Fibronectin precoating wound bed enhances the therapeutic effects of autologous epidermal basal cell suspension for full-thickness wounds by improving epidermal stem cells' utilization

Abstract

Background: Autologous epidermal basal cell suspension therapy has been proven to be one of the most effective treatments for full-thickness wounds. However, we found there remain obvious defects that significantly confined the utilization and function of the epidermal basal cells (EBCs), especially the epidermal stem cells (ESCs) in it. This study investigated whether precoating fibronectin (FN) on the wound bed before spraying EBCs could overcome these defects and further explored its possible mechanisms.

Methods: In the in vitro study, EBCs were isolated from the donor skin of patients who needed skin grafting. Different concentrations of FN were used to precoat culture dishes before cell culture; the adherent efficiency, proliferation and migration ability of ESCs were analyzed and compared with traditional collagen IV precoating. In the in vivo study, Sprague-Dawley (SD) rats with full-thickness skin wounds were selected as full-thickness wounds' model. For the experiment groups, 20 μg/ml FN was precoated on the wound bed 10 min before EBC spray. The quality of wound healing was estimated by the residual wound area rate, wound healing time, and hematoxylin and eosin (H&E) staining. Expression of ESC markers, neovascular markers, inflammation markers, and collagen formation and degradation markers was elucidated by immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and RT-qPCR analysis.

Results: The in vitro study showed that the dishes precoated with 20 μg/ml FN had a similar adherent efficiency and colony formation rate with collagen IV, but it could improve the proliferation and migration of ESCs significantly. Similarly, in the in vivo study, precoating FN on wound bed before EBC spray also significantly promote wound healing by improving ESCs' utilization efficiency, promoting angiogenesis, decreasing inflammations, and regulating collagen formation and degradation.

Conclusion: FN precoating wound bed before EBC spray could significantly promote full-thickness wound healing by improving the utilization and function of the ESCs and further by promoting angiogenesis, decreasing inflammations, and regulating collagen formation and degradation.

Keywords: Autologous EBC suspension therapy; Collagen IV; ESCs; FN; Full-thickness wounds.

Fig. 1

The effects of FN precoating on ESCs’ adhesion, proliferation, and colony formation. a, b Representative pictures of adherent ESCs in different concentrations’ FN-precoated dishes and their adhesion rates. c The proliferation of ESCs in different concentrations’ FN-precoated dishes was analyzed by CCK-8 assay. d, e Representative picture of ESCs’ colony formation and their cloning efficiency in different concentrations FN-precoated dishes. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 5). Scale bar, 200 μm

Fig. 2

The effects of 20 μg/ml FN and 100 μg/ml collagen IV precoating on ESCs’ adhesion, proliferation, and colony formation. a Representative pictures of ESCs’ adhesion and proliferation in 20 μg/ml FN- and 100 μg/ml collagen IV-precoated dishes. b The proliferation of ESCs in 20 μg/ml FN- and 100 μg/ml collagen IV-precoated dishes were analyzed by CCK-8. c, d Representative pictures of ESCs’ colony formation and their cloning efficiency in 20 μg/ml FN- and 100 μg/ml collagen IV-precoated dishes. e, f Representative pictures of ESCs’ scratch-wound assays and their migration rate in 20 μg/ml FN- and 100 μg/ml collagen IV-precoated dishes. g, h Representative pictures of ESCs’ purity and their rates among all the primary adherent cells in 20 μg/ml FN- and 100 μg/ml collagen IV-precoated dishes. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 5). Scale bar, 200 μm

Fig. 3

FN precoating wound bed accelerated wound closure and improved healing quality of SD rats. Full-thickness dermal wounds were created on the dorsal skin of SD rats and treated by PBS (control), EBCs, and FN + EBCs respectively. a Representative wound pictures of rats’ dorsal from each group taken on post-injury days 0, 3, 7, 14, and 21. b, c Residual wound rates and completed wound healing time of each group. The computation was that the indicated area was divided by the initial area. Results represent means ± SEM. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 6), ^#P < 0.05 compared with EBCs value (n = 6)

Fig. 4

Histological features and expression of inflammatory factors of the rats’ dorsal wounds in each group. a Wound tissue sections stained with H&E on post-injury days 3, 7, and 21 showing histological features in rats’ dorsal wound treated with PBS (control), EBCs, and FN + EBCs. b, c Analyses of rate ridges numbers and epidermal thickness of wound tissue sections treated with PBS (control), EBCs, and FN + EBCs on post-injury day 21 showing histological features in rats’ dorsal wound. FN + EBCs treated wound displayed significantly more rate ridges and thicker epidermis than the others. d The number of inflammatory cells on day 3 and day 7 was quantified at per × 40 magnification for five areas randomly, FN + EBCs treated wound displayed significantly lower inflammatory response and fewer inflammatory cells on day 7. e, f Representative western blot and results of densitometric analysis of blots showing relative protein levels of TNF-α, IL-8, and IL-10 for each group on post-injury day 7. g Representative qRT-PCR analysis showing relative mRNA levels of TNF-α, IL-8, and IL-10 for each group on post-injury day 7. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 6), ^#P < 0.05 compared with EBCs value (n = 6). Scale bar, 100 μm

Fig. 5

FN precoating wound bed promotes the adhesion and proliferation of ESCs. a–c Represent area and analysis of wound tissue sections stained with K15 and Ki-67 on post-injury day 7 showing the number of ESCs and proliferating cells in rats’ dorsal wound treated with PBS (control), EBCs, and FN + EBCs. Arrows indicate the positive cells. d Analysis of wound tissue sections double-stained with K15 and Ki-67 on post-injury day 7 showing the number of growing ESCs in rats’ dorsal wound treated with PBS (control), EBCs, and FN + EBCs. e Representative qRT-PCR analysis showing relative mRNA levels of Ki-67 and K15 for each group on day 7. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 6), ^#P < 0.05 compared with EBCs value (n = 6)

Fig. 6

The expression of angiogenesis factors of the rats’ dorsal wounds on day 7 and day 14 in each group was analyzed by IHC. a–c Represent area and analysis of wound tissue sections stained with CD31 on post-injury day 7 and day 14 showing the microvascular regeneration in rats’ dorsal wound treated with PBS (control), EBCs, and FN + EBCs. Arrows indicate the positive cells. d, e Representative qRT-PCR analysis showing relative mRNA levels of CD31 for each group at day 7 and day 14. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 6), ^#P < 0.05 compared with EBCs value (n = 6)

Fig. 7

FN precoating wound bed regulates the collagen formation and degradation. a–c Representative immunoblot and results of densitometric analysis of blots showing relative protein levels of MMP-9, Col I, and Col III for each group on post-injury day 7 and day 21. d, e Representative qRT-PCR analysis showing relative mRNA levels of MMP-9, Col I, and Col III for each group on post-injury day 7 and day 21. The values were analyzed by Graph Prism 7.0. Error bars represent SEM. Student’s t test, *P < 0.05 compared with control value (n = 6), ^#P < 0.05 compared with EBCs value (n = 6)