Poster AA10 - Development of Osteocyte-Based Bone Organoids with Osteonal Structures

Introduction: Osteocytes have come to the forefront of bone biology and are increasingly recognized as a key cell type that integrates hormonal, mechanical and other signals to control bone mass and regulate endocrine homeostasis. Dysfunctional osteocytes disturb bone homeostasis, resulting in bone metabolic diseases such as diabetic bone fragility. As they are deeply embedded in the concentric lamellae of the osteons, their cellular mechanisms in normal or pathological bone tissues in patients remain challenging to study. One way to better understand the inner workings of bone is to perform in vitro studies through bone tissue engineering, especially when animal models fail to recapitulate the human counterparts. However, current bone tissue scaffolds are still insufficient to encapsulate the complexity of bone microenvironment; nevertheless, only limited studies have attempted to address the properties of osteons, which are the chief structural and functional units of the cortical bone and play a crucial role in the biological and mechanical response of bone. Therefore, the development of a bone organoid composed of both native demineralized bone matrix and bone cells such as osteocytes while reproducing the osteonal characteristics may give us a better insight in studying skeletal diseases.

Materials and

Methods: Bone organoid was first fabricated and assessed using bovine bones and mouse cells (Figure 1). Briefly, bovine femurs were obtained from a local slaughterhouse, cut into chunks, and cleaned off bone marrow and soft tissue. Smaller sections cut vertically from the bone were demineralized in 1.2 N hydrochloric acid (HCl), embedded in optimal cutting temperature (OCT) medium, and further sliced into 20-µm thin sheets with a cryostat. Surface morphology such as the lamellar structure of the demineralized bone sheets (DBS) was observed under a microscope to evaluate the intactness of bony extracellular matrix (ECM) structure. DBSs were then arranged longitudinally into a longer strip in a flat tray and fixed in 4% polyvinyl alcohol (PVA) hydrogel after drying overnight. A strip of DBS-PVA film was cut out and rolled around a sterile needle into a three-dimensional (3D) osteonal structure. Murine osteocyte-like MLO-Y4 cells were seeded onto the construct and incubated in culture media (ɑ-MEM supplemented with penicillin/streptomycin at 100 U/ml, 2.5% fetal bovine serum, and 2.5% calf serum). The construct was re-mineralized in 10× simulated body fluid (SBF).

Results, Conclusions, and Discussions: Our investigation yielded preliminary insights into the feasibility of developing such an osteocyte-based bone organoid with osteonal structures. After cleaning and demineralization in HCl for 1 day, the outer layer of the bone turned semitransparent. Demineralization was further confirmed by Raman Spectroscopy, where only low peaks were observed at 427, 577, 958 and 1070 cm-1 that are characteristics of phosphate and carbonate bone mineral (Figure 2). To retain sufficient light transmittance and adequate mechanical durability for handling, 20-µm was found to be a practical thickness set for cryosection. Observations under an inverted microscope showed parallel lamellar structure in DBSs and thus confirmed a well-preserved bony extracellular matrix structure in DBSs (Figure 3). Detailed characterization of surface morphology was performed by scanning electron microscopy (SEM), which showed densely aligned collagen fiber bundles (Figure 4). After the DBSs were arranged into a long strip, fixed in PVA, rolled up and seeded with cells, the resultant construct showed structural stability, cell viability and free-standing ability when either dried in petri dish or submerged in liquid media (Figure 5).

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