Scientific Studies An In Vitro Model for Dissecting Distraction Osteogenesis Francesco Carinci, MD,* Furio Pezzetti, PhD, † Anna Maria Spina, PhD, ‡ Annalisa Palmieri, PhD, § Friedrick Carls, MD, # Gregorio Laino, MD, § Alfredo De Rosa, DMD,  Ernesto Farina, DMD, § Fausto Illiano, DMD, § Giordano Stabellini, PhD,** Lorenzo LoMuzio, MD, †† Vittoria Perrotti, DMD, ‡‡ Adriano Piattelli, MD ‡‡ Distraction osteogenesis (DO) is a mechanotrans- duction process capable of generating viable os- seous tissue by the gradual separation of osteoto- mized bone edges. Several variables are implicated in DO: magnitude of mechanical strain, distraction rate, and type of distracted bone. The combination of these factors acts on different types of cells in- ducing apoptosis, cell proliferation, and differen- tiation. The elucidation of the molecular mecha- nisms has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Pre- vious reports have analyzed several molecules such as extracellular matrix proteins, cytokines, bone morphogenetic proteins, hormones, and angiogenic factors. Moreover, a single protein can have multi- functional roles. With such a huge number of me- chanical, histologic, cellular, and molecular vari- ables, there is the need to have a cell culture model that enables the selection of the effect of a specific strength to a single cell type at different time points and with or without cytokines. The analysis of the genetic profiling of a cell line cultured on an equibiaxial stretch device has such characteristic. Because there is a recruitment and commitment of preosteoblastic cells during bone lengthening and no previous report has focus on them, the authors used a preosteoblast MC3T3-E1 cell line to detect the early molecular effects of distraction on mesen- chymal cells. By using DNA microarrays contain- ing 15,000 clones, the authors identified several genes the expression of which was significantly up- or down-regulated. The differentially expressed genes cover a broad range of biological processes: cell growth, metabolism, morphogenesis, cell com- munication, response to stress, and cell death. The data reported are the first genetic portrait of stretched preosteoblasts. They can be relevant in the better understanding of the molecular mecha- nism of DO and as a model for comparing the effect of distraction on different cell lines and primary cultures, rate and strength of distraction, and with or without cytokines. Key Words: Distraction osteogenesis, equibiaxial stretch device, gene profiling, mesenchymal cell, pre- osteoblast D istraction osteogenesis (DO) is a mechan- otransduction process capable of generat- ing viable osseous tissue by the gradual separation of osteotomized bone edges. The process can be divided into three distinct phases: the lag phase (between osteotomy and the beginning of distraction), the distraction, and the consolidation phase. Several variables are implicated: magnitude of mechanical strain, distraction rate and type of dis- tracted bone. The combination of these factors acts on different types of cells, inducing apoptosis, cell proliferation, and differentiation. These phenomena are responsible for the temporal and spatial organi- zation of growing tissue (bone, cartilage, or fibrous tissue) and govern the type of ossification (ie, in- tramembranous or endochondral ossification). Al- though the histologic and ultrastructural changes have been extensively delineated, the molecular From the *Department of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy; the † Institute of Histology, University of Bologna, Bologna, Italy, and Center of Molecular Genetics, CARISBO Foundation; the ‡ Department of Biochemistry, Second University of Naples, Naples, Italy; the ¶ Institute of Histology, University of Bologna; the # Department of Maxillofacial Surgery, JRH, Oxford, United Kingdom; the Departments of § Oral Surgery, Dental Clinic and  Oral Pathology, Second University of Naples; the **Department of Histology, University of Milano, Milano, Italy; the †† Department of Oral Pathology, University of Ancona; and the ‡‡ Dental Clinic, University of Chieti. Address correspondence and reprint requests to Dr. Francesco Carinci, Chair of Maxillofacial Surgery, School of Medicine, Uni- versity of Ferrara, Arcispedale S. Anna, Corso Giovecca, 203, 44100 Ferrara, Italy. E-mail: crc@.unife.it This work was supported by grants from University of Ferrara, Italy (F.C.), Fondazione CARIFE (F.C.), Guya-bioscience, Ferrara, Italy (F.P.), and Fondazione CARISBO (F.P.). 71