Plastic Surgery Research Council
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Presenter: Adrian McArdle, MD
Co-Authors: Chung MT, Lo DD, Hyun JS, Montoro DT, Paik KJ, Walmsley G, Senarath-Yapa K, Zimmermann AS, Sorkin M, Rennert R, Hu M, Liu C, Chen HH, Chung AS, Longaker MT, Wan DC
Stanford University

Background: Cell sorting has identified subpopulations with enhanced osteogenic potential. However, these markers (CD90, CD105) undergo a phenotypic drift in vitro. Type I BMP receptors play critical roles in the specification of osteoblasts and adipocytes, and may offer a more reliable cell surface marker for prospective sorting. The present study evaluated the potential of a combinatorial approach, employing both FACS and manipulation of the BMP pathway, to enhance bone formation.

Methods: BMPR-IB+, BMPR-IB-, and unsorted ASCs were sorted using FACS. Phenotype was analyzed at Days 0, 7, and 14 using anti-CD90, -CD105, and -BMPR-IB antibodies. Each group was then treated with or without Noggin suppression, and cultured in ODM with or without BMP-2. Alkaline phosphatase and quantification were performed on Day 7, alizarin red staining and quantification on Day 14. Osteogenic gene expression was examined by qRT-PCR. In vivo, critical-sized calvarial defects were created in nude mice, and repair was performed using the abovementioned subpopulations delivered on a HA-PLGA scaffold with or without BMP-2. Healing was monitored using micro-CT scans for eight weeks. Calvaria were harvested at Week 8, and sections were stained with Movat s Pentachrome.

Results: Staining assays and qRT-PCR revealed that BMPR-IB+ ASCs were more osteogenic than BMPR-IB- or unsorted. Suppression of Noggin in ASCs resulted in increased osteogenic gene expression and in vitro osteogenic differentiation, and the addition of BMP-2 to Noggin knockdown even further promoted osteogenesis. In vivo, similar results were found, with Noggin-suppressed BMPR-IB+ ASCs seeded on BMP-2 scaffolds experiencing the greatest skeletal healing compared to the other groups over eight weeks.

Conclusions: Our findings demonstrate that subpopulations of ASCs may be identified with enhanced osteogenic capacity and that ASC-mediated bone formation can be promoted through manipulation of the BMP pathway. Use of BMPR-IB+ as a cell surface marker effectively identified an enriched group of cells which could facilitate more rapid regeneration of skeletal defects.

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