(25) Porous 3D Printed Bioceramic Scaffolds Induce Osteoblastogenesis in Vitro

Abstract: Recently published studies1,2 have shown great bone remodeling properties of the 3D printed bioceramic implant, P3D Bone. CT-imaging and histological analyses clearly visualized that the bioceramic implant was resorbed and replaced by de novo bone over a 6-month period.
Although these are interesting finds, the study does not investigate whether the development of new bone relies on osteoclast presence and signaling or if the implant material and structure alone is sufficient to support new bone formation. Several studies have shown that osteoclasts secrete signaling molecules such as Sphingosine 1 phosphate (S1P), Collagen triple helix repeat containing 1 (CTHRC1) and Complement component C (C3)3, which promotes osteoblastogenesis and subsequently bone formation. One could therefore assume that the remodeling of the P3D Bone implant is reliant on the presence of osteoclasts near the implant to activate residing osteoblasts.
This study aims to examine whether the 3D printed bioceramic material alone is sufficient to differentiate mesenchymal stem cells into osteoblasts and whether these osteoblasts are able to produce extra cellular matrix. To investigate this, we have established a porous 3D cell culture system consisting of 100% β-TCP using Ossiform's proprietary technology, where the mixing of β-TCP powder and fatty acids followed by sintering results in a porous bioceramic. The bioceramic paste was 3D printed in sizes that were suitable for in vitro studies and with a macro-porous size between 600 and 800um organized in a grid structure. Mesenchymal stem cells were cultured on the P3D Scaffolds for two to five weeks in standard maintenance medium and subsequently analyzed using standard laboratory techniques such as qPCR and microscopic analyses for the evaluation of osteogenic marker genes and ECM production.
Data from these studies shows that the osteogenic marker genes ALP and type 1 collagen are upregulated in response to culturing on the P3D Scaffold compared to traditional 2D cell culturing, suggesting that the bioceramic material itself can induce bone formation. Furthermore, UV light microscopy images showed a wide dispersion of the osteoblasts on the scaffold suggesting that the motility of the osteoblasts is not compromised by the surface structure of the P3D Scaffold. SEM imaging showed clear extracellular matrix formation on the P3D Scaffolds within five weeks, which is further validated by confocal microscopy with DAPI staining and collagen antibodies.
Together, these results support that the bone remodeling occurring after implantation of the 3D printed bioceramic implant does not solely rely on the osteoclastic signaling to promote osteoblastogenesis. Instead, it appears that the bioceramic material itself functions as an inducer of the osteogenic gene program, subsequent extracellular matrix production and ultimately de novo bone formation at the implant site.

1. Jensen, M. B. et al. Treating mouse skull defects with 3D-printed fatty acid and tricalcium phosphate implants. Journal of Tissue Engineering and Regenerative Medicine 14, 1858–1868 (2020).
2. Thygesen, T. et al. Comparison of off-the-shelf β-tricalcium phosphate implants with novel resorbable 3D printed implants in mandible ramus of pigs. Bone 159, 116370 (2022).
3. Kim, J. M., Lin, C., Stavre, Z., Greenblatt, M. B. & Shim, J. H. Osteoblast-Osteoclast Communication and Bone Homeostasis. Cells 9, (2020).