Increased Tgf-1 Production by Rat Osteoblasts in the Presence of PepGen P-15 in Vitro Candice Trasatti, DDS, MS, Robert Spears, PhD, James L. Gutmann, DDS, and Lynne A. Opperman, PhD Bone grafting materials may enhance tissue regen- eration after endodontic, periodontal, or implant sur- gery. The differences in physical and biological prop- erties between products may result in different osteoblastic responses. This study was designed to determine whether interleukin-1 and Tgf-1 pro- duction by primary cultures of rat osteoblasts dif- fered when cells were exposed to three grafting materials: BioOss, OsteoGraf N-300, and PepGen P-15. Cells were exposed to materials for 24, 48, and 72 h and were characterized by mineralized nodule formation. Supernatants were collected for Lowry and enzyme-linked immunosorbent assays to assess cytokine production. All groups produced mineral- ized nodules after 14 days. Statistical analysis re- vealed no difference in interleukin-1 production be- tween groups, but a significant increase in Tgf-1 production was noted in the PepGen P-15 group. These results indicate that PepGen P-15 stimulates osteoblasts to express Tgf-1, which may accelerate repair of bone defects created during periradicular or dental implant surgeries. The objective of endodontic or periodontal surgery is to remove diseased tissue and to allow restoration of hard and soft tissue architecture consistent with health and uncompromised function. When the environment is compromised by loss of normal bony architecture, as with extensive periradicular periodontitis or peri- odontal disease, the potential for scar formation and inhibition of bone repair after periradicular surgery is high (1). Prevention of scar tissue formation in the wound by restoration of appropriate bony architecture or bone grafting materials may arguably improve the tooth’s function. The ideal bone replacement system should induce osteogenesis, allow cementogenesis, and facilitate formation of a functional peri- odontal ligament (2). Autogenous grafts achieve true osteogenesis (3), whereas all other materials are either osteoinductive or osteoconduc- tive (4). Xenogenic bone substitutes are transferred from one species to another and include bovine-derived hydroxyapatite (BioOss; Osteohealth, Shirley, NY; and OsteoGraf/N-300; Ceramed, Lake- wood, CO). PepGen P-15 (Ceramed Dental, Lakewood, CO), a newer xenogenic bone grafting material, has shown clinical promise in the regeneration of periodontal tissues (5, 6). The collagenous matrix or hydroxyapatite grafting material can provide a scaffold for three-dimensional cell anchorage, allowing subsequent accumulation and interaction of cells and cytokines. One of the primary cytokines associated with the growth and regulation of repair of bone is Tgf-1. The effects of Tgf-1 may be classified as proliferative, immu- noregulatory, and formative in nature. These effects result from paracrine and autocrine actions, beginning within 24 h after injury and continuing for approximately 10 days (7). Many cell types, including platelets, fibroblasts, and osteoblasts, produce and re- spond to this growth factor. Tgf-1 secreted initially at the wound site by degranulating platelets is chemotactic for osteoblasts (2), fibroblasts (8), and monocytes and macrophages (9). Increased differentiation of osteoblast precursors (10), proliferation of fibro- blasts (11) and osteoblasts (12), and increased collagen synthesis (13) has been shown in the presence Tgf-1 in culture. Tgf-1 production is therefore a measurable marker for repair activity in a wound involving both hard and soft tissues. Inflammation during wound healing may inhibit stromal depo- sition and upregulate tissue-destructive pathways. Examination of proinflammatory cytokine production can be useful in predicting cellular response to a material. During inflammation, interleukin (IL)-1 is one of the primary mediators of bone resorption by activating osteoclasts and inducing production of matrix metallo- proteinases (14). IL-1 is produced mainly by peripheral blood monocytes and elicits a broad range of biologic responses in a large number of cell types (15). Increased production of IL-1 within a wound can delay or prevent regeneration of periradicular tissues. Tgf-1 suppresses IL-1 (16), showing the antagonistic influence these cytokines may have on wound healing. To examine osteoblasts responses to grafting materials, this study was designed to (a) quantify and compare production of Tgf-1 and IL-1 in primary cultures of rat osteoblasts exposed to PepGen P-15, OsteoGraf/N-300, and Bio- Oss, and (b) observe mineralized nodule formation by osteoblasts after exposure to each of the materials in vitro. MATERIALS AND METHODS Isolation of Osteoblasts Nineteen-day pregnant Sprague-Dawley rats (Harlan, Indianap- olis, IN) were kept according to Baylor College of Dentistry- JOURNAL OF ENDODONTICS Printed in U.S.A. Copyright © 2004 by The American Association of Endodontists VOL. 30, NO. 4, APRIL 2004 213