In Vitro Comparison of Macrophage Polarization and Osteoblast Differentiation Potentials between Granules and Block Forms of Deproteinized Bovine Bone Mineral

Abstract

Deproteinized bovine bone mineral (DBBM) bone grafts are commonly utilized for guided bone regeneration (GBR) techniques in regenerative dentistry. It has been hypothesized that different forms (blocks versus particulates) might demonstrate the varying properties of cell behavior during the regenerative process. Therefore, the aim of the present study was to investigate DBBM granules and blocks for their effects on osteoblasts and macrophages (Mφs). DBBM granules and blocks were filled to the same size (φ6.4 mm in diameter × 2.0 mm in height) in cell culture wells and assessed for cell viability and cell differentiation of human osteoblast-like Saos-2 cells, and Mφs derived from human monocyte THP-1 cells. The two groups were first characterized by micro-CT analysis, which demonstrated that DBBM granules had a two-fold greater material volume and a four-fold larger surface area than the blocks. DBBM blocks showed superior viability for both osteoblasts and Mφs. Osteoblast experiments were then utilized to better characterize the influence of DBBM shapes/forms on osteoblast differentiation. Alkaline phosphatase (ALP) staining on the undecalcified frozen sections was observed throughout the DBBM granule surface, yet this staining was only observed on the upper portion of the DBBM blocks. Furthermore, DBBM blocks showed M1-Mφ polarization trends with higher IL-1 and IL-6 mRNA expression in Mφs, while the conditioned media from Mφs cultured on DBBM granules promoted osteoblast differentiation with higher mRNA levels of Runx 2, ALP and osteocalcin. In conclusion, the DBBM granules showed more regenerative effects, lower M1 marker expression, and higher osteoblast differentiation potential when compared with the blocks, which might be related to the larger material volume, higher surface area and greater ability for the cells to penetrate through the scaffold.

The impact of the size of bone substitute granules on macrophage and osteoblast behaviors in vitro

“…In the present study, however, the granules used were standardized throughout the experiments by utilizing the volume-based defect filling size concept instead of the weight-based concept. This approach was intended to increase the similarity of the experimental procedures to the clinical reality of the bone augmentation procedure [15]. The micro-CT imaging analysis revealed that the smaller granules showed higher material volumes and surface areas than the larger granules, as expected (Figs.…”

“…3 and 4). DBBM granules have previously been shown to drive polarization toward the M2 phenotype, as indicated by high expression of CD163 and CD206 in the murinederived macrophage cell line RAW 264.7 and by high expression of IL-10, Arginase 1 (Arg1), and CD206 in THP-1 cellderived Mφs [15,20]. BCP has been reported to upregulate the gene expression of most inflammatory factors and growth factors in RAW 264.7 cells [21,22].…”

“…Throughout the study assays, S-size and L-size granules were compared for DBBM and BCP1, and S-size and M-size granules were compared for BCP2 and CO 3 Ap. All BSs were aseptically prepared at the same size (φ6.4 mm in diameter × 2.0 mm in height) in 96-well plates or cell culture plate inserts (Transwell® inserts, Sigma, St. Louis, MO, USA) to standardize bulk volume, as previously reported [15].…”

Testing active membranes for bone regeneration: A review

“…Primers need to be designed using NCBI-nucleotide library and Primer3-design in order to detect messenger RNA (mRNA) of the targeted genes. In Table 1 a list of the most useful primer sequences for the amplification of cDNA by real-time PCR is presented [50,51]. RT-qPCR can now be performed with the cDNA extracted from the cells and the designed primers.…”

“…RT-qPCR can be used in order to detect differences in the expressions of M1 and M2 markers. For M1 profile; IL-1, IL-6 and TNF-α are normally explored; whereas for M2 markers IL-10, Arg1, CD206 and TGF-β are the selected genes [51,67]. Recommended genes' sequences are presented in Table 2 [51,67].…”