Adipose stem cell supported bone formation may be due factors in addition to VEGF and BMP2

Abstract

Event Abstract Back to Event Adipose stem cell supported bone formation may be due factors in addition to VEGF and BMP2 Shirae Leslie1, David J. Cohen2, Barbara D. Boyan2 and Zvika Schwartz2 1 Virginia Commonwealth University, Chemical Life Science Engineering, United States 2 Virginia Commonwealth University, Biomedical Engineering, United States Introduction: Cell based therapies using adipose derived stem cells (ASCs) have the potential to treat fractures or non-unions. Previous studies in our lab demonstrated rat and human ASCs produce angiogenic factors when grown in MSC-growth medium (GM)[1],[2]. These factors are also released by cells after encapsulation in alginate microbeads (2). In addition microencapsulated rat ASCs express an osteoblast phenotype when cultured in osteogenic medium (OM) and produce osteogenic factors as well[3]. This study examined whether microencapsulated rabbit ASCs (rbASCs) possess this ability and if they can support osteogenesis in vivo. We investigated the multipotency of rbASCs and their ability to induce ectopic bone formation in rabbits following microencapsulation. Methods: Under an IACUC approved protocol, ASCs were isolated from inguinal fat pads of White New Zealand rabbits. Multipotency of rbASCs was determined by incubating confluent cultures in GM, OM, chondrogenic medium (CM) and adipogenic medium (AM) for 18d. mRNA levels were analyzed for RUNX2, OCN, ACAN, COL2, COL10, PPAR-γ, and LEPR. Statistical significance was determined by multi-way ANOVA with post hoc analysis by Bonferroni’s correction to Student’s t-test (n=6 per variable). First passage rbASCs were microencapsulated in alginate in 75mM calcium crosslinker solution containing 1x107 cells/ml alginate using a 6kV electrostatic potential[4],[5]. Microencapsulated rbASCs were cultured in GM or OM for 7d and soluble factors in the conditioned media were quantified. rbASCs were treated with GM or OM for 7d then microencapsulated to form non-degradable or degradable (0.22 U alginate lyase/g alginate) microbeads. 100µl of microencapsulated cells or demineralized bone matrix (DBM) (positive control) were delivered in gelatin capsules into the gastrocenemius and rectus femoris of both legs for 6 wks. MicroCT was done to assess the amount of bone formed. Statistical significance was determined by unpaired t-test (n=7 per variable). Results: rbASCs are able to differentiate along the osteogenic (RUNX2, OCN), chondrogenic (ACAN, COL2, COL10), and adipogenic (PPAR-γ, LEPR) lineages based on mRNA expression. ASCs treated with OM or GM prior to encapsulation secreted comparable levels of VEGF and BMP2 (Fig. 1A, 1B). Radio-opaque “new bone” was observed by microCT in all samples. Microbeads containing cells that were cultured in OM produced more “bone volume” than GM-treated cells or DBM (Fig. 1C). Results were comparable for degradable and non-degradable microbeads. Discussion: The new bone volume evident when OM-treated rbASCs microbeads were implanted heterotopically was not due to the alginate, because all samples had comparable levels of alginate. Production of VEGF and BMP2 was constant between GM and OM samples suggesting other factors were involved. The increase in bone volume by microCT needs to be confirmed histologically.