Effects of Topical Copper Tripeptide Complex on Wound Healing in an Irradiated Rat Model

Introduction

Nonsurgical treatment of head and neck squamous cell carcinoma has increased in the past 3 decades. ¹ Salvage surgery following radiotherapy or chemoradiotherapy increases the risk of soft tissue complications. ² Interventions that improve the healing of radiation-damaged tissues postoperatively might decrease the morbidity and potential mortality of such complications.

Glycyl-histidyl-lysine is a naturally occurring tripeptide first shown in the 1970s to improve hepatocyte growth and survival. ³ In vitro studies of glycyl-histidyl-lysine-copper (GHK-Cu) have shown increased chemo-attraction of repair cells, reduced inflammation, and increased collagen synthesis, fibroblast proliferation, and angiogenesis.^4-5 Irradiated and nonirradiated fibroblasts taken from operative sites treated with GHK-Cu have shown faster population-doubling times and significantly more vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor. ⁶ Thus, GHK-Cu appears to be a beneficial upstream effector to local growth factor and extracellular matrix environments. In vivo rat studies have repeatedly demonstrated improved healing in dorsal soft tissue wounds treated with GHK-Cu,^7-9 but this has never been evaluated in an irradiated model. Randomized, evaluator-blinded, placebo-controlled human clinical trials utilizing topical GHK-Cu have shown improved wound closure and infection rates in diabetic neuopathic ulcers ¹⁰ and higher patient satisfaction in healing results following circumoral CO2 laser resurfacing. ¹¹

For the patient requiring salvage surgery after radiation-induced tissue injury, the continued study of molecules to improve wound healing is vital. This study investigates the ability of topical GHK-Cu to improve wound healing in an irradiated, ischemic flap model in rats, including the evaluation of flap survival and of immunohistochemical (IHC) analyses of 2 markers of angiogenesis, caveolin-1, and VEGF.

Methods

Radiation Protocol

After institutional approval, male Sprague-Dawley rats matched by species, age, weight, and gender were obtained. Animals were anesthetized with 0.15 mg/kg dexmedetomidine and 0.2 mg/kg fentanyl and reversed with 0.5 mg/kg atipamazole, then placed in lateral decubitus position ( Figure 1A ) in a linear accelerator. After plain radiography confirmed avoidance of the spinal canal ( Figure 1B ), a 12 cm length, 0.75 cm width, and 2.5 cm depth radiation field (posterior axillary line to anterior iliac crest) was administered in four 10-Gray (Gy) fractions at 3-day intervals. The effects of this dose are comparable to humans after radiotherapy ¹² and correspond to 66.67 Gy. A 4-week healing period was provided to avoid acute radiation effects. The protocol was based on a previous study. ¹³

OPEN IN VIEWER

Figure 1.

Radiation positioning (A) and x-ray confirmation of spine alignment and radiation exposure confined to dorsal soft tissues (B).

Surgical Procedure

The same anesthetics were used. Preoperative cefazolin was administered. Dorsal skin was shaved and cleansed with povidine-iodine. A centrally located, cranially based 2 cm by 8 cm flap was elevated deep to the panniculus carnosus ( Figure 2 ) based on an ischemic flap model, ¹⁴ then closed with nonabsorbable, monocryl suture. Postoperative pain control included 0.05 to 0.1 mg/kg SQ bupernorphine injections for 24 hours, then per os ibuprofen as needed.

OPEN IN VIEWER

Figure 2.

Animal procedure: premarked 2 × 8 cm incision.

Clinical Evaluation

Digital photographs taken on postoperative day 10 were converted to 8-bit images, and pixel -measurements were scaled according to rulers included in photographs using ImageJ software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, Maryland, http://imagej.nih.gov/ij/, 1997-2011). Conversion to binary images ( Figure 3 ) allowed for objective calculation of total area of necrosis.

OPEN IN VIEWER

Figure 3.

Clinical flap survival: control in vivo (A) converted to binary image for analysis (B).

Histological and Immunohistochemical Evaluation

Digital images of slides were created at 20× magnification using a scanner (ScanScope XT, Aperio Technologies, Vista, California). Using digital software (ImageScope 11.1, Aperio), 4 representative tissue areas (100 um × 100 um) located at the tissue-flap interface were annotated, including the distal end of the flap, 0.5 cm from the distal end, 1.0 cm from the distal end, and 1.5 cm from the distal end. Using image analysis software (Color Deconvolution Algorithm, Aperio) the color vectors for the individual staining components of the Hematoxylin counterstain (blue) and DAB chromagen (brown) were measured. For caveolin-1, analysis software (Microvessel Algorithm, Aperio) identified pixels exceeding intensity threshold limits, then adjusted them to remove background staining and preserve only endothelial-stained cells ( Figure 4A ), which allowed for isolation of pixels arranged in a continuous line, indicating the geometry of blood vessels ( Figure 4B ), and calculation of blood vessel number and area. For VEGF, pixels exceeding threshold limits were quantified as a fraction of total pixels; then the average number of positive pixels expressed as a fraction total was calculated. These methods have been shown to accurately analyze vascular endothelium in animal tissues. ¹⁸

OPEN IN VIEWER

Figure 4.

Caveolin-1 staining: magnified view of blood vessels before (A) and after (B) vessel analysis algorithm.

Results

One animal suffered seizure-like activity following reversal of anesthesia during radiotherapy and recovered with conservative measures. One animal expired following anesthesia administration for radiotherapy, while 6 expired following anesthesia administration for surgery. Ten control group and 13 test group animals survived to the study endpoint. Clinical and immunohistochemical results are listed in Table 1 . Differences in mean ischemic area, vessels number, vessel area, and VEGF intensity scores were not statistically different.

OPEN IN VIEWER

Table 1.

Clinical and immunohistochemical results.

Outcome Measure	Test Mean (SD)	Control Mean (SD)	P-Value
A: Clinical flap survival
Ischemic area (cm2)	5.0 (0.9)	3.8 (1.1)	.011
B: Caveolin-1 analysis
Number of vessels	209.0 (111.1)	207.4 (109.4)	.973
Vessel area (um2)	525.7 (191.0)	422.8 (109.7)	.118
C: Vascular endothelial growth factor analysis
Intensity score (no unit)	0.34 (0.19)	0.54 (0.41)	.169

Note. No differences are considered statistically significant (P < .01).

Discussion

While much attention has been paid to local growth factors and their effects on wound healing, little attention has been paid to upstream molecules that affect the local molecular environment. We sought to perform the first study evaluating the effects of topical GHK-Cu on irradiated soft tissue wound healing and did so using objective analysis of flap survival in an ischemic model and of angiogenesis through caveolin-1 and VEGF analyses. This is the first in vivo study evaluating GHK-Cu as applied topically to closed wounds; prior models used either subcutaneous impregnation in closed wounds^7-9 or topical application to open wounds.^10-12 The topical application was chosen because ultimately, patients at high risk for wound healing complications, such as those treated with radiotherapy or chemoradiotherapy, have closed wounds after salvage surgery.

In the current study, ischemia trended toward being greater in the GHK-Cu group as compared to the test group, but this was not statistically significant. This is contrary to prior animal and human studies reporting improved wound healing.^7-12 We sought to explain the clinical effects at a molecular level through analysis of angiogenesis via caveolin-1 and VEGF expression. We found no difference in caveolin-1 analyses and VEGF expression at the flap-tissue interface, which indicated no improvement in blood vessel formation or in a primary growth factor related to angiogenesis.

Although animal ⁸ and human ¹⁹ studies used GHK-Cu gel topically, it is unclear whether this formulation had a negative impact on the healing of flaps compared to the petroleum-based, hydrophilic ointment used as a control. It is unclear how the topical treatment of such a formulation was affected by tissue absorption. There are also no data reporting the effects of differential frequency of application on the healing of wounds. The study is limited by the survival of animals related to anesthesia during irradiation and the surgical procedures. Perhaps a larger sample size would have allowed for more definitive statistical analysis, while a smaller sample may have led to biased outcomes.

Conclusions

A safe radiation protocol and reproducible animal model was used to evaluate the effect of topical GHK-Cu on wound healing. Objective quantification of flap survival, blood vessel formation, and growth factor expression was performed. Dorsal soft tissue flaps in irradiated rats treated with topical GHK-Cu gel showed no difference in area of clinical ischemia, blood vessel number or vessel area, or VEGF expression compared to controls.

Author Contributions

Noah P. Parker, conception and design, acquisition of data, analysis and interpretation of data, drafting the article and revising it critically, final approval; Farhad Ardeshirpour, acquisition of data, critical revisions of manuscript, final approval; Stephen C. Schmechel, conception and design, critical revisions of manuscript, final approval; Amy Anne D. Lassig, conception and design, critical revisions of manuscript, final approval.

Disclosures

Competing interests: None.

Sponsorships: None.

Funding source: 5M Lions Club Research Grant and NIH grants P30-CA77598 (D Yee), P50-CA101955 (D Buchsbaum), KL2-RR033182 (B Blazar), NCI Contract No. HHSN261200800001E (S Schmechel), funding purchasing of animals, conducting the live animal portions of the study, purchase of appropriate immunohistochemical staining, and conducting the histological and immunohistochemical portion of the study. Funding the digital imaging and analysis of immunohistochemically stained slides.