The combined effect of recombinant human epidermal growth factor and erythropoietin on full‐thickness wound healing in diabetic rat model

Joon Pio Hong; Sung Woo Park

doi:10.1111/j.1742-481X.2012.01100.x

. 2012 Oct 19;11(4):373–378. doi: 10.1111/j.1742-481X.2012.01100.x

The combined effect of recombinant human epidermal growth factor and erythropoietin on full‐thickness wound healing in diabetic rat model

Joon Pio Hong ^1,^✉, Sung Woo Park ¹

PMCID: PMC7950853 PMID: 23078553

Abstract

Diabetic wound is a chronic wound in which normal process of wound healing is interrupted. Lack of blood supply, infection and lack of functional growth factors are assumed as some of the conditions that lead to non‐healing environment. Epidermal growth factor (EGF) acts primarily to stimulate epithelial cell growth across wound. Erythropoietin (EPO) is a haematopoietic factor, which stimulates the production, differentiation and maturation of erythroid precursor cells. This study hypothesised combining these two factors, non‐healing process of diabetic wound will be compensated and eventually lead to acceleration of wound healing compared with single growth factor treatment. A total of 30 diabetic Sprague–Dawley rats were divided into three treatment groups (single treatment of rh‐EPO or rh‐EGF or combined treatment on a full‐thickness skin wound). To assess the wound healing effects of the components, the wound size and the healing time were measured in each treatment groups. The skin histology was examined by light microscopy and immunohistochemical analysis of proliferating markers was performed. The combined treatment with rh‐EPO and rh‐EGF improved full‐thickness wound significantly (P < 0·05) accelerating 50% healing time with higher expression of Ki‐67 compared with single growth factor‐treated groups. The combined treatment failed to accelerate the total healing time when compared with single growth factor treatments. However, the significant improvement were found in wound size reduction in the combined treatment group on day 4 against single growth factor‐treated groups (P < 0·05). This study demonstrated that the combined treatment of rh‐EPO and rh‐EGF improved the wound healing possibly through a synergistic action of each growth factor. This application provides further insight into combined growth factor therapy on non‐healing diabetic wounds.

Keywords: Diabetes, Epidermal growth factor, Erythropoietin, Wound healing

Introduction

The wound healing process is a complex biological event that includes inflammation, proliferation and migration of different cell types (1). This complex process involves broad spectrum of cascades like matrix synthesis, angiogenesis, collagen deposition leading to reepithelialisation, neovascularisation and formation of granulation tissue (2). In chronic wounds such as diabetes, this normal process is interrupted by many conditions such as lack of blood supply, infection, disorientation of appropriate cytokines and lack of functional growth factors ³^,⁴^,⁵.

Epidermal growth factor (EGF), which is a growth factor produced by platelets, macrophages and monocytes, interacts with the EGF receptor on epidermal cells and fibroblasts (6). EGF acts primarily to stimulate epithelial cell growth across the wound, and also acts on fibroblasts and smooth muscle cells. Many studies had proven the clinical effects of EGF on wound healing by shortening the healing time. Also, these studies reported that EGF increased the tensile strength and reduced unfavourable tissue events such as failure of adequate extracellular matrix (ECM) protein production ⁷^,⁸.

Erythropoietin (EPO) is a haematopoietic factor and it stimulates the production, differentiation and maturation of erythroid precursor cells (9). The effects of EPO have been observed on myocardial, vascular smooth muscle and mesangial cells. Recently, novel biological functions of EPO were found ¹⁰^,¹¹^,¹². Endogenous EPO signalling exists in non‐haematopoietic tissues and exogenous EPO modulate organ functions and cellular responses to diverse types of injury. Consequently, EPO stimulates endothelial cell mitosis and motility, which can contribute to neovascularisation in wounds ¹³^,¹⁴.

The microenvironment of diabetic foot ulcer showed the alteration of growth factors, influenced by hyperglycaemia and local inflammatory responses (15). Thus, it may be hypothesised that the addition of growth factors may enhance wound healing. Furthermore, considering the use of various growth factors in microenvironment of wound healing, multiple growth factor treatment can be hypothesised to be beneficial to single growth factor treatment. The purpose of this study is to evaluate the combined treatment of rh‐EGF and rh‐EPO in full‐thickness diabetic wounds and to investigate whether the synergistic effect exists over single growth factor treatment.

Materials and methods

Animals

Thirty Sprague–Dawley rats, aged 7–8 weeks and weighing between 180 and 220 g, were caged with free access to standard hamster food and water, at room temperature (23 ± 1°C) and humidity of 55 ± 5%, with 12:12 dark : light cycle under ambiguous light (150–300 lux). All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Asan Life Science Research Center animal review board.

After 1 week of the adjustment period, intraperitoneal injection of streptozotocin (70 mg/kg) was given to induce acute diabetic conditions. Diabetic condition was evaluated by daily blood glucose level and the value over 300 µg/dl was diagnosed as diabetic condition. When the blood glucose level was over 450 µg/dl, it was controlled with 2 units of insulin. After 14 days, rats with stable diabetic condition underwent surgical procedure to make full‐thickness wounds.

The animals were divided into three groups of ten animals each. All groups had two equal‐sized wounds where one wound was selectively treated and one wound remained as control. Each rats in group 1 were topically applied with 20 IU/day recombinant human EPO (Eposis®;, Daewoong Pharmaceutical Company, Seoul, Korea), group 2 with 20 µg/day recombinant human EGF (Easyef®;, Daewoong Pharmaceutical Company) and group 3 with combined 20 IU/day rh‐EPO and 20 µg/day rh‐EGF. Treatment was carried out two times a day with a medical sprayer and the volume of each spray was 50 µl approximately.

Surgical procedure

All procedures were performed aseptically. Animals were anaesthetised using intraperitoneal injections of 40 mg/kg Zoletil®; (Zolazepam + Tiletamine, Virbac Laboratories; Carros, France). After shaving dorsum for a clean surgical procedure, 2 × 2 cm² sized full‐thickness wounds were made symmetrically on both side of dorsal flank. The left wound was only treated with same dressing material and remained as control and the right wound was treated with each application according to the designated treatment group (Supporting information, Figure S1).

Dressing

A hydrocolloid dressing (Duoderm®;, Convatec, NY) was used as a marginal framework to keep the growth factor from escaping the wound site. Both wounds were covered with a film (Opsite®;, Smith & Nephew, London, England) after application and the torso was wrapped with a bandage (Peha‐haft®;, Paul Hartmann, Heidenheim, Germany). Drug application and dressing were performed twice a day with a 12‐hour interval until the wound was fully epithelialised.

Wound assessment

Starting from day 0, digital photograph was taken once every 3 days (day 4, 7, 10, 13, 16) and at full epithelialisation. Wound size was measured by an image analyser (image measurement standard v4.01, Bersoft, Puerto Plata, Dominican Republic) to assess the size changes during healing periods. The results were analysed with relative wound size difference compared with each non‐treated sites. Sizes of wounds at initial day 0 were standardised as 0%, which indicates no reepithelialisation was observed. And, the longitudinal wound healing process was assessed by the reduction of the wound size and expressed as a percentage from baseline (the wound size of day 0). HT50 (half healing time) calculated as the time point when the wound size was reduced to 50% of the initial wound size.

Histological and immunohistochemical analysis

Full‐thickness biopsy specimens of dorsal skin including periwound margin were obtained at days 4, 7, 10 and the end day of the observation. Each specimen was fixed in 10% formalin and embedded in paraffin. The sections were stained with standard haematoxylin and eosin (H&E) stain and examined by light microscopy.

Immunohistochemical analysis was performed on the sections obtained at the endpoints. The sections were stained with mouse anti‐rat Ki‐67 antibody (Dako, Zug, Switzerland, 1:50), visualised using horse radish peroxidase‐conjugated secondary antibodies and examined by light microscopy. The staining intensity was quantified with the digital images taken under ×200 magnification using an image analyser (image measurement standard v4_01, Bersoft).

Statistical analysis

Data were presented as means ± standard deviation. Statistical analyses were performed by the analysis of relative wound size difference with control by SAS 9.1 using a linear mixed model. P value of <0·05 was taken as the limit of significance. To evaluate the expression for Ki‐67, statistical analysis was performed by SAS version 9.1 GLM procedure and values were expressed as means ± standard deviation. P value of <0·05 was taken as the limit of significance.

Results

The combined treatment of EPO and EGF accelerated wound healing

Between the non‐treated wounds among all three groups (P > 0·05) the average time for 50% epithelialisation was 7·5 ± 0·52 days. However, the wound size was reduced by 50% (HT50) in rh‐EPO and rh‐EGF‐treated groups at 5·7 ± 0·92 and 5·6 ± 0·90 days, respectively. Moreover, the HT50 in the combined treatment group was 4·8 ± 0·86 days, which was significantly faster than those of single treatment groups (P < 0·01) (Table 1).

Table 1.

rh‐EPO and rh‐EGF combined treatment group shows shorter time to reach reepithelialisation especially in early proliferative phase of wound healing. ^*

Group	HT 50 (days) (mean ± 2 SD)	N
Control	7·5 ± 0·52	10
EPO (20 IU/day)	5·7 ± 0·92	10
EGF (20 µg/day)	5·6 ± 0·90	10
EPO & EGF (20 IU, 20 µg/day)	4·8 ± 0·86	10

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EGF, epidermal growth factor; EPO, erythropoietin; rh‐EPO, recombinant human EPO; rh‐EGF, recombinant human EGF.

^*The HT50 (healing time till 50% epithelialisation) in the combined rh‐EPO and rh‐EGF‐treated group was 4·8 ± 0·86 days being significantly 2·7 days shorter than control wounds and about 1 day shorter than single growth factor‐treated groups. All treatment groups show significantly shorter healing period compared with control group (P < 0·01).

Epithelialisation analysis

Wound size was evaluated at days 1, 4, 7, 10, 13, 16 and 18 for longitudinal study. All treatment groups significantly healed faster the contralateral control wound (P < 0·05). However, findings at day 4 showed the most significant epithelialisation rate between groups. The rh‐EPO‐treated group showed 33·83% of reepithelialisation, rh‐EGF‐treated group show 36·58% and the combined treatment group at 44·27%.(P < 0·05) (Figure 1).

(A) Longitudinal analysis wound healing in each group shows faster reepithelialisation when treated with rh‐EPO and rh‐EGF compared with single treatment groups (P < 0·01). Dotted lines are landmarks for HT 50, which indicate time to reach 50% of initial wound size that is used as time horizon of early proliferative phase. All treatment groups show significantly faster reepithelialisation compared with control group (P < 0·01). (B) Gross size changes of wounds in each group at days 1, 6 and 16 show noticeable size changes, especially in rh‐EPO and rh‐EGF combined treatment group. D1, day 1; D4, day 4; D16, day 16.

Histology

H&E analysis of wound site after full reepithelialisation showed no microscopic differences between all treatment groups. All three treatment groups showed fully recovered stratum corneum and maturing epidermal cells. And, margins at the edge of granulation and normal tissue were noticed in each group samples (Figure 2). Although timing differences existed in wound healing of each groups, final composition of healed wounds was similar to non‐disrupted control group. Comparatively indicating EGF or EPO enhances chronic wound healing by compensation of normal wound healing process, not by arousing extra effects noticed in microscopic level.

Haematoxylin and eosin staining of skin sample harvested at the end of observation showing merely distinguishable differences in each groups. Samples from all groups show fully recovered stratum corneum and maturing epidermal cells when it reached final phase of wound healing. (A) Con, control group; (B) EPO, rh‐EPO treatment group, (C) EGF, rh‐EGF treatment group, (D) EPO, EGF, rh‐EPO and rh‐EGF combined treatment group. Arrows indicates margins between normal skin and granulation tissue. HF, hair follicles; SG, sweat glands.

Immunohistochemical staining

Detecting Ki‐67 positive cells in basal layer was analysed to assess cellular proliferation between each groups (Figure 3). The combined treatment group showed significantly highest proportional detection of positive cells with average value of 11·03%, whereas the rh‐EPO‐treated group and rh‐EGF‐treated group expressed 7·83 and 8·74%, respectively (P < 0·05). All treatment groups showed significantly higher proportional value against control wound that showed 3·28% of positive cell area (P < 0·05).

Discussion

Chronic wound becomes even more complex because normal healing process is altered due to break in homoeostasis of cytokines, growth factors, multiple cells, inflammatory mediators, nitric oxide and various other factors ¹^,²^,³^,⁴^,⁵^,¹⁶. To put the healing back on the right track, debridement of necrotic tissue, infection control, moist wound environment and abundant oxygenation to the tissue are essential. Despite these efforts, chronic wounds may still be resistant to treatment. One reason may be the lack of growth factors that may perpetuate chronic diabetic wounds (15). Thus the addition of multiple growth factors can be hypothesised to enhance healing in chronic wound such as diabetic wounds. In this study, rh‐EGF and rh‐EPO were selected based on the characteristics of diabetic wound where epithelialisation and angiogenesis are impaired. According to other studies, the application of EGF was found to enhance the healing capacity and protect from wound disruption whereas EPO has been reported for its non‐haematopoietic effect in various organs ¹⁷^,¹⁸^,¹⁹. The importance of adequate reperfusion and oxygenation on tissue site has been emphasised and EPO can be expected to elicit neovascularisation and to develop better oxygenation when applied on wound ¹⁷^,¹⁸^,¹⁹^,²⁰^,²¹^,²²^,²³. It was hypothesised that by adding the two growth factors that play a different role to enhance healing, it will elicit a synergistic effect.

In this study, diabetic wound healed faster, especially in initial proliferative phase when rh‐EPO and rh‐EGF were combined for treatment. The HT50 (healing time till 50% epithelialisation) in the combined rh‐EPO and rh‐EGF‐treated group was 4·8 ± 0·86 days being significantly 2·7 days shorter than control wounds and about 1 day shorter than single growth factor‐treated groups. However, days taken until full reepithelialisation (HT100) did not show significant differences between treatment groups. The reason for this observation may be because of the reduction of deepithelialised skin at the end of wound healing making delivery of growth factors difficult. This is supported by observation during the later stage of healing when stall in wound size reduction was apparent. For example, during early proliferation phases at day 4–7, the rate of healing is rapid leading up to 20–30% wound size change in each group. But during the later days from 13 to 16, changes in wound size is reduced from 3% to 7%. Another possible explanation for this rapid healing during the early phase is possibly because of the fact that growth factors have significant influence during this time. Once healing has reached its later phases by rich granulation and near epithelialisation of the defect, the need for growth factors would decrease. But despite the overall healing having similar outcomes, one must consider that chronic wound itself is difficult to engage in the proper healing process and this early engagement may allow to enhanced healing.

H&E staining of histological samples from days 4, 7, 10 and at the endpoint show corresponding findings to wound healing phase. Granulation tissues are noticed in samples which were harvested at day 4, 7 in each group, when proliferative functions are most active. However, samples at the endpoint show merely distinguishable microscopic characteristics between each group, indicating EGF or EPO enhances chronic wound healing by compensation of normal wound healing process, not by arousing extra effects noticed in microscopic level.

Immunohistochemical results show more functional capacity of proliferation occurs when rh‐EPO and rh‐EGF are combined for treatment. The Ki‐67 protein is a cellular marker for proliferation. During the interphase, the Ki‐67 antigen can be exclusively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. Ki‐67 protein is present during all active phases of the cell cycle (G1, S, G2 and mitosis), but is absent from resting cells (G0). The Ki‐67 antigens, marker to determine the growth fraction of a given cell population, was measured by calculating relative fractional area. The combined treatment group expressed significantly higher value of 11·03% compared to rh‐EPO (7·83%) and rh‐EGF (8·74%)‐treated groups.

The hypothesis that there would be a synergistic effect of rh‐EPO and rh‐EGF treatment was shown to be successful in achieving enhanced healing, especially emphasised during the early phase of diabetic wound healing through different mechanisms. Further study with advanced diabetic mouse models, larger wounds, different dosage and administration is warranted to find the ideal combination of EGF and EPO based on this first novel approach. And, studies to determine action of these two growth factors on wound healing mechanisms will be required. Reaction with other growth factors may also provide further insight in understanding the orchestrated growth factors effect over healing.

Acknowledgements

We acknowledge Ms. Ahn Hyung Mi for helpful discussion and comments on statistical analysis. This work was supported by the Student Research Grant (09‐000) of University of Ulsan College of Medicine, Seoul, Korea. We thank Daewoong Pharmaceutical Company for their donation of rh‐EGF and rh‐EPO.

This paper was presented during the 20th Annual Meeting of Wound Healing Society on April 17–20, 2010.

Supporting information

SUPPORTING INFORMATION

The following Supporting information is available for this article:

Figure S1. Surgical preparation for full‐thickness wounds. Two full‐thickness wounds are made on dosum of each rats partitioned by vertebra and hydrocolloid dressing. The left wound [1] is treated with nothing but only with same dressing materials for control and right wound [2] is treated with drug solvent on each groups.

Click here for additional data file.^{(1.5MB, tif)}

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SUPPORTING INFORMATION

The following Supporting information is available for this article:

Click here for additional data file.^{(1.5MB, tif)}

[b1] 1. Singer AJ , Clark RA. Cutaneous wound healing. N Engl J Med 1999. ; 341 : 738 – 46. [DOI] [PubMed] [Google Scholar]

[b2] 2. Greenhalgh DG. The role of growth factors in wound healing. J Trauma 1996. ; 41 : 159 – 67. [DOI] [PubMed] [Google Scholar]

[b3] 3. Brown GL , Curtsinger L , Jurkiewicz MJ , Nahai F , Schultz G. Stimulation of healing of chronic wounds by epidermal growth factor. Plast Reconstr Surg 1991. ; 88 : 189 – 94 ; discussion 95 – 6. [PubMed] [Google Scholar]

[b4] 4. Tsang MW , Wong WK , Hung CS , Lai KM , Tang W , Cheung EY , Kam G , Leung L , Chan CW , Chu CM , Lam EK. Human epidermal growth factor enhances healing of diabetic foot ulcers. Diabetes Care 2003. ; 26 : 1856 – 61. [DOI] [PubMed] [Google Scholar]

[b5] 5. Bennett SP , Griffiths GD , Schor AM , Leese GP , Schor SL. Growth factors in the treatment of diabetic foot ulcers. Br J Surg 2003. ; 90 : 133 – 46. [DOI] [PubMed] [Google Scholar]

[b6] 6. Nanney LB. Epidermal and dermal effects of epidermal growth factor during wound repair. J Invest Dermatol 1990. ; 94 : 624 – 9. [DOI] [PubMed] [Google Scholar]

[b7] 7. Brown GL , Curtsinger L 3rd , Brightwell JR , Ackerman DM , Tobin GR , Polk HC Jr , George‐Nascimento C , Valenzuela P , Schultz GS. Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. J Exp Med 1986. ; 163 : 1319 – 24. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b8] 8. Epstein JB , Gorsky M , Guglietta A , Le N , Sonis ST. The correlation between epidermal growth factor levels in saliva and the severity of oral mucositis during oropharyngeal radiation therapy. Cancer 2000. ; 89 : 2258 – 65. [DOI] [PubMed] [Google Scholar]

[b9] 9. Fisher JW. Erythropoietin: physiology and pharmacology update. Exp Biol Med (Maywood) 2003. ; 228 : 1 – 14. [DOI] [PubMed] [Google Scholar]

[b10] 10. Digicaylioglu M , Lipton SA. Erythropoietin‐mediated neuroprotection involves cross‐talk between Jak2 and NF‐kappaB signalling cascades. Nature 2001. ; 412 : 641 – 7. [DOI] [PubMed] [Google Scholar]

[b11] 11. Moon C , Krawczyk M , Ahn D , Ahmet I , Paik D , Lakatta EG , Talan MI. Erythropoietin reduces myocardial infarction and left ventricular functional decline after coronary artery ligation in rats. Proc Natl Acad Sci U S A 2003. ; 100 : 11612 – 7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b12] 12. Spandou E , Tsouchnikas I , Karkavelas G , Dounousi E , Simeonidou C , Guiba‐Tziampiri O , Tsakiris D. Erythropoietin attenuates renal injury in experimental acute renal failure ischaemic/reperfusion model. Nephrol Dial Transplant 2006. ; 21 : 330 – 6. [DOI] [PubMed] [Google Scholar]

[b13] 13. Parsa CJ , Kim J , Riel RU , Pascal LS , Thompson RB , Petrofski JA , Matsumoto A , Stamler JS , Koch WJ. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts. J Biol Chem 2004. ; 279 : 20655 – 62. [DOI] [PubMed] [Google Scholar]

[b14] 14. Buemi M , Vaccaro M , Sturiale A , Galeano MR , Sansotta C , Cavallari V , Floccari F , D'Amico D , Torre V , Calapai G , Frisina N , Guarneri F , Vermiglio G. Recombinant human erythropoietin influences revascularization and healing in a rat model of random ischaemic flaps. Acta Derm Venereol 2002. ; 82 : 411 – 7. [DOI] [PubMed] [Google Scholar]

[b15] 15. Gary Sibbald R , Woo KY. The biology of chronic foot ulcers in persons with diabetes. Diabetes Metab Res Rev 2008. ; 24 ( 1 Suppl ): S25 – 30. [DOI] [PubMed] [Google Scholar]

[b16] 16. Schaffer CJ , Nanney LB. Cell biology of wound healing. Int Rev Cytol 1996. ; 169 : 151 – 81. [DOI] [PubMed] [Google Scholar]

[b17] 17. Hockel M , Schlenger K , Doctrow S , Kissel T , Vaupel P. Therapeutic angiogenesis. Arch Surg 1993. ; 128 : 423 – 9. [DOI] [PubMed] [Google Scholar]

[b18] 18. Jelkmann W. Biology of erythropoietin. Clin Investig 1994. ; 72 ( 6 Suppl ): S3 – 10. [PubMed] [Google Scholar]

[b19] 19. Goodnough LT , Monk TG , Andriole GL. Erythropoietin therapy. N Engl J Med 1997. ; 336 : 933 – 8. [DOI] [PubMed] [Google Scholar]

[b20] 20. Alvarez Arroyo MV , Castilla MA , Gonzalez Pacheco FR , Tan D , Riesco A , Casado S , Caramelo C. Role of vascular endothelial growth factor on erythropoietin‐related endothelial cell proliferation. J Am Soc Nephrol 1998. ; 9 : 1998 – 2004. [DOI] [PubMed] [Google Scholar]

[b21] 21. Risau W. Mechanisms of angiogenesis. Nature 1997. ; 386 : 671 – 4. [DOI] [PubMed] [Google Scholar]

[b22] 22. Zachary I. Vascular endothelial growth factor. Int J Biochem Cell Biol 1998. ; 30 : 1169 – 74. [DOI] [PubMed] [Google Scholar]

[b23] 23. Choi K , Kennedy M , Kazarov A , Papadimitriou JC , Keller G. A common precursor for hematopoietic and endothelial cells. Development 1998. ; 125 : 725 – 32. [DOI] [PubMed] [Google Scholar]

PERMALINK

The combined effect of recombinant human epidermal growth factor and erythropoietin on full‐thickness wound healing in diabetic rat model

Joon Pio Hong

Sung Woo Park

Abstract

Introduction