Inflammation-Related Gene Profile Histology and Immunohistochemistry of Soft Connective Tissue Covering Bone Grafted with Deproteinized Bovine Bone Mineral and Demineralized Freeze-Dried Bone Allograft

Abstract

Purpose: To determine the profile of gene expression of soft connective tissue covering bone grafted with deproteinized bovine bone mineral (DBBM) or demineralized freeze-dried bone allograft (DFDBA) in comparison to that without grafting.

Materials and methods: Calvaria defects of mice were created and treated as follows: (1) defect without a graft as a control, (2) grafted with DBBM, or (3) grafted with DFDBA. After 1 month, the animals were sacrificed. Soft connective tissue covering the defect area was collected by a punch technique. RNA was isolated and processed to cDNA. Gene expression was evaluated with the microarray technique. Pathway analyses were performed via the PANTHER Overrepresentation test and WikiPathway analysis. Inflammatory marker genes were chosen for mRNA expression using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Tissue sections were used for the histologic and immunohistologic evaluation.

Results: The numbers of genes that were significantly differently expressed were 312 (DBBM vs control), 82 (DFDBA vs control), and 113 (DBBM vs DFDBA). Inflammation-related genes were upregulated in DBBM vs control (16 genes), DFDBA vs control (3 genes), and DBBM vs DFDBA (15 genes). Two of these genes, Bcl2a1 and Cxcl9, were significantly upregulated in both the DBBM and DFDBA groups compared with the control. Pathway analysis indicated that Bcl2a1 and Cxcl9 are dominantly expressed in inflammation-related pathways. RT-qPCR and immunohistochemistry showed upregulation of Bcl2a1 and Cxcl9 in DBBM compared with the control and DFDBA groups. Cxcl9 showed a significantly higher expression in the DBBM group. Bcl2a1 at the protein level was equally expressed in all groups. Any sign of inflammation, however, was not seen by histology in any of the groups.

Conclusion: After a 1-month healing period, soft tissue covering bone grafted with DBBM expressed a higher number of inflammation-related genes compared with those non-grafted or grafted with DFDBA. DFDBA resulted in a decreased expression of inflammation-related genes.