Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts

Abstract

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK-as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-β (TGF-β) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-β immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-β nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-β receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-β are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts.

Keywords: AREG; C11orf96; IL11; RNAseq; bone allograft; bone augmentation; bone regeneration; gingival fibroblasts.

Figure 1

RNAseq analysis of gingival fibroblasts exposed to ALBA. Gingival fibroblasts were exposed to acid lysates of bone allografts (ALBA). RNAseq revealed a panel of highly regulated genes (>50-fold) that are considered biomarkers to reflect the response of local mesenchymal cells. Data are shown by STRING analysis.

Figure 2

RT-PCR analysis of gingival fibroblasts exposed to ALBA. Gingival fibroblasts were incubated with acid lysates of bone allografts (ALBA). RT-PCR analysis confirmed the strong increase in expression changes in ALBA-induced gingival fibroblasts. Data points represent four independent experiments. Data were normalized to untreated control cells with x-fold changes compared to the untreated cells. The statistical analysis was based on the Friedmann test with uncorrected Dunn’s test.

Figure 3

RT-PCR analysis of gingival fibroblasts in the presence of SB431542. RT-PCR analysis of gingival fibroblasts incubated with acid lysates of bone allograft (ALBA) with and without the TGF-β RI kinase inhibitor SB431542. Expression analysis showed that the blocking of TGF-β signaling reduced ALBA-induced expression of IL11, AREG, and C11orf96 in gingival fibroblasts. Data points represent four independent experiments. Data were normalized against untreated control cells with x-fold changes compared to the untreated cells. The analysis was based on a paired t-test.

Figure 4

Immunoassay analysis of gingival fibroblasts in the presence of SB431542. Immunoassay of gingival fibroblasts exposed to acid lysates of bone allograft (ALBA) with and without the TGF-β RI kinase inhibitor SB431542. Immunoassay indicated that blocking of TGF-β signaling reduced ALBA-induced expression of IL11 but not STC1 in gingival fibroblasts. Data points represent four and three independent experiments, respectively. The analysis was based on a ratio-paired t-test.

Figure 5

Immunostaining of gingival fibroblasts for smad2/3 nuclear translocation. Immunostaining of gingival fibroblasts exposed to acid lysates of bone allograft (ALBA) or TGF-β1. The staining pattern revealed a strong nuclear translocation of smad2/3 when gingival fibroblasts are exposed to recombinant TGF-β1 but not to ALBA. The scale bars represent 100 µm.

Figure 6

STRING analysis of bone allograft proteome. STRING analysis of the 114 proteins common to all batches identified. Please note the cluster related to the collagen matrix and non-collagenous proteins.