BASIC SCIENCE RESEARCH Osteoblast Differentiation Is Enhanced in Rotary Cell Culture Simulated Microgravity Environments Y. Joon Ko, DDS, MS; 1,2 Rebecca S. Zaharias, BA; 3 Denise A. Seabold, BA; 3 John Lafoon, BS; 3 and Galen B. Schneider, DDS, PhD 3,4 Purpose: As the aging population increases, more people will become reliant on regenerative dental medicine for implant therapy. The objective of this study was to test the hypothesis that 3D rotary cell culture (RCC) environments created by simulated microgravity would enhance osteogenic gene expression using integrin mediated pathways. Materials and Methods: Human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre- osteoblasts were cultured in either RCC to create 3D environments or in 2D monolayers for 72 hours. Gross phenotypic analysis was performed using Alizarin Red S staining for calcium and microscopy. Real-time PCR analysis was used to detect differences in osteoblast gene expression. Aggregates developed in 3D RCC environments were treated with or without antibody to the collagen-I integrin receptor α2β1 to determine whether this molecular pathway might contribute to the development of a mineralized matrix. Results: Microscopic analysis demonstrated that RCC environments promoted 3D aggregate forma- tion by 72 hours without any scaffold. The mass appeared osseous-like with a white, shiny, translucent surface. The center was amorphous with areas of vacuolization, tubule-like structures, and fibrous-like extensions. Real-time PCR data showed that 3D environments enhanced osteogenic gene expression as compared with 2D monolayer culturing conditions. At 72 hours, changes in levels of osteogenic gene expression were noted. Cbfa1, a necessary transcription factor for osteoblast differentiation, was expressed 33% higher (p = 0.26); Collagen 1, 69% higher (p = 0.05); Osterix, 49% higher (p = 0.001); and BSPII, 54% higher (p = 0.001) than osteoblasts cultured for 72 hours in standard 2D monolayer conditions. When cultured in the presence of collagen α2β1 integrin receptor antibody, 3D aggregates had decreased levels of mineralization as compared with non-treated aggregates. Conclusion: RCC enhances osteoblast differentiation using integrin mediated pathways. J Prosthodont 2007;16:431-438. Copyright C  2007 by The American College of Prosthodontists. INDEX WORDS: tissue engineering, dental implants, osseointegration, bone, rotary cell culture A S THE aging population increases, more people will become reliant on regenerative 1 Assistant Professor, Department of Prosthodontics, College of Den- tistry, University of Iowa, Iowa City, IA. 2 John J. Sharry Finalist, 2nd Place, 2005 ACP Meeting, Los Angeles, CA. 3 Dows Institute of Dental Research, College of Dentistry, University of Iowa, Iowa City, IA. 4 Associate Professor, Department of Prosthodontics, College of Den- tistry, University of Iowa, Iowa City, IA. Accepted March 21, 2006. Correspondence to: Dr. Galen B. Schneider, Department of Prosthodontics and Dows Institute of Dental Research, College of Den- tistry, University of Iowa, N402 DSB, Iowa City, IA 52242. E-mail: galen-schneider@uiowa.edu Copyright C  2007 by The American College of Prosthodontists 1059-941X/07 doi: 10.1111/j.1532-849X.2007.00204.x dental medicine. More people are being defined as partially edentulous, and their treatment options have expanded to include the use of dental im- plants. For the best biologic, biomechanical, and esthetic results of these implant rehabilitations, proper implant placement is essential. This type of therapy requires special consideration with re- spect to the quality and quantity of bone available at the surgical site. The placement of an implant into a defective osseous site not only prevents ad- equate positioning of the final prosthetic restora- tion, but also results in poor osseointegration and subsequently, a poor prognosis for the therapeutic outcome. Bone augmentation is a subject of intensive investigation in dentistry. Current approaches in bone reconstruction use biomaterials, autografts, or allografts, although restrictions on all these Journal of Prosthodontics, Vol 16, No 6 (November-December), 2007: pp 431-438 431