Description
- Abstract:
- Objective: Grafted, cultured stromal vascular fraction (SVF) cells have been shown to improve wound healing in a diabetic murine model, however retention and viability of the cells at the wound site is low. Furthermore, cell culturing poses several problems: it is a time-consuming process and use of serum increases risk for contamination or infection. It is hypothesized that the use of Nanosheets with directly harvested SVF cells would improve wound healing while addressing the shortcomings of cell culturing. This study focuses on whether 1) Nanosheets exhibit improved retention and survival rate of cultured, grafted cells in a healthy murine model; 2) how Nanosheets impact wound healing in a diabetic murine model; and 3) the combined effect of Nanosheets and direct, uncultured SVF cells on wound healing in a diabetic murine model as measured by cell proliferation, collagen deposition, angiogenesis, wound area, epithelialization, and growth factors. Approach: Very thin, porous poly(D,L,-lactic acid) (PDLLA) films were created (the Nanosheet). To address the first arm, wounds were created on the dorsum of C57BL/6 mice. GFP-labeled stromal vascular fraction (SVF) cells were grafted with or without the Nanosheet (n=6 per group) on the day of the operation. On post operation day (POD) 10, the wound site was assessed with bioimaging. Intensity of GFP was measured by ImageJ and cell survival rate was calculated for both groups using Grafted Cell Retention % (GCR%) (high intensity area on POD 10 / high intensity area on POD 0). For the second and third arms, wounds were created on the dorsum of 12-week-old male BKS.Cg-Dock7m +/+ Leprdb /J “diabetic” mice. These mice were then treated with either directly harvested SVF cells, Nanosheet, no treatment, or directly harvested SVF cells and Nanosheet (n=8 per group) and followed for 10 days. Gross examination of the wound sites occurred on 0, 5, and 10 POD. On 10 POD, the mice were sacrificed and histological evaluations of collagen deposition, angiogenesis, wound area, epithelialization length, growth factors, and cell proliferation were performed using immunohistochemistry and polymerase chain reaction. Results: In the preliminary experiment (Nanosheet Increases Spreading Study), the Nanosheet spread 1 µl of blood to an area (360.9 ± 18.9 mm², p<0.0001) 80 times larger than that of Tegaderm (36.7 ± 6.4 mm²). For the first aim (achieved through the Cell Survival Test), cell survival rate as measured by Grafted Cell Retention % (GCR%) was significantly higher on 10 POD (p=0.0136) for GFP-labeled adipose-derived mesenchymal cells (GFP+ADSCs) grafted with the Nanosheet (36.6 ± 23.3%) than cells without the Nanosheet (7.49 ± 5.33%) (Fig. 3c,d). In the second and third arms (in vivo Rapid Transformation study), 4 cohorts were observed: uncultured SVF cell group (S group), Nanosheet group (N group), group without treatment (Control), and uncultured SVF cells with the Nanosheet group (SN). SN group showed a significantly smaller Remainder Wound Area % (RWA%) on 10 POD (37.4 ± 11.2%, p<0.01) compared to the control (66.6 ± 17.8%). Angiogenesis as indicated by number of CD31+ capillaries was significantly different between the SN group (18.6 ± 5.6, p=0.02) and control (10.9 ± 5.7). For cell proliferation, the S group (0.26 ± 0,07, p<0.01) and the SN group (0.23 ± 0.07, p<0.01) had significantly higher ratios of Ki-67 positive nucleus/total nuclei compared to the control (0.153 ± 0.07). Collagen deposition was significantly increased for the SN group (63636 ± 20525.3) compared to the control (p <p<0.01); N group (p=0.0114); and S group (p<0.001). For reepithelialization length, the SN group (2.44 ± 0.673 μm) had significantly greater reepithelialization compared to the control (1.65 ± 0.327 μm, p=0.01). Innovation: This is the first reported attempt at using nano-thin sheets with direct, uncultured SVF cells to overcome low cellular survival rate at the wound site and low amounts of harvested cells in cell therapy. Nanosheet with uncultured SVF cell transplantation has a significant high survival rate of grafted cells on wounded diabetic mice through at least 10 days compared to SVF cell transplantation alone. Conclusion: While grafting cultured SVF cells have been shown to improve impaired healing in chronic wounds, the cell culturing is time-costly. Residual grafted cell survival at the wounded site is suboptimal. This study addresses both issues by demonstrating uncultured, direct SVF cells with Nanosheet have a significantly smaller Remainder Wound Area % (37.4 ± 11.2%) 10 POD compared to control cells (p<0.01), significantly increased collagen deposition compared to SVF cells alone (p<0.001); and significantly increased epithelialization length compared to control cells (1.65 ± 0.327 μm, p=0.01).
- Notes:
- Scholarly concentration: Translational Research in Medicine
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Citation
Shimpo Aoki, , Yori Endo, Cynthia Guo, Mengfan Wu, Megumi Takuma, Toshinori Fujie, Audrey Kim, Indranil Sinha,
"Application of Autologous Non-Cultured Direct-to-Clinic Stromal Vascular Fraction Cells and Nanosheet for the Treatment of Diabetic Wounds in a Murine Model"
(2022).
Warren Alpert Medical School Academic Symposium.
Brown Digital Repository. Brown University Library.
https://repository.library.brown.edu/studio/item/bdr:juej932m/
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Warren Alpert Medical School Academic Symposium
The Warren Alpert Medical School Academic Symposium is an annual event at Warren Alpert Medical School of Brown University that provides Year II medical students a venue to present their summer research in a poster format. Participation in the Symposium …...