GelMA and Biomimetic Culture Allow the Engineering of Mineralized, Adipose, and Tumor Tissue Human Microenvironments for the Study of Advanced Prostate Cancer In Vitro and In Vivo. Adv. Healthc. Mater. 2201701 (2023) doi:10.1002/adhm.202201701
  4. Thrivikraman, G. et al. Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization. Nat. Commun. 10, 3520 (2019)