Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro Jianbo Peng, MD, DDS;* ,‡,¶ Michael Nemec, MD; †,‡ Eliane Brolese, DDS;* ,‡ Dieter D. Bosshardt, PhD; †,§ Benoit Schaller, MD, DDS;* Daniel Buser, DMD; † Reinhard Gruber, PhD †,‡ ABSTRACT Background and Purpose: Autografts are used for bone reconstruction in regenerative medicine including oral and maxillofacial surgery. Bone grafts release paracrine signals that can reach mesenchymal cells at defect sites. The impact of the paracrine signals on osteogenic, adipogenic, and chondrogenic differentiation of mesenchymal cells has remained unclear. Material and Methods: Osteogenesis, adipogenesis, and chondrogenesis were studied with murine STβ osteoblast progenitors, γTγ-L1 preadipocytes, and ATDC5 prechondrogenic cells, respectively. Primary periodontal fibroblasts from the gingiva, from the periodontal ligament, and from bone were also included in the analysis. Cells were exposed to boneconditioned medium (BCM) that was prepared from porcine cortical bone chips. Results: BCM inhibited osteogenic and adipogenic differentiation of STβ and γTγ-L1 cells, respectively, as shown by histological staining and gene expression. No substantial changes in the expression of chondrogenic genes were observed in ATDC5 cells. Primary periodontal fibroblasts also showed a robust decrease in alkaline phosphatase and peroxisome proliferator-activated receptor gamma (PPARȖ) expression when exposed to BCM. BCM also increased collagen type 10 expression. Pharmacologic blocking of transforming growth factor (TGF)-ȕ receptor type I kinase with SB4γ154β and the smad-γ inhibitor SISγ at least partially reversed the effect of BCM on PPARȖ and collagen type 10 expression. In support of BCM having TGF-ȕ activity, the respective target genes were increasingly expressed in periodontal fibroblasts. Conclusions: The present work is a pioneer study on the paracrine activity of bone grafts. The findings suggest that cortical bone chips release soluble signals that can modulate differentiation of mesenchymal cells in vitro at least partially involving TGF-ȕ signaling. KEY WORDS: ADAM19, adipocytes, adipogenic differentiation, ALPL, autografts, autologous bone grafts, bone regeneration, COL10, chondrogenic differentiation, conditioned medium, differentiation, graft consolidation, IL-11, MAPK, mesenchymal cells, osteoblasts, osteocytes, osteogenic differentiation, paracrine, periodontal fibroblasts, PPARȖ, Sema7a, smad-γ, supernatant, TGF-ȕ INTRODUCTION Autologous bone grafts (ABGs) are assumed to have osteoconductive, osteoinductive, and osteogenic properties. This means that new bone can grow on the surface of the bone graft, the growth factors stored in the bone can initiate the process of de novo bone formation at ectopic sites, and the transplanted cells can form bone at the defect site. 1–γ This trinity of biological properties supports the claim that ABG are the “gold standard” for bone augmentation in oral and maxillofacial surgery 4–6 and orthopedics. 7 There is evidence that autologous bone is osteoconductive, whereas surprisingly, little *Department of Cranio-Maxillofacial Surgery, Inselspital, University of Bern, Bern, Switzerland; † Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Bern, Bern, Switzerland; ‡ Laboratory of Oral Cell Biology, School of Dental Medicine, University of Bern, Bern, Switzerland; § Robert K. Schenk Laboratory of Oral Histology, School of Dental Medicine, University of Bern, Bern, Switzerland; ¶ College of Stomatology, GuangXi Medical University, GuangXi, China Corresponding Author: Reinhard Gruber, PhD, Laboratory of Oral Cell Biology, School of Dental Medicine, University of Bern, Freiburgstrasse 7, Bern γ010, Switzerland; e-mail: reinhard.gruber @zmk.unibe.ch