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Novel Targeting Of The Alk-2 Receptor Using The Cre/lox System To Enhance Osseous Regeneration By Mesenchymal Stem Cells
Cameron Brownley, BS, Shailesh Agarwal, MD, Jonathan Peterson, BS, Eboda Oluwatobi, BS, Kavitha Ranganathan, MD, Stewart C. Wang, MD, PhD, Yuji Mishina, PhD, Steven R. Buchman, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
Purpose: There is a significant need for readily available autogenous tissue to aid in bone regeneration without causing a donor-site defect. Most studies focus on the ability of bone morphogenetic protein ligands (BMP-2 and 7) to stimulate the Bmpr1b receptor (ALK-6). The ACVR1 gene is often overlooked, but provides instructions for making the activin receptor type I protein (ALK-2), a protein member of the Bmpr1 family. When the ALK-2 receptor is overexpressed globally, patients develop fibrodysplasia ossificans progressive. We believe this highly osteogenic phenotype can be harnessed in adipose derived stem cells (ASCs) to improve bone tissue engineering. Our goal here was to demonstrate that ALK-2 may serve as a novel target to 1) improve in vitro ASC osteogenic differentiation and 2) enhance in vivo bone regeneration and calvarial healing.
Methods: Transgenic mice were designed using the Cre/lox system to express constitutively active ALK2 (ca-ALK2) after exposure to tamoxifen. Exposure to tamoxifen results in expression of the Cre enzyme, which splices out a floxed nucleotide sequence in the regulatory domain of the ALK-2 receptor, resulting in constitutive activation. ASCs were collected from these mice, seeded and cultured in osteogenic differentiation medium +/- tamoxifen. These wells were assessed for markers of osteogenic differentiation including histologic staining for mineral and bone deposition (alkaline phosphatase and alizarin red) and transcription of pro-osteogenic genes (alkaline phosphatase, osteocalcin, osteopontin, Runx2, and collagen-1). Next, ASCs collected from these transgenic mice were cultured in osteogenic differentiation medium +/- tamoxifen and impregnated into 4mm hydroxyapatite-coated PLGA scaffolds (200,000 cells/scaffold). 4mm-diameter calvarial defects were created in the parietal bone of C57BL/6 mice; the impregnated scaffolds were then implanted into the defect. The defects were monitored by serial micro-CT scans.
Results: We found that ASCs from our transgenic mice with ALK-2 over-expression had 4 times as much early bone signaling (alkaline phosphatase staining) as ASCs with normal ALK-2 expression (n=4, p<0.05). We similarly found that ALK-2 overexpressing ASCs had 5 times as much bone deposition (Alizarin red stain) compared to control (Fig 1). Transcription of pro-osteogenic genes at day 7 was significantly higher in ASCs from ALK-2 overexpressing mice: Alkaline phosphatase was 50 times higher, osteocalcin 4 times higher, osteopontin 10 times higher, Runx2 3 times higher, and collagen-1 2 times higher than from ASCs not over-expressing ALK-2 (n=4, p<0.05). Finally, based on in vivo micro-CT imaging and histology, we found that bone growth within the calvarial defect was significantly increased in mice which had received PLGA scaffolds impregnated with ALK-2 over expressing ASCs (n=4, p<0.05).
Conclusions: Using a novel transgenic mouse model, we show that constitutive activity of the ALK-2 receptor results in significantly increased osteogenic differentiation of ASCs. Furthermore, we show that this increased differentiation can be harnessed to improve calvarial healing. Future studies to target the ALK-2 receptor to enhance both endogenous and implanted tissues are underway to improve bone tissue engineering.
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