21.09 Delivery of Monocyte Lineage Cells in a Biomimetic Scaffold Enhances Tissue Repair

G. G. Walmsley^1,2, M. S. Hu¹, K. Weiskopf², R. C. Rennert¹, M. Januszyk¹, Z. N. Maan¹, D. Duscher¹, K. Senarath-Yapa¹, A. J. Whittam¹, R. Tevlin¹, D. A. Atashroo¹, I. L. Weissman², H. P. Lorenz¹, G. C. Gurtner¹, M. T. Longaker^{1,2  1}Stanford University School Of Medicine,Department Of Surgery, Division Of Plastic & Reconstructive Surgery,Stanford, CA, USA ²Stanford University School Of Medicine,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA

Introduction:
Macrophages are thought to play a critical regulatory role in many stages of wound healing, including angiogenesis, reepithelialization, and remodeling. Evidence for the importance of macrophages in these processes comes from experiments demonstrating impaired wound healing in mice following DTR-based ablation of macrophages, genetic knockout of G/M-CSF, or administration of anti-macrophage antiserum. We have previously shown that transplantation of macrophages into excisional wounds on wild type (FVB/NJ) mice significantly increases the rate of wound healing. Here, we expand the analysis to include diabetic wound healing and monocyte transplantation.

Methods:
Macrophages derived from the bone marrow of L2G (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J) were seeded on pullulan-collagen hydrogels and transplanted onto splinted excisional wounds on the dorsum of diabetic (FVB.BKS(D)-Lepr^db/ChuaJ) mice. Human monocytes isolated from drawn blood were similarly transplanted on pullulan-collagen hydrogels onto splinted excisional wounds on the backs of immunodeficient nude (Foxn1^nu) mice. Histologic analysis allowed for in vivo tracking of the survival, localization, and phenotype of transplanted macrophages and monocytes. Microfluidic single-cell gene expression analysis of transplanted L2G macrophages (GFP⁺Luc⁺) FACS-isolated on the basis of GFP expression from cutaneous wounds provided further insight into macrophage phenotype and behavior during wound healing.

Results:
L2G macrophage-seeded hydrogels improved wound healing compared to un-seeded hydrogel controls on days 4-20 (*p<0.01) in diabetic mice. The average time for complete wound healing was 17.2 days in the macrophage group versus 20.3 days in the control group (*p<0.001). IVIS imaging revealed survival of transplanted macrophages in diabetic wounds through day 20 of wound healing. Microfluidic single-cell gene expression analysis revealed that macrophages transplanted into wounds displayed a predominantly M2 phenotype after being in the wound environment for 24 hours. Human monocyte-seeded hydrogels significantly improved healing compared to un-seeded control hydrogels. The average time to complete healing was 17.8 days in the monocyte group versus 21 days in the control group (*p<0.005). Histologic analysis of monocyte treated wounds showed that transplanted monocytes differentiate in vivo to a predominantly M2 phenotype after 48 hours in the wound environment. Importantly, scar size and quality was not affected in wounds receiving either monocyte or macrophage transplant as compared to controls.

Conclusion:
Here we demonstrate that by increasing the number of monocyte lineage cells in the wound site above physiologic levels in diabetic and nude mice the rate of wound healing can be significantly accelerated with no adverse impact on the quality of repair. These findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.