R. Kosaraju1, R. Rennert1, J. Barrera1, A. J. Whittam1, Z. N. Maan1, M. Rodrigues1, D. Duscher1, M. Januszyk1, G. C. Gurtner1 1Stanford University,Department Of Surgery, Division Of Plastic Surgery,Palo Alto, CA, USA
Introduction:
Biomaterials that promote neovascularization represent a promising treatment of 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, resulting in upregulation of genes related to stemness and 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 the recruitment of endogenous progenitor cells by ASCs seeded on our hydrogel bioscaffold.
Methods:
ASCs were either seeded into hydrogels or plated in standard growth medium. After 48 hours of incubation in hypoxia, conditioned medium (CM) from the ASC-seeded hydrogels and from the plated ASCs was harvested. The effects of CM from ASC-seeded hydrogels on the functionality of bone-marrow mesenchymal progenitor cells (BM-MPCs), a cell population defined by our laboratory to be crucial to neovascularization, were assessed using qPCR, ELISAs, immunocytochemistry to measure cell proliferation, a migration assay, and a tubulization assay. In vivo, we parabiosed green-fluorescent protein positive (GFP+) donor mice and wild-type recipient mice. An excisional wound model on the recipient mouse was treated with a control saline injection, injection of ASCs, or ASC-seeded hydrogels, and wounds were harvested four days post-wounding for fluorescence-activated cell sorting (FACS) and microfluidic single-cell analysis.
Results:
In vitro, we found that ASC-seeded hydrogel CM significantly upregulated the functionality of BM-MPCs. In particular, 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). Furthermore, 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). In vivo, microfluidic single-cell analysis identified a subpopulation of GFP+/Lineage-/CD45- cells, putative BM-MPCs, defined by the expression of pro-vasculogenic genes, including Ang, Cd248, and Sca1, that was increased in wounds treated with ASC-seeded hydrogels (45.8% of GFP+/Lin- cells) compared to injected ASCs and PBS controls (32.7% and 22.8% of GFP+/Lin- cells), respectively. FACS quantification of GFP+/Lin-/CD45-/Sca1+ cells within the wounds demonstrated an increase of 38% in recruited BM-MPCs when wounds were treated with ASC-seeded hydrogels compared to ASC injection.
Conclusion:
ASC-seeded hydrogels upregulate BM-MPC functionality, and enhance BM-MPC recruitment, to effect greater neovascularization and accelerate wound healing.