DEVELOPMENTAL BIOLOGY Animal Cells and Systems Vol. 16, No. 6, December 2012, 469478 a a b c d e Dong-Mok Lee $, Abdul Roouf Bhat $, Yong-Woon Kim , Dong Hoon Shin , Joo-Young Kim , Keuk-Jun Kim , f g h a Ki-Ho Lee , Yong-Pil Cheon , Taehoon Chun and Inho Choi * a b School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Korea; Department of Physiology, College of Medicine, Yeungnam University, Daegu, Korea; Department of Dermatology, College of Medicine, Yeungnam University, Daegu, Korea; d e Department of Anatomy, College of Medicine, Yeungnam University, Daegu, Korea; Department of Clinical Pathology, Tae Kyeung College, Gyeongsan 712-749, Korea; Department of Biochemistry and Molecular Biology and Medical Sciences Research Institute, Eulji University, Daejeon 301-746, Korea; Department of Biology, Sungshin Women’s University, Seoul 136-742, Korea; School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea (Received 22 June 2012; received in revised form 28 August 2012; accepted 29 August 2012) Sex hormones have long been considered to play an important role in bone turnover rate, periodontal diseases, and wound healing. We have studied the effect of porcine testis steroid extract (PTSE), an extract of porcine testes, which holds a good ratio of 19-nortestosterone (nandrolone), testosterone, androstenedione, 17b-estradiol, and estrone, on the healing rate of a standardized full-thickness linear wound on the back of the rat. Skin punch or carbon dioxide (CO ) laser methods were used to create the deep skin injury in two groups of animals. The animals were treated with the PTSE cream, control cream and Vaseline (control) to find out the effect in re-epithelialization, contraction, and formation of granulation and scar tissues. Histological examination after 21 days showed 100, 87.4, and 80.5% recovery of epidermis, dermis, and hypodermis, respectively in the PTSE-treated animals. Similarly, on the 15th day of treatment, complete healing of intact skin was observed in the PTSE cream-treated animals among the laser radiation group. Even though the beginning of re-epithelialization phase and completion of serum crust formation was also observed in the base cream- and Vaseline-treated animals respectively, the complete healing cycle was observed only in the PTSE-treated group. The white blood cell count in the PTSE-treated group showed that PTSE cream is nontoxic to animals. Keywords: collagen; PTSE; skin injuries; wound healing Introduction (Sibilia 2003; Campagnolo et al. 2008). There are numerous animal and clinical studies of cutaneous Wound healing is a complex biological process invol- wounds, and the positive wound-healing effects of ving temporal interactions among extracellular matrix anabolic steroids have been described (Demling and molecules, soluble factors, and numerous types of cells DeSanti 1997; Beiner et al. 1999; Spungen et al. 1999). such as platelets, macrophages, fibroblasts, epithelial The effect of anabolic steroids appears due to an cells, and endothelial cells. In addition to the various increase in tensile strength or re-epithelialization cellular interactions, healing is also influenced by the (Pitkow et al. 1988; Nandi et al. 1986; Kowalewski action of proteins and glycoproteins such as cytokines, 1996). Increased protein synthesis has been considered chemokines, growth factors, inhibitors, and their as a proposed mechanism of the healing. However, the receptors (Stadelmann et al. 1998; Holmdahl and anabolic process of protein synthesis with new tissue Ivarsson 1999; Rajkumar et al. 2006; Darby and formation requires the action of anabolic hormones Hewitson 2007; Xu et al. 2010). The wounds are (Uehara et al. 1999; Zhang 2002; Demling 2005; normally healed in an orderly and efficient manner Gaillard et al. 2008; Miller and Wolf 2008). Anabolic and progress smoothly through four distinct phases: steroids exert their effect when bound to androgen hemostasis, inflammation, proliferation, and remodel- receptors with activation of deoxyribonucleic acid ing (Kumara Swamy et al. 2007). Improved wound care (DNA) and ribonucleic acid (RNA) polymerase, lead- is the subject of intensive research. A number of ing to increase in protein synthesis in skeletal muscle organic, inorganic, and natural methods have been (Carson-Jurica and Schrader 1990; Demling 2000). We employed for centuries to aid the healing process. have reported on the abundance of the natural anabolic Steroids in the form of bile acids, adrenal, and sex hormone 19-nortestosterone in the blood and in some hormones are synthesized by our bodies to regulate a tissues such as the testes of piglets (Choi et al. 2007). vast number of physiological, immune, and metabolic The decanoate ester of 19-nortestosterone is an effec- processes. Steroids have been broadly used due to their tive drug for muscle growth, appetite stimulation, and anti-inflammatory and immunomodulatory properties treatment of anemia and osteoporosis. Clinical studies *Corresponding author. Email: firstname.lastname@example.org $The authors equally contributed to this work. ISSN 1976-8354 print/ISSN 2151-2485 online # 2012 Korean Society for Integrative Biology http://dx.doi.org/10.1080/19768354.2012.726645 http://www.tandfonline.com 470 D.-M. Lee et al. have shown it to be effective in treating neoplasia PTSE cream preparation including breast cancer and its efficacy as a progestin- Polysorbate (15 g) was dissolved in 200 g of water. based contraceptive. PTSE (0.07 g) was solubilized at 808C. NaH PO (5 g) 2 4 By understanding the direct or indirect relationship and disodium edentate (0.5 g) were mixed in the of anabolic steroids with healing process, we developed solution and refluxed at 708C. A mixture of stearyl a strategy to deduce the effect of porcine testis steroid alcohol (4% w/v), octyl alcohol (4% w/v), olive wax extract (PTSE), which has high content of sex steroids, (1.7% w/v), isopropyl myristate (6.8% w/v), and on cutaneous wounds generated by the skin punching propylene glycol (3.6% w/v) were added to the refluxed and laser radiation in a rat wound model. In addition solution. Distilled water was added to make a final to the natural and nontoxic behavior of extract, use of volume of 1 kg solution. The mixture was agitated at waste byproduct of meat industry in a useful way is an 10,000 rpm to obtain the final cream. This cream was important significance of this study. subjected to the hormone analysis using the steroidal hormone kits; the results are summarized in Table 1. Another cream of similar composition except for the Methods absence of PTSE was prepared as the control. Porcine testes were collected from a local slaughter house (Samse, Yeong Cheon, Korea). The solvents used In vivo analysis for extraction were purchased from Sigma-Aldrich (St. Wound treatment Louis, MO, USA) and all were of analytical grade. Enzyme-linked immunosorbent assay kits for analyzing Sixty rats were divided into two groups: control group steroid concentrations were purchased from DRG (group 1) and experimental group (group 2) (n30 per Instruments GmbH (Marburg, Germany). The cream group). Four full-thickness skin wounds were prepared base was provided by MILAE Resources (Seoul, by biopsy punch skin punches (diameter 4 mm and Korea). Animal handling was done according to the 15 mm) on the dorsum of each rat under aseptic approved protocols of Yeungnam University Medical conditions. The rats were anesthetized with 30 mg/kg College Animal Studies. One hundred and twenty male intramuscular injection of Zoletil 50 (Virba Animal Sprague-Dawley rats weighing 55095 g and 10 weeks Health, Seoul, Korea). The skin over the dorsal area old were used in this study. The rats were caged was completely shaved before punching. The cream separately and housed in 23928C with humidity without PTSE was applied to the control group, and maintained at 55910%. The rats were provided with the PTSE cream was applied to the experimental group. chow diet and water ad libitum. Each rat was supplemented with 1 gm of cream twice a For the confirmation of type I collagen distribu- day up to 21 days. Before the application of creams, tions in the dermis of the rats’ skin, goat serum, rabbit wounds were washed three times with sterile saline anti-collagen I antibody (primary antibody), goat solution. Both groups were scrutinized for a maximum polyclonal secondary antibody to rabbit IgG-H & L of 21 days after application of the creams. Body weight (HRP), and DAB substrate kit were purchased from was measured daily, and symptoms of skin irritation Abcam (Cambridge, MA, USA). The primary anti- were monitored daily. Blood samples were collected body has cross reactivity with the mouse, rat, horse, from the heart of each rat on day 0, 7, 14, and 21 of the cow, and human. treatment for measurements of WBCs and hemoglobin. Laser burns Extraction of PTSE Another set of experiments were performed to find the The tissue was immersed in 100 ml of chloroform/ effect of PTSE cream on burns induced by laser methanol (50/50 v/v) per 10 g of tissue, homogenized radiation. Sixty rats were divided into three groups: a for five minutes, and filtered through Whatman No. 2 paper. The filtrate was washed with equal volume of Table 1. Hormone concentration per gram of PTSE cream. 0.9% NaCl. The hydrophobic phase was dried in vacuo using an EYELA Rotary Evaporator (Rikakikai, Steroid PTSE Cream (ng/g) Tokyo, Japan). The dried material was re-dissolved in 19-nortestosterone (Nandrolone) 70 an equal volume of 85% ethanol and 5 M NaOH and Testosterone 28 incubated at 808C for 45 minutes. After cooling, the pH Androstenedione 2 was adjusted to 23 by the addition of 6N HCl. The 17ß-Estradiol 9 solution was treated with ether, and the ether layer was Estrone 2 collected and dried in vacuo. Animal Cells and Systems 471 control group, an experimental group, and a group Statistical analysis treated with Vaseline (as control). The laser burns were All values represent the mean9SEM. The means were introduced in the 10-week-old rats with a measured compared by Tukey’s Studentized Range (HSD) to area of 4 cm . Four sites were selected on the back detect significant differences at pB0.05, pB0.001, and of each rat and de-epithelialization was performed pB0.0001. All analyses were performed with the SAS using a CO laser with flashscanner (Sharplan 40C software package, ver. 9.0 (SAS Institute INC., Cary, TM SilkTouch ; Lasser Labs, Tampa, FL, USA). A 22- NC, USA). cm square-shaped wound was made with the 4 mm spot diameter probe (F200) using 25 W power for 0.2 s (Park et al. 2010). The process was repeated five times. Results After the injury, the rats were divided into three groups. The method used for extraction of a steroidal mixture First group was treated with control cream, the second from the testes was similar to that described previously group with Vaseline, and the third group with PTSE cream. Each group of animals was treated for 21 days. (Lee et al. 2010). Two creams were prepared using the After the treatment with corresponding creams and same composition, one with PTSE and another without Vaseline, one rat from each group was anesthetized on PTSE (control cream). The PTSE cream was analyzed days 3, 7, 14 and 21, and 5-mm sized biopsy samples for hormones, and the presence of different concentra- were obtained, embedded in paraffin, and stained for tions of 19-nortestosterone (nandrolone), testosterone, histological studies (Ross et al. 1997). androstenedione, progesterone, 17b-estradiol, and es- trone was confirmed. Porcine testes are distinguished by their unique possession of nandrolone, which is also present in the highest concentration compared to the Wound size measurement other steroidal hormones (Table 1). A scanscope CS system (Aperio, Vista, CA, USA) was WBC counts were measured for the analysis of cell used to measure the wound size at different time toxicity following the application of PTSE cream. intervals. Each rat was sacrificed, and the skin area Blood samples collected at different times were tested; under investigation was collected from the rat. The skin no significant change was observed in the WBC count was stained with hematoxylin and eosin (H&E) and of PTSE-treated rats compared to the rats treated with analyzed using the scanscope to measure the size of the control cream. According to the World Health epidermis, dermis, and hypodermis. Organization toxicity criterion, WBC level differs in toxic conditions (Doroshow et al. 1990). All the animals were carefully observed for physical para- Tissue processing, histological analysis, and meters like body weight, food consumption, eating immunohistochemistry (IHC) disorder, appetite, emotional and mental state disor- On days 3, 7, 14, and 21 following application of the ders, skin irritation, and rashes due to allergy. Abrupt control or PTSE-containing cream, one rat from each decrease in body weight shortly after the injury was control and experimental group was sacrificed and skin attributed to the trauma experienced by the rats. This samples were collected. The central portion of each was consistent with the observations of weight regain sample was taken and fixed with 10% buffered formalin after the first week. Experimental rats treated with the embedded in paraffin blocks. The 4-mm thick sections PTSE cream regained weight markedly faster than the were prepared and H&E stained to evaluate the rats treated with control cream (Figure 1). Skin rashes recuperative percentage of the wound. Revitalization were absent in both groups. These findings suggested of the collagen fibers of the dermis of wound were that PTSE was nontoxic and safe for topical applica- confirmed by Masson trichrome staining. Therapeutic tion. Wound area inflammation was compared; in rats effects on the four distinct layers of wounded area were treated with PTSE, inflammation resolved within the recorded by light microscopy at 20magnification and first three days, while inflammation was still present in analyzed by pixel-based CellSens software (Olympus, control animals. Persistent inflammation is a major Tokyo, Japan). Similar experiments were repeated factor that interferes with collagen formation and with the three groups of animals treated for the laser- deposition. As soon as the inflammation ends, cell induced injuries. The immuno-staining procedures of proliferation phase begins, which results in a high paraffin sections for investigating the distributions deposition of fibroblasts, collagen, epithelial cells, and of the type I collagen of the dermis were followed endothelial cells. In animals treated with PTSE cream, by the IHC-paraffin protocol provided with Abcam the faster resolution of inflammation could be the key (Cambridge, MA, USA). These sections were counter- stained by Harris hematoxylin. regulatory mechanism behind the faster recovery of 472 D.-M. Lee et al. Figure 1. Quantitative analysis of diameter in wound area. (A) Wounds on the back of a rat made by skin punch. (B, C) Posttreatment wound on back of rat on day 21; (B) control (cream base) and (C) PTSE cream. (D) The effect of cream on the WBC count. (E) The comparison of weight change in the rats after the injury. epidermis, dermis, and hypodermis in the first week of when compared on day 7. The data were found treatment (Table 2). statically significant. Epidermal measurements indi- Figures 1AC depicts the size of wound (expressed cated 67 times faster recovery in rats treated with PTSE cream than in those treated with the control as percentage of initial wound size) before and after cream. Similarly, there was a marked increase in the treatment using PTSE and control cream. The size of thickness of both the dermis and epidermis in the wound was smaller when treated with the PTSE PTSE-treated group. cream (Figure 1C) than the control cream (Figure 1B). However, differences in the epidermal recovery of Table 2 represents the data on the percentage of healing the two groups became progressively similar with time. area on treatment days 7, 14, and 21. The percentile This may have reflected a self-healing characteristic of healing area of the epidermis, dermis, and hypodermis the rats, which aided the recovery of the control of the two animal groups showed a great difference animals. But, comparative data for the dermis and hypodermis revealed a fast recovery in rats treated with Table 2. Effect of porcine testis steroid extract on epidermal, PTSE cream. dermal, and hypodermal tissue regeneration. The data of the histological examinations of wounds from all rats on days 7, 15, and 21 of treatment Group Epidermis (%) Dermis (%) Hypodermis (%) are presented in Figures 2 and 3. Masson trichrome stain was used to determine the collagen formation or Day 7 Control 4.591.2 3.691.3 1.790.6 tensile strength of the reconstructed tissue. Compared PTSE 27.894.2 29.395.9 25.497.0 to PTSE-treated animals, unevenness of epidermis near Day 14 the wound, decrease in collagen, and higher inflamma- Control 75.693.4 58.792.3 40.997.1 tion was found in control animals. Masson trichrome PTSE 83.094.6 71.199.2 57.398.7 stains collagen and yields a blue color, while the red Day 21 color denotes the cytoplasm, red blood cells, and Control 99.591.2 65.395.1 54.297.0 muscle. The pattern of staining intensity corresponds PTSE 100.090.0 87.494.8 80.592.5 to the relative quantity of collagen fiber deposit, which Animal Cells and Systems 473 Figure 2. H&E staining. (A) H&E staining used to determine the regeneration of dermis and muscle after the treatment at days 3, 7, and 14. Comparison of the posttreatment diameter of wound dermis (B) and hypodermis (C). changes in the processes of synthesis, degradation, and evident in the Vaseline-treated group, whereas re- remodeling of the lesions. Presently, the density of blue epithelialization followed by the formation of serum color increased with the posttreatment days and was crust was noted in the PTSE group. The treatment was denser in PTSE-treated rats than in the control rats continued and on day 14, one rat from each group was (Figure 3). These results demonstrated that collagen again anesthetized; samples were collected and stained formation and, ultimately, tensile strength was higher for histological examination. In the control group, in PTSE-treated animals than the control rats. beginning of serum crust formation was observed. In Another set of rats were used to find the effect of the Vaseline-treated group, although re-epithelialization PTSE cream on the burns or injury induced by the laser as well as serum crust formation was observed, a cleft treatment. Rats were treated with PTSE cream, control was found in the dermal-epidermal (D-E) junction. In cream, and Vaseline to discern the effect of PTSE on the PTSE-treated group, generally re-epithelialization the injury (Ross et al. 1997). Figures 4AD provides the and normal skin structure was observed. The treatment quantitative data concerning the effect of the treat- was continued and on day 21, the re-epithelialization ments. The three groups of rats were monitored daily phase begins in the control rats treated with the base for the recovery. Figure 5A illustrates the change in cream. Similarly, serum crust formation was complete epidermis, dermis, and hypodermis laser burns due to in the control group treated with the Vaseline. However, the application of PTSE cream, control cream, and the re-epithelialization phase continued with the D-E Vaseline. After seven days of topical application of the junction cleft was still present. In the PTSE cream- cream, ulcer formation was observed in the control treated animals, intact skin structure was observed. group, and early stage of re-epithelialization was The collagen content was further measured by IHC. 474 D.-M. Lee et al. Figure 3. The density of collagen ﬁbers (blue colored in trichrome stain). Trichrome stain used to measure the amount of collagen. The collagen shows a clear difference when measured on days 7, 14, and 21. However, on day 21, the collagen formation (blue color density) is higher in PTSE-treated animals than the control one. Type I collagens were stained with brown color by injury. Anticortisol property of anabolic steroids de- IHC. They were much more abundant in the reticular creases the catabolic response of cortisol without layer of the dermis than the papillary layer. Compared altering its protective anti-inflammatory response with control cream groups (without PTSE), the stain- (Ziegler and Wilmore 1994; Biols et al. 1997; Saito ing intensities of PTSE cream groups were relatively 1998; Wray et al. 2002). Second process is the accent- stronger. The staining intensities of PTSE-treated uation of anabolic activity. Human growth hormone, groups were much stronger than control groups, insulin-like growth factor 1 (IGF-1), insulin, and especially, on day 21. Blue-colored structures such as testosterone (and its analogue, anabolic steroids) are epidermis, hair follicles, and fibroblasts counter stained four major anabolic hormones that indirectly or by Harris hematoxylin were easily observed in PTSE- directly affect wound healing (Fleming et al. 1992; treated groups (Figure 5B). Woerman et al. 1992; Demling and Orgill 2000). Although each of them has specific mode of action, they bear a considerable interrelationship. Discussion PTSE, an extract of porcine testes holds a good ratio of anabolic steroids viz 19-nortestosterone, tes- The restoration/regeneration of the wound healing is tosterone, androstenedione, 17b-estradiol, and estrone. directly proportional to improvement in the net protein synthesis. This depends on two important factors: one, These sex hormones are known to play an important role in bone turnover rate, periodontal diseases, and attenuation of the catabolic hormonal response to the Animal Cells and Systems 475 Figure 4. Quantitative analysis healed laser burn/injury. (A) Wounds on the back of a rat made by laser. (B, C, D) Posttreatment wound on back of rat on day 15; (B) control (cream base), (C) Vaseline, and (D) PTSE cream. wound healing. Anabolic hormones are necessary to (Knighton and Fiegel 1993). Collagen is one of the maintain the necessary protein synthesis required for abundant proteins in animals, and collagen fiber forms maintaining lean body mass including wound healing, a major component of connective tissues. The enzyme assuming the presence of adequate protein intake lysyl oxidase acts on the collagen to form stable cross- (Forbes 1985). However, endogenous levels of these links. As the collagen becomes older and matures, hormones are decreased in acute and chronic illness increasingly more of these intramolecular and inter- and with increasing age, especially in the presence of a molecular cross-links develop in the molecule. These large wound. Anabolic steroids have direct effect on the stable cross-linkage steps maintain collagen strength regulation of growth factors like IGF-1(19) and and stability and are measured as the tensile strength of transforming growth factor beta TGF-b (Falanga the tissue. Thus, collagen provides the tensile strength et al. 1998). Growth factors play an important role in of the healed area in the animals. Tennenbaum and cell division, migration, differentiation, protein expres- Skhlar (1970) reported an increase in the synthesis of sion, and enzyme production. They also have a bone, collagen, matrix, and epidermis in a standardized potential ability to heal wounds by stimulating angio- wound of the oral cavity using anabolic steroids. genesis and cellular proliferation, affecting the produc- Several studies reported an increase in wound tensile tion and degradation of the extracellular matrix, and strength as a measure of healing, which depended upon by their chemotactic activity for inflammatory cells and the matrix deposition, cell migration, and collagen fibroblasts (Roberts 1995; O’Kane and Ferguson 1997). deposition (Sharath et al. 2009). Topical application of a mixture of various growth The anabolic steroids are known to have direct factors that included platelet-derived growth factor, effect on the wound healing, and the mode of action is TGF-b, platelet-derived angiogenesis factor, platelet believed to be through activation of the androgenic factor 4, and platelet-derived epidermal growth factor receptors present in higher concentrations in myocytes had shown positive wound-healing effects over controls and skin fibroblasts. Some populations of epithelial 476 D.-M. Lee et al. Figure 5. H&E staining and immunohistochemistry for type 1 collagen ﬁber showing re-epithelialization with PTSE treatment on skin injury induced by laser radiation. (A) H&E staining used to determine the revitalization of dermis and muscle after the treatment at day 6, 12, and 15. The cream base (control), Vaseline, and PTSE cream was applied to the injured area. SE cream was applied to the injured area. (B) IHC stain used to identify the location of type I collagen ﬁbers. These ﬁbers are located mostly in the reticular layer of the dermis. The intensities of type I collagen of PTSE-treated groups show clear differences when comparing with control groups at days 7, 14, and 21, respectively. The brown-colored collagens are much stronger in PTSE-treated groups than control groups, especially, on day 21. Blue-colored structures counter stained by Harris hematoxylin are easily observed. cells also contain these receptors. The stimulation of high, showing increased tensile strength. The WBC androgenic receptors results in decrease in efflux of count of rats treated with the PTSE cream was found in amino acids and an increase in influx into the cells. accordance with the control- or Vaseline-treated ani- Activation of intracellular DNA and DNA polymerase mals, which indicates the nontoxic nature of the PTSE and decrease in fat mass occurs due to the activation of cream. In previous studies, results showed that a androgenic receptors. All anabolic steroids have andro- chronic use of synthetic anabolic steroids produced side effects (Kuhn 2002). However, while using PTSE in genic or masculinizing effect and are known to increase the overall protein synthesis and new tissue formation. the rat model, we have not found any serious side Demling has found the use of oxandrolone, an anabolic effects. This suggests that either the pig have evolved steroid that enhances the healing of a cutaneous wound and developed a body system that can avoid the side in the rat (Demling 2000). effects that are caused by high amounts of endogen- In this study, we explored the use of PTSE on ously synthesized anabolic steroids or a combination of wounds caused by the skin puncture and laser light. several anabolic steroids naturally found in the pig Wounds treated with PTSE cream showed faster somehow prevents these side effects. 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Animal Cells and Systems
– Taylor & Francis
Published: Dec 1, 2012
Keywords: collagen; PTSE; skin injuries; wound healing