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. 2019 Jun 13;14(6):e0217965.
doi: 10.1371/journal.pone.0217965. eCollection 2019.

Delivery of silver sulfadiazine and adipose derived stem cells using fibrin hydrogel improves infected burn wound regeneration

Jaideep Banerjee  1 , Shanmuganathan Seetharaman  1 , Nicole L Wrice  1 , Robert J Christy  1 , Shanmugasundaram Natesan  1
Affiliations

Delivery of silver sulfadiazine and adipose derived stem cells using fibrin hydrogel improves infected burn wound regeneration

Jaideep Banerjee et al. PLoS One. .

Abstract

Infection control is necessary for improved burn wound regeneration. In this study contact burn wounds were induced on the dorsum of the rats and were infected with Pseudomonas aeruginosa (107cfu/ml of saline) and left overnight (12-14 hours) to establish the infection. After 12 hours, the wounds were treated with PEGylated fibrin hydrogel containing 50 mgs of silver sulfadiazine (SSD) loaded chitosan microsphere (SSD-CSM-FPEG). On day 9, SSD-CSM-FPEG treated burn wounds further received adipose derived stem cell (5×104 ASCs cells/ml) embedded in PEGylated fibrin hydrogel. Wounds were assessed for the healing outcomes such as neovascularization, granulation tissue formation, wound closure and collagen maturation. Analysis of bacterial load in the burn wound biopsies, demonstrated that SSD-CSM-FPEG significantly reduced bacterial infection, while overt infection was still observed in the untreated groups on day 14. Sequential treatment of infected wounds with SSD-CSM-FPEG followed by ASC-FPEGs (SSD-CSM-ASC-FPEG) significantly reduced bacterial colonization (9 log reduction) and pro-inflammatory cytokine (TNF-α) expression. A significant increase in neovascularization markers; NG2 and vWF was also observed. Histological analysis indicated the wounds treated with SSD-CSM-ASC-FPEG increased amount of dermal collagen matrix deposition, a thicker granulation tissue on day 21 and more mature collagen on day 28. This work demonstrates that the sequential treatment of infected burn wounds with SSD-CSM-FPEG followed by ASC-FPEG reduces bacterial infection as well as promotes neo-vascularization with improved matrix remodeling.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
A) Burn device, B) Photographic image of the burn wound, C) Timeline of experiment, D) Histology: unburnt skin—All layers of skin is intact and viable E) Histology: 10s burn–Epidermis and most of dermis is lost (partial thickness burn).
Fig 2
Fig 2. SSD-CSM-FPEG reduces bacterial load.
(A) Bacterial growth kinetics upon treatment with SSD-CSM-FPEG. (B) Gram staining demonstrating presence of bacteria (indicated by arrows) in infected saline samples while absent in SSD-CSM-FPEG treated samples. (n = 4, p<0.05).
Fig 3
Fig 3. SSD-CSM-FPEG reduces inflammation.
(A-G) SSD-CSM-FPEG exhibits increased anti-inflammatory marker IL-10. Day 7 wound tissue exhibits increased expression of IL-10 (green, white arrows) in wound tissue treated with SSD-CSM-FPEG as compared to infected saline treated samples. (n = 4, p<0.05). (H-N) SSD-CSM-FPEG exhibits decreased pro-inflammatory marker TNF-α. Day 7 wound tissue exhibits decreased expression of TNF-α (green, white arrows) in wound tissue treated with SSD-CSM-FPEG as compared to infected saline treated samples. (n = 4, p<0.05).
Fig 4
Fig 4. SSD-CSM-ASC-FPEG facilitates neo-vascularization.
Day 21 SSD-CSM-ASC-FPEG treated samples exhibit A-C) pericyte marker NG2 expression, indicating neo-vascularization, and D-F) endothelial cell marker von-Willebrand factor (vWF) expression. These markers were not expressed in the infected saline treated samples (n = 4, p<0.05).
Fig 5
Fig 5. Percentage wound closure observed over 28 days in infected/ non-infected groups treated with SSDM-CSM-FPEG with or without ASCs.
(n = 4).
Fig 6
Fig 6. Wound closure characteristics.
Representative Masons trichrome showing wound histology on day 7, day 21 and day 28. Interpretation of Masons trichrome staining is as follows: red: keratin and muscle fibers; blue or green: collagen and bone; light red or pink: cytoplasm; and dark brown to black: cell nuclei. SSD-CSM-ASC-FPEG treated samples was observed to have a significantly thicker granulation tissue (n = 4).
Fig 7
Fig 7
Masson’s trichrome stained tissue sections of wounds treated with SSDM-CSM-FPEG without (A-D) or with ASCs (F-E) on day 14. B-D are zoomed in views from A and F-H are zoomed in areas from E.
Fig 8
Fig 8. Wound closure characteristics.
Representative Movatt’s pentachrome staining showing wound histology on (A) day 7, (B) day 14 (C) day 21 and (D) day 28. Interpretation of Pentachrome staining is as follows: blue: ground substance, mucin; bright red: fibrin; red: muscle; yellow: collagen and reticular fibers; black: cell nuclei. SSD-CSM-ASC-FPEG treated samples was observed to have a significantly thicker granulation tissue. (n = 4).
Fig 9
Fig 9. Maturation of collagen.
Picrosirius staining on day 28 infected burn wounds treated with (A) saline or (B) SSD-CSM-ASC-FPEG. Zoomed images from A shows (C) pre-dominantly thinner (green) regrowing fibers in infected saline control samples, (D) more number of thicker, arranged mature collagen fibers (red) in wounds treated with SSD-CSM-ASC-FPEG, and a basket like mature collagen in the (E) unburnt skin.
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This study was supported by funding from a DRMRP Grant (W81XWH-09-1-0607) to RJ Christy.