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Novel regulation of human adipose derived stromal cell osteogenesis through Noggin knockdown and a BMP-2 slow releasing scaffold
Benjamin Levi, MD1, Emily R. Nelson, BS1, Shuli Li, PhD1, Jeong Hyun, MD1, Jason Glotzbach, MD1, George Commons, MD1, Min Lee, PhD, DDS2, MIchael T. Longaker, MD, MBA1.
1Stanford University, Stanford, CA, USA, 2UCLA, Los Angeles, CA, USA.
PURPOSE: There remains a pressing need for a suitable alternative to currently available techniques for bone tissue repair. We have previously demonstrated the osteogenic potential of human adipose derived stromal cells (hASCs), however, we believe we can enhance hASC osteogenic capability by modulating the BMP pathway in vitro and in vivo. We hypothesize that by knocking down noggin on a genetic level, we can increase the osteogenic capability of hASCs and that along with a novel bmp loaded biomimetic scaffold, we can greatly improve skeletal tissue engineering.
hASCs were harvested from lipoaspirate of 5 female patients. After 48 hrs in vitro expansion, the cells were transfected with Noggin shRNA. Gene and protein knockdown were confirmed. Osteogenesis was assessed by staining and qRT-PCR. Cells were seeded on an osteoinductive scaffold with or without BMP and implanted into a 4mm critical size calvarial defect in athymic mice. In vivo calvarial defect healing was assessed by microCT and histology.
hASCs with confirmed noggin knockdown displayed significantly enhanced osteogenic differentiation in vitro as compared to control, shown by an up-regulation of alkaline phosphatase activity, mineralization and numerous gene markers (RUNX2, OCN and BMPR1B, *p<0.05). This enhanced osteogenesis was observed in vivo as well, as calvarial defects seeded with noggin knock down cells had significantly increased boney healing when compared to those seeded with control hASCs defined as calcification on Micro CT analysis and histology (*p<0.05) (Figure 1). BMP loaded scaffolds further enhanced in vivo healing of hASCs in a calvarial defect.
We demonstrate that hASCs can be enriched for a more osteogenic subpopulation by manipulating the BMP pathway. By removing a BMP inhibitor, we have created a population of hASCs that possess superior osteogenic potential in vitro and in vivo. Creating a hASC population that demonstrates a robust ability to differentiate along an osteogenic lineage, we hope to optimize the potential utility of cell-based therapies in skeletal regeneration.
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