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. 2016 Nov:45:234-246.
doi: 10.1016/j.actbio.2016.08.053. Epub 2016 Aug 31.

Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells

P Brouki Milan  1 , N Lotfibakhshaiesh  2 , M T Joghataie  3 , J Ai  4 , A Pazouki  5 , D L Kaplan  6 , S Kargozar  7 , N Amini  8 , M R Hamblin  9 , M Mozafari  10 , A Samadikuchaksaraei  11
Affiliations

Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells

P Brouki Milan et al. Acta Biomater. 2016 Nov.

Abstract

There is an unmet clinical need for novel wound healing strategies to treat full thickness skin defects, especially in diabetic patients. We hypothesized that a scaffold could perform dual roles of a biomechanical support and a favorable biochemical environment for stem cells. Human umbilical cord perivascular cells (HUCPVCs) have been recently reported as a type of mesenchymal stem cell that can accelerate early wound healing in skin defects. However, there are only a limited number of studies that have incorporated these cells into natural scaffolds for dermal tissue engineering. The aim of the present study was to promote angiogenesis and accelerate wound healing by using HUCPVCs and decellularized dermal matrix (DDM) in a rat model of diabetic wounds. The DDM scaffolds were prepared from harvested human skin samples and histological, ultrastructural, molecular and mechanical assessments were carried out. In comparison with the control (without any treatment) and DDM alone group, full thickness excisional wounds treated with HUCPVCs-loaded DDM scaffolds demonstrated an accelerated wound closure rate, faster re-epithelization, more granulation tissue formation and decreased collagen deposition. Furthermore, immunofluorescence analysis showed that the VEGFR-2 expression and vascular density in the HUCPVCs-loaded DDM scaffold treated group were also significantly higher than the other groups at 7days post implantation. Since the rates of angiogenesis, re-epithelization and formation of granulation tissue are directly correlated with full thickness wound healing in patients, the proposed HUCPVCs-loaded DDM scaffolds may fulfil a role neglected by current treatment strategies. This pre-clinical proof-of-concept study warrants further clinical evaluation.

Statement of significance: The aim of the present study was to design a novel tissue-engineered system to promote angiogenesis, re-epithelization and granulation of skin tissue using human umbilical cord perivascular stem cells and decellularized dermal matrix natural scaffolds in rat diabetic wound models. The authors of this research article have been working on stem cells and tissue engineering scaffolds for years. According to our knowledge, there is a lack of an efficient system for the treatment of skin defects using tissue engineering strategy. Since the rates of angiogenesis, re-epithelization and granulation tissue are directly correlated with full thickness wound healing, the proposed HUCPVCs-loaded DDM scaffolds perfectly fills the niche neglected by current treatment strategies. This pre-clinical study demonstrates the proof-of-concept that necessitates clinical evaluations.

Keywords: Decellularization; Dermal tissue engineering; Scaffold; Stem cell; Wound healing.

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Figures

Fig. 1
Fig. 1
Histological assessment of human dermis before and after decellularization. H&E and Masson’s trichrome staining demonstrate that there are no cells or cellular debris and skin appendages (hair follicles, sweat glands, endocrine glands etc.) in DDM following the decellularization process. The DAPI staining shows that the DDM scaffold is free of cellular nuclei, while it retains structural integrity during processing. The Masson’s trichrome stained light micrographs illustrate that the DDM scaffolds preserved the normal collagen bundling pattern and normal collagen orientation.
Fig. 2
Fig. 2
The effect of decellularization process on DNA content. (A) The PCR fragments in the fresh skin and DDM scaffolds identified by agarose gel electrophoresis. The red arrow shows the 250 kb band in the standard ladder. The PCR analysis illustrated that the samples had no detectable copies of β-actin. (B) The dsDNA concentration of the fresh skin and DDM were determined by using a Quanti-iT PicroGreen® dsDNA assay. The dsDNA concentration significantly decreased in the DDM scaffolds after processing (*P<0.001). Points are means ± SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Cellular interaction of the DDM scaffolds with HUCPVCs. (A) Representative SEM micrographs of the DDM scaffold showing that the HUCPVCs cells were attached and distributed on the surface of the scaffolds after 5 days. The augmented cells attached to each other and to the surface of the DDM scaffolds. The extended lamellipodia of the HUCPVCs can be observed in the DDM scaffolds. (B) The MTT photograph revealed that there was a significant difference between the experimental groups and control group after 7 days. These results suggested that the DDM scaffolds promoted the HUCPVCs proliferation and had no cytotoxic effects. (P values of the fresh dermis group and DDM group are *P < 0.05, **P < 0.01 respectively). (C) Histological evaluation of DDM scaffold and HUCPVCs loaded–DDM scaffold after 7 days in vitro culture.
Fig. 4
Fig. 4
Tensile strength of the DDM scaffolds. The results of uniaxial tensile testing showed no significant differences under the conditions tested. For the fresh dermis and DDM scaffolds of similar thickness, the ultimate tensile strength (UTS) of the fresh dermis was approximately 0.77 Mpa greater than the DDM scaffolds, which was not a statistically significant difference. The breakdown pattern was observed to be similar between the fresh dermis and the DDM scaffolds, indicating that the DDM scaffold had strong mechanical properties similar to fresh skin.
Fig. 5
Fig. 5
Wound healing effects of DDM alone and HUCPVCs loaded-DDM in a diabetic rat model of cutaneous wounds. (A) Representative photographs of the wound closure rate among the three groups at days 0, 7, 14 and 21 after grafting. (B) Quantitative analysis indicated that the wound size was reduced in the HUCPVCs loaded-DDM group compared with the control and DDM groups after 21 days (P < 0.001). The results showed that wound closure rate in the DDM group was higher than control group, and significant differences were observed at 7 and 14 days post-wounding (P < 0.001). The HUCPVCs loaded-DDM group demonstrated no significant improvement in wound healing compared with the DDM treated groups at 7 days. Moreover, the HUCPVCs loaded-DDM scaffolds had much higher wound healing rate and contraction ability than the other groups.
Fig. 6
Fig. 6
Representative photomicrographs showing the histology for the structure of epithelial and dermal layer and healing status in each group over 21 days after transplantation (H&E). After 7 days, the control, DDM and HUCPVCs loaded-DDM groups showed a number of inflammatory cells. In the HUCPVCs loaded-DDM group, some collagen fibers and fibroblasts appeared in the wound area, which related to the migration phase of wound healing process, indicating that the HUCPVCs loaded-DDM scaffold had a significant effect on the wound healing process compared with other groups. The moderate inflammation remained in the control group until 14 days. The results also show that the epidermal layer was completely formed in the HUCPVCs loaded-DDM group and covered the entire wound site after 2 weeks. However, the re-epithelization process was slow in the other groups. The HUCPVCs loaded-DDM group showed better wound healing compared with the control and DDM groups, demonstrating no sign of immunorejection over 21 days post transplantation.
Fig. 7
Fig. 7
Formation of granulation tissue, epidermal thickness and wound maturity. (A) Area of granulation tissue of wounds treated with the HUCPVCs loaded-DDM scaffold were much higher than DDM and control groups (*P < 0.001). (B) The HUCPVCs-loaded DDM scaffold treated wounds demonstrated an increased thickness of the newly regenerated epidermis compared with the other groups at 7 days (*P < 0.001, ***P < 0.0001). Furthermore, the epithermal layer thickness in the DDM scaffold treated groups was significantly higher than control group (***P < 0.0001). (C) Wound maturity was measured in margin and central parts of different implanted groups, showing that the wound maturity was significantly higher in the HUCPVCs loaded-DDM treated group, especially in the central part of the wound site compared to DDM and control groups (*P < 0.001).
Fig. 8
Fig. 8
Masson’s trichrome staining of the control, DDM and HUCPVCs-DDM groups at 14 and 21 days post implantation. The collagen bundles started to appear in the wound treated with DDM and HUCPVCs-loaded DDM scaffolds after 14 days, after which a better remodeling progress was observed for the HUCPVCs-loaded DDM scaffolds. However, more collagen accumulation and deposition was observed in the control group after 21 days.
Fig. 9
Fig. 9
(A) The length of the newly formed epithelium measured in three groups at days 7 and 14, indicating that the length of the neo-epithelium was statically significant in the HUCPVCs-loaded DDM group compared with that of DDM and control groups after 14 days (P < 0.001). While there was no significant difference between the DDM and control groups. (B) The measurement of the percentage of collagen deposition was higher in control groups after 21 days (P < 0.001). There was no significant difference between all the groups at day 14. (E, EG and GT refer to epithelization, epithelial gap and granulation tissue, respectively.).
Fig. 10
Fig. 10
The angiogenic effect of DDM and HUCPVCs-loaded DDM scaffolds in diabetic wounds are shown in H&E sections. The sections clearly show blood vessels (black arrows) in the control, DDM and HUCPVCs-DDM scaffolds treated groups. In addition, the capillary density increased in the HUCPVCs loaded-DDM scaffold treated group.
Fig. 11
Fig. 11
The angiogenesis activity of the scaffolds were detected by immunofluroscence staining for VEGFR-2 expression (red) in wound tissue are shown for different groups at the day 7 post implantation. The HUCPVCs-loaded DDM group showed a higher number of VEGFR-2-positive endothelial cells. (PE refers to phycoerythrin). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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