ECM remodelling components regulated during jaw periosteal cell osteogenesis Dorothea Alexander 1 *, Nina Ardjomandi*, Adelheid Munz*, Bjo ¨ rn Friedrich { and Siegmar Reinert* * Department of Oral and Maxillofacial Surgery, University Hospital Tu ¨ bingen, Tu ¨ bingen, Germany { Department of Internal Medicine IV, University Hospital Tu ¨ bingen, Tu ¨ bingen, Germany Abstract Human JPCs (jaw periosteal cells) are a promising source for the engineering of cell-based osteoinductive grafts in oral surgery. For this purpose, cell characteristics of this stem cell source should be elucidated in detail. Analysis of gene expression profiles may help us to evaluate key factors and cellular targets of JPC osteogenesis. Because little is known about the interplay of osteogenic-related components, we analysed the expression of different collagen types reflecting important players for extracellular matrix assembly and of TIMPs (tissue inhibitors of metalloproteinases) responsible for the inhibition of matrix degradation. Gene expression analyses using microarrays and quantitative RT–PCR (reverse transcription–PCR) during JPC osteogenesis revealed the induction of several collagen types’ expression (VII, VIII, XI and XII), and some of them (types I, VIII and XI) seemed to be susceptible to BMP-2 (bone morphogenetic protein-2) that is known to be a potent osteogenic inducer of periosteal cells. Among the TIMPs, only TIMP-4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) expressions were strongly up-regulated during JPC osteogenesis. Proteome profiler analysis of supernatants from untreated and differentiated JPCs confirmed the gene expression data in terms of TIMP expression. In summary, we identified new collagen types and TIMPs that seem to play important roles during the osteogenesis of jaw periosteal progenitor cells. Keywords: collagen; gene expression; jaw periosteal cell; osteogenesis; tissue inhibitor of metalloproteinases 1. Introduction The use of autograft bone has several disadvantages, including limited tissue available for transplantation and increased patient morbidity at the site of harvest. Autogenous bone grafting could be replaced by tissue engineering, which combines the use of autologous cells with bioresorbable three-dimensional scaffolds to repair lost bone tissue function in oral and maxillofacial surgery. MSC (mesenchymal stem cells) from bone marrow are capable of undergoing osteoblastic differentiation (Jaiswal et al., 1997; Pittenger et al., 1999). Several studies have described the use of periosteal cells for implantation in animal models to fill bone defects (Sakata et al., 2006). Periosteum-derived cells show stem cell differentiation potential by their capacity to form multiple tissues in vitro and in vivo (De Bari et al., 2001, 2006; Nakahara et al., 1990a, 1991). Several animal models have already been established based on the application of periosteal cells into bone defects, but knowledge of the molecular level of this dynamic process is sparse (Kawase et al., 2009; Sakata et al., 2006). Little is understood about the interplay of the osteoblastic-related genes or their contextual role. Bone formation involves strong proliferation of progenitor cells followed by a differentiation phase that ends with the mineralization of the ECM (extracellular matrix). Understanding the mineralization process involves identifying the relevant ECM remodelling molecules. The ECM surrounds the cells, plays key instructive and structural roles in tissue architecture and provides a reservoir of cytokines and growth factors. By its nature, the ECM is permanently undergoing changes in both structure and composition in response to different cellular stimuli. ECM remodelling involves two key processes: the synthesis and deposition of ECM components, on the one hand, and their proteolytic breakdown, on the other hand. In most organs, the principal proteinaceous components of the ECM are collagens, which provide the scaffold necessary for the attachment and organization of cells. MMP (matrix metalloproteinases) cleave the protein components of the ECM, but they are stringently controlled by endogenous inhibitors, the so-called TIMPs (tissue inhibitors of metalloproteinases). In addi- tion to the TIMPs, a novel class of membrane-anchored proteins has been discovered that shows negative regulation of MMP acti- vity and that was subsequently found to be a potent inhibitor of tumour metastasis, the RECK (reversion-inducing cysteine-rich protein with Kazal motifs) (Clark et al., 2007). To optimize tissue engineering applications using this stem cell type, we previously sought to identify specific osteoprogenitor markers for mineralizing and non-mineralizing JPCs (jaw periosteal cells; Alexander et al., 2009, 2010) and to elucidate the osteogen- esis process. Herein, we analysed the expressions of multiple collagen types and TIMPs including RECK involved in and regu- lated during JPC osteogenesis. Since BMP-2 (bone morphogenetic protein-2) is known to play an important role in the early stages of bone repair by recruiting progenitors within periosteum and by determining their differentiation towards the osteogenic lineage 1 To whom correspondence should be addressed (email dorothea.alexander@med.uni-tuebingen.de). Abbreviations: BMP-2, bone morphogenetic protein-2; DEPC, diethyl pyrocarbonate; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; FCS, foetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JPC, jaw periosteal cell; MMP, matrix metalloproteinase; MSC, mesenchymal stem cell; OB, osteogenically differentiated; RECK, reversion-inducing cysteine-rich protein with Kazal motifs; RT–PCR, reverse transcription–PCR; TIMP, tissue inhibitor of metalloproteinases; WT, wild-type. Cell Biol. Int. (2011) 35, 973–980 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2011 Portland Press Limited Volume 35 (10) N pages 973–980 N doi:10.1042/CBI20100836 N www.cellbiolint.org 973