3D human osteon mimic using a rolled demineralized bone paper strip

Introduction: Compact bone is a vascularized mechanical tissue. An osteon, consisting of a central blood vessel surrounded by a mineralized lamellar collagen matrix embedded with osteocytes, is a structural and functional unit that supports blood perfusion while providing mechanical support. While the mechanical aspects of compact bone have been well documented, its vascular biology remains poorly understood due to the intrinsic challenge of accessing blood vessels. Mouse models have been widely used for studying bone biology, but their bones do not include osteons, which only appear in larger vertebrates. The development of 3D osteon models is imperative to understand vascular biology in compact bone and its turnover processes. Here, we report a tissue-engineered 3D osteon mimic using demineralized bone paper (DBP) that preserves the intrinsic collagen fiber structure, providing a suitable matrix for osteoblast proliferation and mineralization.

Materials and

Methods: Bovine compact bone was demineralized in 1.2M HCl and sliced into 20 µm sections using a cryostat. A DBP strip was prepared by laminating DBP with polyvinyl alcohol, cut into rectangular templates (5 x 20 mm) and weaved them into a longer strip (5 x 80 mm). After dissolving polyvinyl alcohol, the sterilized DBP strip was seeded with human osteoblasts differentiated from bone marrow stromal cells. The DBP strip was rolled around an aluminum rod (D=5mm) and cultured for 3 weeks under mechanical vibration to promote mineral synthesis and differentiation of osteoblasts into osteocytes. The prepared 3D human bone model mimics concentric lamellar bone structure in the osteon. Finally, human umbilical vein endothelial cells (HUVECs) were seeded inner surface of cylindrical 3D bone mimic. This engineered 3D human osteon mimic was cultured with shear flow directed into the inner cavity and out, creating a shear stress to support the development of mature capillary-like structures.

Results, Conclusions, and Discussions: DBP preserves the intrinsic collagen fiber structure, providing a suitable matrix for osteoblast proliferation and mineralization. PVA lamination allows tailoring an identical complex shape of DBP, which can be weaved to make a long DBP strip. DBP supports osteoblasts adhesion and mineralization. When rolled DBP, osteoblasts layered between DBPs differentiate into osteocytes. Mechanical vibration increased osteoblastic mineral deposition. After 3 weeks of culture, it forms a free-standing cylindrical 3D lamellar bone model.
Our initial studies using a 2D model showed that HUVECs seeded on DBP formed initial tubule-like structures, both in monoculture and co-culture with human osteoblasts. Imaging confirmed these structures, indicating DBP provides a support matrix for HUVEC organization. Confocal imaging revealed that HUVECs formed a separate layer on top of the osteoblasts, maintaining distinct endothelial and osteoblastic layers. Given the limitations of 2D models in replicating the complex architecture of human osteons, we aim to develop a three-dimensional (3D) osteon model. By manipulating DBP into a 3D cylindrical structure, we mimic natural bone's dense, osteon-shaped architecture. In this 3D configuration, osteoblasts on DBP will differentiate into osteocytes, and HUVECs subjected to shear flow will form mature capillary-like structures. This approach replicates the capillaries within the osteon model, which are crucial for nutrient and waste exchange in bone tissue. Our 3D osteon model aims to fill the gap in understanding vascular dynamics in compact bone, thereby advancing our knowledge of bone remodeling and disease progression. This model is a valuable tool for studying osteoporosis and offers potential drug testing and bone tissue engineering applications.
In conclusion, we created the 3D lamellar bone model by rolling a DBP strip seeded with human osteoblasts. Subsequently, introduced HUVECs emulate the multicellular complexity of an osteon in an anatomically relevant configuration. This 3D human osteon model is expected to advance our understanding of bone vascular biology, bone mechanobiology, as well as compact bone remodeling.

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