Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Pullulan-Collagen Hydrogels Seeded with Adipose-Derived Mesenchymal Stem Cells Enhance Progenitor Cell Recruitment and Functionality
Janos Barrera, BS; Revanth Kosaraju, BS Candidate; Robert C. Rennert, MD; Zeshaan N. Maan, MBBS, MRCS; Dominik Duscher, MD; Alexander J. Whittam, BA; Melanie Rodrigues, PhD; Michael Januszyk, MD; Jayakumar Rajadas, PhD; Geoffrey C. Gurtner, MD, FACS
Stanford University, Stanford, CA, USA.

PURPOSE: Biomaterials that promote neovascularization represent a promising treatment for chronic wounds, particularly in combination with stem cells. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest, due to their pro-regenerative function and ease of harvest. Our laboratory has demonstrated the benefits of delivering ASCs seeded on a pullulan-collagen hydrogel bioscaffold, with upregulation of genes related to neovascularization and accelerated wound closure in vivo. More recently, it has been shown that mesenchymal stem cells (MSCs) enhance the recruitment of endogenous progenitor cells, likely through cytokine secretion. Therefore, we investigated whether ASCs seeded on our hydrogel bioscaffold modulate the recruitment and/or functionality of endogenous progenitor cells, specifically bone-marrow mesenchymal progenitor cells (BM-MPCs), a cell population previously defined by our laboratory to be crucial to neovascularization.
METHODS: Parabiosis, of fluorescent (FP+) donor mice and wild-type recipient mice, was performed. Excisional wounds on the recipient mouse were treated with either control saline injection, ASC injection, or ASC-seeded hydrogels at one day and four days post-wounding. Wounds were harvested 24 hours after the second treatment for fluorescence-activated cell sorting (FACS) and microfluidic single-cell analysis. In vitro, ASCs were either seeded into hydrogels or plated in standard growth medium. After 48 hours of incubation in hypoxia, conditioned medium (CM) was collected. qPCR, ELISAs, immunocytochemistry for Ki67, a transwell assay, and a tubulization assay were used to assess the effects of CM from ASC-seeded hydrogels on the functionality of BM-MPCs.
RESULTS: In vivo, microfluidic single-cell analysis identified a subpopulation of FP+/Lineage-/CD45- cells, putative BM-MPCs, defined by the expression of pro-vasculogenic genes, including Ang, Cd248, and Sca1. These BM-MPCs were increased in wounds treated with ASC-seeded hydrogels (45.8% of GFP+/Lin- cells) compared to those treated with injected ASCs and PBS controls (32.7% and 22.8% of FP+/Lin- cells, respectively). FACS quantification of FP+/Lin-/CD45-/Sca1+ cells within the wounds demonstrated that BM-MPCs comprised 23% of the FP+/Lin- cell population, corresponding to circulating progenitor cells, in wounds treated with ASC-seeded hydrogels, as opposed to 8.4% of FP+/Lin- cells in wounds treated with ASC injection (p=0.017), and 2.1% of such cells in wounds treated with PBS (p=0.0012). In vitro, we found that BM-MPCs exposed to ASC-seeded hydrogel CM displayed a significant increase of 44% in cell migration (p=0.019) and a fourfold increase in cell proliferation (p=0.0027). Also, qPCR and ELISAs revealed the upregulation of several angiogenic genes, including Hgf and Mmp3, and a significant increase in tubulization of BM-MPCs exposed to ASC-seeded hydrogel CM (p=0.045).
CONCLUSION: ASC-seeded hydrogels enhance BM-MPC recruitment to the wound and upregulate BM-MPC functionality, to effect greater neovascularization and accelerate wound healing.


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