59. Tissue-Harvest Procedure has no Effect on Osteogenic Differentiation of Adipose-Derived Stromal Cells in Vitro

Abstract

Introduction: Bone tissue engineering mediated by adipose-derived stromal cells (ASCs) is a particularly interesting prospect due to the clinical burden of craniofacial skeletal defects and drawbacks of current techniques including limited supply and donor site morbidity of autogenous bone for grafting and demise of alloplastic materials. Current studies of ASCs largely employ the use of cells isolated from tissue harvested by suction-assisted lipoaspiration (SAL). However, ultrasound-assisted lipoaspiration (UAL) is gaining clinical popularity as the immediate benefits to the patient include reduced tissue trauma and bleeding and faster recovery. Therefore, there is a widening dichotomy between clinical principle and research focus. No current study has rigorously examined the potential for UAL-derived ASC-mediated osteogenesis. The current study investigates the in vitro osteogenic potential of UAL-derived ASCs with regard to their proliferative and differentiative capacities compared to more commonly used SAL-derived ASCs. Methods: UAL- and SAL-derived ASCs were harvested from lipoaspiration specimens and established in primary culture. After a brief period of expansion, cells were trypsinized and replated for proliferation and osteogenic assays. Cell proliferation was assessed by seeding equal numbers of first passage UAL- and SAL-derived ASCs in side-by-side culture dishes in triplicate. Cell counting was performed by trypan blue exclusion and hemacytometer 1, 3 and 7 days after seeding to obtain growth curves of these cells. Osteogenic differentiation was also assayed for UAL- and SAL-derived cells. First passage UAL- and SAL-derived ASCs were seeded in equal numbers in side-by-side culture dishes in triplicate. After a brief period of expansion to subconfluence, both cell types were induced with osteogenic differentiation media (ODM) for a period of 10 days. Cells were harvested 3, 7 and 10 days after induction for analysis of osteogenic gene expression analysis by RNA extraction and quantitative RT-PCR. Finally, UAL- and SAL-derived ASCs were assayed for their ability to form a mineralized extracellular matrix in vitro by staining for alkaline phosphatase activity (early) and Alizarin Red (terminal differentiation) which was normalized to protein content. Results: UAL- and SAL-derived ASCs exhibited similar proliferative capacities. There were no significant differences in cell numbers 1, 3 or 7 days after seeding. Furthermore, there was no significant difference in rate of cell death by trypan blue exclusion. Osteogenic differentiation by gene expression revealed similar numbers of RUNX2, osteopontin and osteocalcin transcripts that were not significantly different during the differentiation period. Finally, osteogenesis by staining revealed alkaline phosphatase activity (3 days of ODM) and Alizarin Red staining (7 and 10 days of ODM) that were not significantly different when normalized to protein content. Conclusions: With growing popularity and clinical benefits of UAL, research efforts should elect to further explore the biology of these cells in bone tissue engineering applications. Preliminary observations have revealed that UAL-derived ASCs may exhibit equal in vitro proliferative and osteogenic differentiative capacities compared to ASCs collected from conventional SAL. Future directions will include an in vivo assay of these cells to further investigate their potential in bone tissue engineering.