Acid bone lysate activates TGFβ signalling in human oral fibroblasts

Abstract

Demineralized bone matrix is a widely used allograft from which not only the inorganic mineral but also embedded growth factors are removed by hydrochloric acid (HCl). The cellular response to the growth factors released during the preparation of demineralized bone matrix, however, has not been studied. Here we investigated the in vitro impact of acid bone lysate (ABL) prepared from porcine cortical bone chips on oral fibroblasts. Proteomic analysis of ABL revealed a large spectrum of bone-derived proteins including TGF-β1. Whole genome microarrays and RT-PCR together with the pharmacologic blocking of TGF-β receptor type I kinase with SB431542 showed that ABL activates the TGF-β target genes interleukin 11, proteoglycan 4, and NADPH oxidase 4. Interleukin 11 expression was confirmed at the protein level by ELISA. Immunofluorescence and Western blot showed the nuclear localization of Smad2/3 and increased phosphorylation of Smad3 with ABL, respectively. This effect was independent of whether ABL was prepared from mandible, calvaria or tibia. These results demonstrate that TGF-β is a major growth factor that is removed upon the preparation of demineralized bone matrix.

Figure 1

Acid bone lysate (ABL) contains a large spectrum of proteins including TGF-β. Panther analysis of molecular function (a) cellular component (b) and class (c) of proteins in ABL. (d) TGF-β1 immunoassay of different bone regions: mandible, calvaria and tibia. N = 3–5. Data represent the mean ± SD.

Figure 2

STRING representation of a network involving the proteins detected in ABL. Different line colors represent the types of evidence for the association between proteins.

Figure 3

Viability of primary oral fibroblast exposed to acid bone lysate (ABL). Cell viability staining of primary oral fibroblast upon exposure to ABL was tested by MTT assay (a) and Live-Dead staining (b). The results from these experiments demonstrated that stimulation with ABL at 5% is highly biocompatible with oral fibroblast. Live-Dead staining was done with viable cells appearing in green and dead cells in red. N = 3–5. Data represent the mean ± SD relative to the control. *P < 0.05, by Kruskal-Wallis test with Dunn’s multiple comparisons correction.

Figure 4

Acid bone lysate (ABL) provokes changes in gene expression based on gene arrays. Eighteen genes were at least 10-fold up- and down-regulated by ABL (Table 2). Panther analysis of molecular function (a) biological process (b) and cellular component (c) of most regulated genes in oral fibroblasts by ABL.

Figure 5

Acid bone lysate (ABL) changes the expression of selected genes via TGF-β signaling. Addition of TGF- β receptor 1 kinase antagonist SB431542 to ABL blocks the expression of the selected genes (a). Immunoassay of IL11 supports the previous findings at the protein level (b). N = 3–5. Data represent the mean ± SD. *P < 0.05, ***P < 0.001, by two-tailed Mann-Whitney test.

Figure 6

Acid bone lysate (ABL) activates TGF-β-Smad2/3 signaling in primary oral fibroblasts. Incubation of gingival fibroblasts with ABL also caused an increased phosphorylation of Smad3 (a). Representative immunofluorescence confirmed the translocation of Smad2/3 into the nucleus upon stimulation with ABL (b). Treatment with 10 ng/ml of TGF-β was used as a positive control.

Figure 7

Acid bone lysate (ABL) obtained from mandible, calvaria and tibia produces an equivalent gene expression and TGF-β activity is highly dependent on hydrochloric acid concentration. ABL obtained from different sources (mandible, calvaria and tibia) produces a similar response on selected genes (a). ABL prepared with 0.1 N hydrochloric acid induces a higher TGF-β activity at mRNA level (b). Immunoassay of IL11 confirmed the higher activity of TGF-β at protein level with 0.1 N ABL (c). N = 3–5. Data represent the mean ± SD. P > 0.05, by Kruskal-Wallis test with Dunn’s multiple comparisons correction, *P < 0.05, **P < 0.01, by two-tailed Mann-Whitney test.

Figure 8

Acid bone lysate (ABL) decreases osteogenic and adipogenic differentiation. Exposure of osteogenic calvaria cells and adipogenic 3T3-L1 cells to ABL caused a considerable decrease of the marker genes alkaline phosphatase (ALP) and osteocalcin (OC) (a), as well as PPARγ and C/EBP (b), respectively. Chondrogenic ATDC5 cells showed a moderate increase of SOX9 and COL10 (c). Histochemical staining of alkaline phosphatase activity confirmed the findings from gene expression in osteogenic calvaria cells (d). N = 3–5. Data represent the mean ± SD.