Acid bone lysate activates TGFβ signalling in human oral fibroblasts
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
... ectrum 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. Bone grafts are regularly used for augmentation in implant dentistry, oral and maxillofacial surgery, besides other medical fields including orthopedics and traumatology dealing with bone reconstructions 1,2 . Freshly prepared bone autografts are considered gold standard in reconstructing large and complex bone defects due to the osteoconductive surface and the presence of osteogenic cells that can contribute to bone formation 3 . Furthermore, growth factors released upon autograft resorption are supposed to support bone regeneration, even though evidence to support this claim is poor. Similar to the resorption of autografts by osteoclasts, demineralization of allografts by hydrochloric acid does not only remove the mineral phase 4 . Hydrochloric acid also removes a fraction of growth factors intrinsic to bone. The biological activity of the respective acid bone lysate (ABL), which are discarded upon the preparation of demineralized bone matrix, has not been characterized so far. Bone is a rich source of growth factors including TGF-β1 5,6 . Pioneer work of purification and characterization of TGF-β1 released by hydrochloric acid and other methods dates back to the 1980s 6-8 . With the introduction of proteomics, bone extraction protocols were refined 9 still including demineralization of bone by hydrochloric acid 10 . The concentration of TGF-β1 with around 0.5 ng/ml in bone lysates is conserved among skeletal areas and gender 5 . In vivo, TGF-β1 is stored in a latent form and can be released and activated by osteoclasts 11-13 . TGF-β1 released during bone remodeling induces migration of mesenchymal stem cells 14,15 and targets osteoclasts 16 . However, the activity of TGF-β1 and other growth factors in ABL has not been studied recently. Bioassays with TGF-β-responsive cells are appropriate to determine TGF-β activity in ABL. We previously used oral fibroblasts to detect TGF-β1 activity in supernatants of freshly prepared bone chips 17 and enamel matrix derivatives 18 . Moreover, the adsorption of TGF-β1 from these preparations to collagen matrices commonly used for guided bone regeneration was determined by gene expression changes of oral fibroblasts 19,20 . The selection of genes was based on proteomic analysis and a whole genome microarray resulting in a panel of TGF-β target genes including interleukin 11 (IL11), proteoglycan4 (PRG4), and NADPH oxidase 4 (NOX4) 21, 22 . Further support for activation of TGF-β signaling comes from phosphorylation and translocation of Smad3 into the nucleus 23 .