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.
Highlights
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]
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), proteoglycan[4] (PRG4), and NADPH oxidase 4 (NOX4)[21,22]
Bone is a rich source of growth factors that are released by osteoclasts during remodeling[14,15], fracture healing[26], and during the preparation of demineralized bone matrix
Summary
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]. Similar to the resorption of autografts by osteoclasts, demineralization of allografts by hydrochloric acid does remove the mineral phase[4]. Hydrochloric acid 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. With the introduction of proteomics, bone extraction protocols were refined[9] still including demineralization of bone by hydrochloric acid[10]. We report the protein composition of ABL and determine the respective biological activity for oral fibroblasts. Based on a whole genome microarray and RT-PCR approach including the pharmacologic blocking of TGF-β receptor type I kinase with SB43154224, ABL caused a robust activation of TGF-β signaling. Our findings suggest that TGF-β is among the major growth factors being discarded upon the demineralization of bone matrix
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