Skin regeneration in deep second-degree scald injuries either by infusion pumping or topical application of recombinant human erythropoietin gel

Abstract

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.

Keywords: neovascularization; re-epithelialization; scald wound; segmentation; skin regeneration; vascularization.

Figure 1

Diagrammatic representation of EPO treated and untreated mouse model for skin regeneration. Notes: Scald mouse model (ages, 6–8 weeks; weights, 20–25 g), EPO treated (A, a–e). The dorsum was centered by a 2×3 cm window and then immersed in 80°C water for 6 seconds to produce a second-degree scald wound. A 2×3 cm deep dermal scald injury defect was created in the backs of the animals. This group was treated with rHuEPO (352 U/kg/day) by infusion pump, an attempt to mimic the in vivo circulation process. Scald mouse model (ages, 6–8 weeks; weights, 20–25 g), untreated EPO (B, a–e). The dorsum was centered by a 2×3 cm window and then immersed in 80°C water for 6 seconds to produce a second-degree scald wound. A 2×3 cm deep dermal scald injury defect was created in the backs of the animals. This group was treated with distilled water (control model) only by infusion pump. Abbreviations: EPO, erythropoietin; rHuEPO, recombinant human erythropoietin.

Figure 2

Flow chart of image analysis methods. Notes: First, a series of steps to enhance the image, remove noise from background of the image, and track the edges of the vessels for blood vessel segmentation were applied. Quantification was also applied. Second, in attempting to locate a clear view of blood vessels for the quantitative analysis, we have followed a number of steps including smoothing, thresholding, skeletonizing, and distance transformation.

Figure 3

Quantitative image analysis of blood vessels. Notes: Comparative vessel length over area (A) and thickness measurements (B) in rHuEPO-treated and untreated groups. Overall vessel thickness for treated rHuEPO-treated animals was 586.13 μm and for untreated animals, 260.10 μm. Median value for rHuEPO-treated animals was 91.96 μm and for untreated animals, 72.70 μm. Image processing pipeline went through the following steps (C): the target tissue (rHuEPO-treated) was extracted (a); selections of regions of interest (ROI) (b); converted to grayscale (c); high-pass filtering (d); anisotropic diffusion filtering (e); global contrast enhancement (f); binarization (g); unwanted segment removal (h); skeletonization (i); pruning (j); distance transformation (k); skeleton-over-distance transformation (l); skeleton-over-ROI (m). Image processing pipeline then went through the following steps (D): the target tissue (untreated rHuEPO) was extracted (a); selection of ROI (b); converted to grayscale (c); high-pass filtering (d); anisotropic diffusion filtering (e); global contrast enhancement (f); binarization (g); unwanted segment removal (h); skeletonization (i); pruning (j); distance transformation (k); skeleton-over-distance transformation (l); skeleton-over-ROI (m). E1 and E2 are two sample specimens, taken from regenerated skins of rHUEPO treated animals for analysis of vessel length measurement and vessel thickness measurements. C1 and C2 are two sample specimens, taken from skins of untreated rHUEPO animals for analysis of vessel length measurement and vessel thickness measurements. Abbreviations: rHuEPO, recombinant human erythropoietin; EPO, erythropoietin; w/o, without; E1, treated rHuEPO (day 14); E2 treated rHuEPO (day 14); C1, untreated rHuEPO (day 14); C2, untreated rHuEPO (day 14).

Figure 3

Quantitative image analysis of blood vessels. Notes: Comparative vessel length over area (A) and thickness measurements (B) in rHuEPO-treated and untreated groups. Overall vessel thickness for treated rHuEPO-treated animals was 586.13 μm and for untreated animals, 260.10 μm. Median value for rHuEPO-treated animals was 91.96 μm and for untreated animals, 72.70 μm. Image processing pipeline went through the following steps (C): the target tissue (rHuEPO-treated) was extracted (a); selections of regions of interest (ROI) (b); converted to grayscale (c); high-pass filtering (d); anisotropic diffusion filtering (e); global contrast enhancement (f); binarization (g); unwanted segment removal (h); skeletonization (i); pruning (j); distance transformation (k); skeleton-over-distance transformation (l); skeleton-over-ROI (m). Image processing pipeline then went through the following steps (D): the target tissue (untreated rHuEPO) was extracted (a); selection of ROI (b); converted to grayscale (c); high-pass filtering (d); anisotropic diffusion filtering (e); global contrast enhancement (f); binarization (g); unwanted segment removal (h); skeletonization (i); pruning (j); distance transformation (k); skeleton-over-distance transformation (l); skeleton-over-ROI (m). E1 and E2 are two sample specimens, taken from regenerated skins of rHUEPO treated animals for analysis of vessel length measurement and vessel thickness measurements. C1 and C2 are two sample specimens, taken from skins of untreated rHUEPO animals for analysis of vessel length measurement and vessel thickness measurements. Abbreviations: rHuEPO, recombinant human erythropoietin; EPO, erythropoietin; w/o, without; E1, treated rHuEPO (day 14); E2 treated rHuEPO (day 14); C1, untreated rHuEPO (day 14); C2, untreated rHuEPO (day 14).

Figure 3

Quantitative image analysis of blood vessels. Notes: Comparative vessel length over area (A) and thickness measurements (B) in rHuEPO-treated and untreated groups. Overall vessel thickness for treated rHuEPO-treated animals was 586.13 μm and for untreated animals, 260.10 μm. Median value for rHuEPO-treated animals was 91.96 μm and for untreated animals, 72.70 μm. Image processing pipeline went through the following steps (C): the target tissue (rHuEPO-treated) was extracted (a); selections of regions of interest (ROI) (b); converted to grayscale (c); high-pass filtering (d); anisotropic diffusion filtering (e); global contrast enhancement (f); binarization (g); unwanted segment removal (h); skeletonization (i); pruning (j); distance transformation (k); skeleton-over-distance transformation (l); skeleton-over-ROI (m). Image processing pipeline then went through the following steps (D): the target tissue (untreated rHuEPO) was extracted (a); selection of ROI (b); converted to grayscale (c); high-pass filtering (d); anisotropic diffusion filtering (e); global contrast enhancement (f); binarization (g); unwanted segment removal (h); skeletonization (i); pruning (j); distance transformation (k); skeleton-over-distance transformation (l); skeleton-over-ROI (m). E1 and E2 are two sample specimens, taken from regenerated skins of rHUEPO treated animals for analysis of vessel length measurement and vessel thickness measurements. C1 and C2 are two sample specimens, taken from skins of untreated rHUEPO animals for analysis of vessel length measurement and vessel thickness measurements. Abbreviations: rHuEPO, recombinant human erythropoietin; EPO, erythropoietin; w/o, without; E1, treated rHuEPO (day 14); E2 treated rHuEPO (day 14); C1, untreated rHuEPO (day 14); C2, untreated rHuEPO (day 14).

Figure 4

Difference in vessel density ratio. Notes: The average wound vessel density on day 7 and day 14 was 18.14% and 13.90%, respectively, while averages were 6.77% and 8.07%, respectively, in the control group. Significant statistical difference at both time points was evident (P<0.05). Values indicate the mean ± SD of each group. *P<0.05 vs control group. Abbreviations: SD, standard deviation; Ctrl, control; vs, versus; d, day; EPO, erythropoietin.

Figure 5

Re-epithelialization of skin in rHuEPO-treated and untreated groups. Notes: Re-epithelialization level at day 4 and day 7 (A): histological scoring of the results of scald regeneration in rats treated with rHuEPO or vehicle at different healing times (4 and 7 days). Representative photomacroscopic images of wounds directly after tangential excision (day 0), and on day 4 and 7 after wounding with (B) EPO gel treated and (C) control (no EPO gel). In the rHuEPO group, we noticed a significant decrease in wound size and quicker wound closure than in the control group at day 7. In addition, we found increased epithelial covering in the rHuEPO treatment group than in the control group. A considerably better skin regeneration, re-epithelialisation, and wound closure, and visibly less scar formation was found on day 4 in rats with topical rHuEPO treatment as compared to controls. The final score for epithelialization of skin regeneration for each animal specimen is demonstrated as the sum of the three partial score values. Values are the mean ± standard error of the mean of each group. *P < 0.05 versus control group. Abbreviations: EPO, erythropoietin; rHuEPO, recombinant human erythropoietin.

Figure 6

Diagrammatic representation of EPO treated and untreated rat model for skin regeneration. Notes: Model of tangential excision in 6–8-week-old female rats weighing 200–250 g EPO treated (A, a–e). Each animal received four dorsolateral skin defects (1×1.5 cm) via tangential excision of 0.2 mm using a dermatome (Aesculap Inc., Center Valley, PA, USA). Every second day the rats were treated with rHuEPO hydrogel over the dermal wound bed (150 IU rHuEPO/g, dose of 1 g/cm²). Model of tangential excision in 6–8-week-old female rats weighing 200–250 g untreated with EPO (B, a–e). Each animal received four dorsolateral skin defects (1×1.5 cm) via tangential excision of 0.2 mm. These rat models are controls (no topical application of rHuEPO). Abbreviations: EPO, erythropoietin; rHuEPO, recombinant human erythropoietin.

Figure 7

Representative immunostaining of scalded skin tissue specimens from rats on day 7 after wounding and topical application of rHuEPO gel or its vehicle (control gel). Notes: (A) control group without rHuEPO (vehicle only), rat number 22 (a, c, d, e), rat number 23 (b, f); (B) rHuEPO group, rat number 21 (a, c, d, e), rat number 24 (b, f). Staining to assess CD 31 (a, b); CD 71 (c); CD 90 (d); nestin (e, f). Arrows indicate the location of expression of these different markers. Magnification 100×, scale bar is 200 μm. Abbreviations: EPO, erythropoietin; rHuEPO, recombinant human erythropoietin.