Effects of porcine testis extract on wound healing in rat
Dong-Mok Leea$, Abdul Roouf Bhata$, Yong-Woon Kimb, Dong Hoon Shinc, Joo-Young Kimd, Keuk-Jun Kime, Ki-Ho Leef, Yong-Pil Cheong, Taehoon Chunh and Inho Choia*
a
School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Korea;bDepartment of Physiology, College of Medicine,
Yeungnam University, Daegu, Korea;cDepartment of Dermatology, College of Medicine, Yeungnam University, Daegu, Korea;
d
Department of Anatomy, College of Medicine, Yeungnam University, Daegu, Korea;eDepartment of Clinical Pathology, Tae
Kyeung College, Gyeongsan 712-749, Korea;fDepartment of Biochemistry and Molecular Biology and Medical Sciences Research
Institute, Eulji University, Daejeon 301-746, Korea;gDepartment of Biology, Sungshin Women’s University, Seoul 136-742, Korea;
hSchool 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
(CO2) 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
Wound healing is a complex biological process invol-ving temporal interactions among extracellular matrix molecules, soluble factors, and numerous types of cells such as platelets, macrophages, fibroblasts, epithelial cells, and endothelial cells. In addition to the various cellular interactions, healing is also influenced by the action of proteins and glycoproteins such as cytokines, chemokines, growth factors, inhibitors, and their receptors (Stadelmann et al. 1998; Holmdahl and Ivarsson 1999; Rajkumar et al. 2006; Darby and Hewitson 2007; Xu et al. 2010). The wounds are normally healed in an orderly and efficient manner and progress smoothly through four distinct phases: hemostasis, inflammation, proliferation, and remodel-ing (Kumara Swamy et al. 2007). Improved wound care is the subject of intensive research. A number of organic, inorganic, and natural methods have been employed for centuries to aid the healing process. Steroids in the form of bile acids, adrenal, and sex hormones are synthesized by our bodies to regulate a vast number of physiological, immune, and metabolic processes. Steroids have been broadly used due to their anti-inflammatory and immunomodulatory properties
(Sibilia 2003; Campagnolo et al. 2008). There are numerous animal and clinical studies of cutaneous wounds, and the positive wound-healing effects of anabolic steroids have been described (Demling and DeSanti 1997; Beiner et al. 1999; Spungen et al. 1999). The effect of anabolic steroids appears due to an increase in tensile strength or re-epithelialization (Pitkow et al. 1988; Nandi et al. 1986; Kowalewski 1996). Increased protein synthesis has been considered as a proposed mechanism of the healing. However, the anabolic process of protein synthesis with new tissue formation requires the action of anabolic hormones (Uehara et al. 1999; Zhang 2002; Demling 2005; Gaillard et al. 2008; Miller and Wolf 2008). Anabolic steroids exert their effect when bound to androgen receptors with activation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerase, lead-ing to increase in protein synthesis in skeletal muscle (Carson-Jurica and Schrader 1990; Demling 2000). We have reported on the abundance of the natural anabolic hormone 19-nortestosterone in the blood and in some tissues such as the testes of piglets (Choi et al. 2007). The decanoate ester of 19-nortestosterone is an effec-tive drug for muscle growth, appetite stimulation, and treatment of anemia and osteoporosis. Clinical studies
*Corresponding author. Email: inhochoi@ynu.ac.kr $The authors equally contributed to this work.
DEV ELO PME N TA L BIO L OG Y
Vol. 16, No. 6, December 2012, 469478
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
have shown it to be effective in treating neoplasia including breast cancer and its efficacy as a progestin-based contraceptive.
By understanding the direct or indirect relationship of anabolic steroids with healing process, we developed a strategy to deduce the effect of porcine testis steroid extract (PTSE), which has high content of sex steroids, on cutaneous wounds generated by the skin punching and laser radiation in a rat wound model. In addition to the natural and nontoxic behavior of extract, use of waste byproduct of meat industry in a useful way is an important significance of this study.
Methods
Porcine testes were collected from a local slaughter house (Samse, Yeong Cheon, Korea). The solvents used for extraction were purchased from Sigma-Aldrich (St. Louis, MO, USA) and all were of analytical grade. Enzyme-linked immunosorbent assay kits for analyzing steroid concentrations were purchased from DRG Instruments GmbH (Marburg, Germany). The cream base was provided by MILAE Resources (Seoul, Korea). Animal handling was done according to the approved protocols of Yeungnam University Medical College Animal Studies. One hundred and twenty male Sprague-Dawley rats weighing 55095 g and 10 weeks old were used in this study. The rats were caged separately and housed in 23928C with humidity maintained at 55910%. The rats were provided with chow diet and water ad libitum.
For the confirmation of type I collagen distribu-tions in the dermis of the rats’ skin, goat serum, rabbit anti-collagen I antibody (primary antibody), goat polyclonal secondary antibody to rabbit IgG-H & L (HRP), and DAB substrate kit were purchased from Abcam (Cambridge, MA, USA). The primary anti-body has cross reactivity with the mouse, rat, horse, cow, and human.
Extraction of PTSE
The tissue was immersed in 100 ml of chloroform/ methanol (50/50 v/v) per 10 g of tissue, homogenized for five minutes, and filtered through Whatman No. 2 paper. The filtrate was washed with equal volume of 0.9% NaCl. The hydrophobic phase was dried in vacuo using an EYELA Rotary Evaporator (Rikakikai, Tokyo, Japan). The dried material was re-dissolved in an equal volume of 85% ethanol and 5 M NaOH and incubated at 808C for 45 minutes. After cooling, the pH was adjusted to 23 by the addition of 6N HCl. The solution was treated with ether, and the ether layer was collected and dried in vacuo.
PTSE cream preparation
Polysorbate (15 g) was dissolved in 200 g of water. PTSE (0.07 g) was solubilized at 808C. NaH2PO4(5 g)
and disodium edentate (0.5 g) were mixed in the solution and refluxed at 708C. A mixture of stearyl alcohol (4% w/v), octyl alcohol (4% w/v), olive wax (1.7% w/v), isopropyl myristate (6.8% w/v), and propylene glycol (3.6% w/v) were added to the refluxed solution. Distilled water was added to make a final volume of 1 kg solution. The mixture was agitated at 10,000 rpm to obtain the final cream. This cream was 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 absence of PTSE was prepared as the control.
In vivo analysis Wound treatment
Sixty rats were divided into two groups: control group (group 1) and experimental group (group 2) (n 30 per group). Four full-thickness skin wounds were prepared by biopsy punch skin punches (diameter 4 mm and 15 mm) on the dorsum of each rat under aseptic conditions. The rats were anesthetized with 30 mg/kg intramuscular injection of Zoletil 50 (Virba Animal Health, Seoul, Korea). The skin over the dorsal area was completely shaved before punching. The cream without PTSE was applied to the control group, and the PTSE cream was applied to the experimental group. Each rat was supplemented with 1 gm of cream twice a day up to 21 days. Before the application of creams, wounds were washed three times with sterile saline solution. Both groups were scrutinized for a maximum of 21 days after application of the creams. Body weight was measured daily, and symptoms of skin irritation were monitored daily. Blood samples were collected from the heart of each rat on day 0, 7, 14, and 21 of the treatment for measurements of WBCs and hemoglobin.
Laser burns
Another set of experiments were performed to find the effect of PTSE cream on burns induced by laser radiation. Sixty rats were divided into three groups: a
Table 1. Hormone concentration per gram of PTSE cream.
Steroid PTSE Cream (ng/g)
19-nortestosterone (Nandrolone) 70
Testosterone 28
Androstenedione 2
17ß-Estradiol 9
control group, an experimental group, and a group treated with Vaseline (as control). The laser burns were introduced in the 10-week-old rats with a measured area of 4 cm2. Four sites were selected on the back of each rat and de-epithelialization was performed using a CO2 laser with flashscanner (Sharplan 40C
SilkTouchTM; Lasser Labs, Tampa, FL, USA). A 2
2-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. After the injury, the rats were divided into three groups. First group was treated with control cream, the second group with Vaseline, and the third group with PTSE cream. Each group of animals was treated for 21 days. After the treatment with corresponding creams and Vaseline, one rat from each group was anesthetized on days 3, 7, 14 and 21, and 5-mm sized biopsy samples were obtained, embedded in paraffin, and stained for histological studies (Ross et al. 1997).
Wound size measurement
A scanscope CS system (Aperio, Vista, CA, USA) was used to measure the wound size at different time intervals. Each rat was sacrificed, and the skin area under investigation was collected from the rat. The skin was stained with hematoxylin and eosin (H&E) and analyzed using the scanscope to measure the size of epidermis, dermis, and hypodermis.
Tissue processing, histological analysis, and immunohistochemistry (IHC)
On days 3, 7, 14, and 21 following application of the control or PTSE-containing cream, one rat from each control and experimental group was sacrificed and skin samples were collected. The central portion of each sample was taken and fixed with 10% buffered formalin embedded in paraffin blocks. The 4-mm thick sections were prepared and H&E stained to evaluate the recuperative percentage of the wound. Revitalization of the collagen fibers of the dermis of wound were confirmed by Masson trichrome staining. Therapeutic effects on the four distinct layers of wounded area were recorded by light microscopy at 20 magnification and analyzed by pixel-based CellSens software (Olympus, Tokyo, Japan). Similar experiments were repeated with the three groups of animals treated for the laser-induced injuries. The immuno-staining procedures of paraffin sections for investigating the distributions of the type I collagen of the dermis were followed by the IHC-paraffin protocol provided with Abcam (Cambridge, MA, USA). These sections were counter-stained by Harris hematoxylin.
Statistical analysis
All values represent the mean9SEM. The means were compared by Tukey’s Studentized Range (HSD) to detect significant differences at p B0.05, pB0.001, and p B0.0001. All analyses were performed with the SAS software package, ver. 9.0 (SAS Institute INC., Cary, NC, USA).
Results
The method used for extraction of a steroidal mixture from the testes was similar to that described previously (Lee et al. 2010). Two creams were prepared using the same composition, one with PTSE and another without PTSE (control cream). The PTSE cream was analyzed for hormones, and the presence of different concentra-tions of 19-nortestosterone (nandrolone), testosterone, 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 other steroidal hormones (Table 1).
WBC counts were measured for the analysis of cell toxicity following the application of PTSE cream. Blood samples collected at different times were tested; no significant change was observed in the WBC count of PTSE-treated rats compared to the rats treated with the control cream. According to the World Health Organization toxicity criterion, WBC level differs in toxic conditions (Doroshow et al. 1990). All the animals were carefully observed for physical para-meters like body weight, food consumption, eating disorder, appetite, emotional and mental state disor-ders, skin irritation, and rashes due to allergy. Abrupt decrease in body weight shortly after the injury was attributed to the trauma experienced by the rats. This was consistent with the observations of weight regain after the first week. Experimental rats treated with the PTSE cream regained weight markedly faster than the rats treated with control cream (Figure 1). Skin rashes were absent in both groups. These findings suggested that PTSE was nontoxic and safe for topical applica-tion. Wound area inflammation was compared; in rats treated with PTSE, inflammation resolved within the first three days, while inflammation was still present in control animals. Persistent inflammation is a major factor that interferes with collagen formation and deposition. As soon as the inflammation ends, cell proliferation phase begins, which results in a high deposition of fibroblasts, collagen, epithelial cells, and endothelial cells. In animals treated with PTSE cream, the faster resolution of inflammation could be the key regulatory mechanism behind the faster recovery of
epidermis, dermis, and hypodermis in the first week of treatment (Table 2).
Figures 1AC depicts the size of wound (expressed as percentage of initial wound size) before and after treatment using PTSE and control cream. The size of wound was smaller when treated with the PTSE cream (Figure 1C) than the control cream (Figure 1B). Table 2 represents the data on the percentage of healing area on treatment days 7, 14, and 21. The percentile healing area of the epidermis, dermis, and hypodermis of the two animal groups showed a great difference
when compared on day 7. The data were found statically significant. Epidermal measurements indi-cated 67 times faster recovery in rats treated with PTSE cream than in those treated with the control cream. Similarly, there was a marked increase in the thickness of both the dermis and epidermis in the PTSE-treated group.
However, differences in the epidermal recovery of the two groups became progressively similar with time. This may have reflected a self-healing characteristic of the rats, which aided the recovery of the control animals. But, comparative data for the dermis and hypodermis revealed a fast recovery in rats treated with PTSE cream.
The data of the histological examinations of wounds from all rats on days 7, 15, and 21 of treatment are presented in Figures 2 and 3. Masson trichrome stain was used to determine the collagen formation or tensile strength of the reconstructed tissue. Compared to PTSE-treated animals, unevenness of epidermis near the wound, decrease in collagen, and higher inflamma-tion was found in control animals. Masson trichrome stains collagen and yields a blue color, while the red color denotes the cytoplasm, red blood cells, and muscle. The pattern of staining intensity corresponds to the relative quantity of collagen fiber deposit, which
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.
Table 2. Effect of porcine testis steroid extract on epidermal, dermal, and hypodermal tissue regeneration.
Group Epidermis (%) Dermis (%) Hypodermis (%)
Day 7 Control 4.591.2 3.691.3 1.790.6 PTSE 27.894.2 29.395.9 25.497.0 Day 14 Control 75.693.4 58.792.3 40.997.1 PTSE 83.094.6 71.199.2 57.398.7 Day 21 Control 99.591.2 65.395.1 54.297.0 PTSE 100.090.0 87.494.8 80.592.5
changes in the processes of synthesis, degradation, and remodeling of the lesions. Presently, the density of blue color increased with the posttreatment days and was denser in PTSE-treated rats than in the control rats (Figure 3). These results demonstrated that collagen formation and, ultimately, tensile strength was higher in PTSE-treated animals than the control rats.
Another set of rats were used to find the effect of PTSE cream on the burns or injury induced by the laser treatment. Rats were treated with PTSE cream, control cream, and Vaseline to discern the effect of PTSE on the injury (Ross et al. 1997). Figures 4AD provides the quantitative data concerning the effect of the treat-ments. The three groups of rats were monitored daily for the recovery. Figure 5A illustrates the change in epidermis, dermis, and hypodermis laser burns due to the application of PTSE cream, control cream, and Vaseline. After seven days of topical application of the cream, ulcer formation was observed in the control group, and early stage of re-epithelialization was
evident in the Vaseline-treated group, whereas re-epithelialization followed by the formation of serum crust was noted in the PTSE group. The treatment was continued and on day 14, one rat from each group was again anesthetized; samples were collected and stained for histological examination. In the control group, beginning of serum crust formation was observed. In the Vaseline-treated group, although re-epithelialization as well as serum crust formation was observed, a cleft was found in the dermal-epidermal (D-E) junction. In the PTSE-treated group, generally re-epithelialization and normal skin structure was observed. The treatment was continued and on day 21, the re-epithelialization phase begins in the control rats treated with the base cream. Similarly, serum crust formation was complete in the control group treated with the Vaseline. However, the re-epithelialization phase continued with the D-E junction cleft was still present. In the PTSE cream-treated animals, intact skin structure was observed. The collagen content was further measured by IHC.
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).
Type I collagens were stained with brown color by IHC. They were much more abundant in the reticular layer of the dermis than the papillary layer. Compared with control cream groups (without PTSE), the stain-ing intensities of PTSE cream groups were relatively stronger. The staining intensities of PTSE-treated groups were much stronger than control groups, especially, on day 21. Blue-colored structures such as epidermis, hair follicles, and fibroblasts counter stained by Harris hematoxylin were easily observed in PTSE-treated groups (Figure 5B).
Discussion
The restoration/regeneration of the wound healing is directly proportional to improvement in the net protein synthesis. This depends on two important factors: one, attenuation of the catabolic hormonal response to the
injury. Anticortisol property of anabolic steroids de-creases the catabolic response of cortisol without altering its protective anti-inflammatory response (Ziegler and Wilmore 1994; Biols et al. 1997; Saito 1998; Wray et al. 2002). Second process is the accent-uation of anabolic activity. Human growth hormone, insulin-like growth factor 1 (IGF-1), insulin, and testosterone (and its analogue, anabolic steroids) are four major anabolic hormones that indirectly or directly affect wound healing (Fleming et al. 1992; Woerman et al. 1992; Demling and Orgill 2000). Although each of them has specific mode of action, they bear a considerable interrelationship.
PTSE, an extract of porcine testes holds a good ratio of anabolic steroids viz 19-nortestosterone, tes-tosterone, androstenedione, 17b-estradiol, and estrone. These sex hormones are known to play an important role in bone turnover rate, periodontal diseases, and
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.
wound healing. Anabolic hormones are necessary to maintain the necessary protein synthesis required for maintaining lean body mass including wound healing, assuming the presence of adequate protein intake (Forbes 1985). However, endogenous levels of these hormones are decreased in acute and chronic illness and with increasing age, especially in the presence of a large wound. Anabolic steroids have direct effect on the regulation of growth factors like IGF-1(19) and transforming growth factor beta TGF-b (Falanga et al. 1998). Growth factors play an important role in cell division, migration, differentiation, protein expres-sion, and enzyme production. They also have a potential ability to heal wounds by stimulating angio-genesis and cellular proliferation, affecting the produc-tion and degradaproduc-tion of the extracellular matrix, and by their chemotactic activity for inflammatory cells and fibroblasts (Roberts 1995; O’Kane and Ferguson 1997). Topical application of a mixture of various growth factors that included platelet-derived growth factor, TGF-b, platelet-derived angiogenesis factor, platelet factor 4, and platelet-derived epidermal growth factor had shown positive wound-healing effects over controls
(Knighton and Fiegel 1993). Collagen is one of the abundant proteins in animals, and collagen fiber forms a major component of connective tissues. The enzyme lysyl oxidase acts on the collagen to form stable cross-links. As the collagen becomes older and matures, increasingly more of these intramolecular and inter-molecular cross-links develop in the molecule. These stable cross-linkage steps maintain collagen strength and stability and are measured as the tensile strength of the tissue. Thus, collagen provides the tensile strength of the healed area in the animals. Tennenbaum and Skhlar (1970) reported an increase in the synthesis of bone, collagen, matrix, and epidermis in a standardized wound of the oral cavity using anabolic steroids. Several studies reported an increase in wound tensile strength as a measure of healing, which depended upon the matrix deposition, cell migration, and collagen deposition (Sharath et al. 2009).
The anabolic steroids are known to have direct effect on the wound healing, and the mode of action is believed to be through activation of the androgenic receptors present in higher concentrations in myocytes and skin fibroblasts. Some populations of epithelial
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.
cells also contain these receptors. The stimulation of androgenic receptors results in decrease in efflux of amino acids and an increase in influx into the cells. Activation of intracellular DNA and DNA polymerase and decrease in fat mass occurs due to the activation of androgenic receptors. All anabolic steroids have andro-genic or masculinizing effect and are known to increase the overall protein synthesis and new tissue formation. Demling has found the use of oxandrolone, an anabolic steroid that enhances the healing of a cutaneous wound in the rat (Demling 2000).
In this study, we explored the use of PTSE on wounds caused by the skin puncture and laser light. Wounds treated with PTSE cream showed faster recovery than controltreated wounds, at the epider-mal, derepider-mal, and hypodermal levels. The collagen level in the tissue of animals treated with PTSE cream was
high, showing increased tensile strength. The WBC count of rats treated with the PTSE cream was found in accordance with the control- or Vaseline-treated ani-mals, which indicates the nontoxic nature of the PTSE cream. In previous studies, results showed that a chronic use of synthetic anabolic steroids produced side effects (Kuhn 2002). However, while using PTSE in the rat model, we have not found any serious side effects. This suggests that either the pig have evolved and developed a body system that can avoid the side effects that are caused by high amounts of endogen-ously synthesized anabolic steroids or a combination of several anabolic steroids naturally found in the pig somehow prevents these side effects. Our current research is focused on delineating the molecular mechanism by which the PTSE enhanced the wound healing in the rat.
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.
Acknowledgements
This study was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agricul-ture, Forest and Fisheries), Ministry for Food, AgriculAgricul-ture, Forest and Fisheries, Republic of Korea.
References
Beiner JM, Joki P, Cholewicki J, Panjabi MM. 1999. The effect of anabolic steroids and corticosteroids on healing of muscle contusion injury. Am J Sports Med. 27(1):29. Biols G, Toigo G, Ciocechi B. 1997. Metabolic response to injury and sepsis: changes in protein metabolism. Nutri-tion. 13(Suppl. 9):5257.
Campagnolo AM, Tsuji DH, Sennes LU, Imamura R. 2008. Steroid injection in chronic inflammatory vocal fold disorders, literature review. Rev Bras Otorrinolaringol. 74:926932.
Carson-Jurica M, Schrader W. 1990. Steroid receptor family; structure and functions. Endocrine Rev. 11(2):20220. Choi NJ, Hyun JH, Choi JM, Lee EJ, Cho KH, Kim Y,
Chang J, Chung B, Chung CS, Choi I. 2007. Testicular expression of steroidogenic enzyme genes is related to a transient increase in serum 19-nortestosterone during neonatal development in pigs. Asian-Aust J Anim Sci. 20(12):18321842.
Darby IA, Hewitson TD. 2007. Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol. 257:143179. Demling RH. 2000. Oxandrolone, an anabolic steroid, enhances the healing of a cutaneous wound in the rat. Wound Repair Regen. 8(2):97102.
Demling RH. 2005. The role of anabolic hormones for wound healing to catabolic states. J Burns Wounds. 4:4662.
Demling RH, DeSanti L. 1997. Oxandrolone, an anabolic steroid, significantly increases the rate of weight gain in the recovery phase after major bums. J Trauma. 43(1): 4751.
Demling R, Orgill D. 2000. The anticatabolic and wound healing effects of the testosterone analog, oxandrolone, after severe burn injury. J Crit Care Med. 15(1):1218. Doroshow JH, Multhauf P, Leong L, Margolin K, Litchfield
T, Akman S, Carr B, Bertrand M, Goldberg D, Blayney D. 1990. Prospective randomized comparison of fluor-ouracil versus fluorfluor-ouracil and high-dose continuous infusion leucovorin calcium for the treatment of ad-vanced measurable colorectal cancer in patients pre-viously unexposed to chemotherapy. J Clin Oncol. 8(3):491501.
Falanga V, Greenberg AS, Zhou L, Ochoa SM, Roberts AB, Falabella A, Yamaguchi Y. 1998. Stimulation of collagen ynthesis by the anabolic steroid stanozolol. J Investigative Dermatology. 111(6):11931197.
Fleming R, Rutan R, Jahoor F. 1992. Effect of recombinant human growth hormone in catabolic hormones and free fatty acids following thermal injury. J Trauma. 698(6):703707.
Forbes GB. 1985. The effect of anabolic steroids on lean body mass: the dose response curve. Metabolism. 34(6): 571573.
Gaillard C, Alix E, Boirie Y, Berrut G, Ritz P. 2008. Are elderly hospitalized patients getting enough protein? J Am Geriatr Soc. 56(6):10451049.
Holmdahl L, Ivarsson ML. 1999. The role of cytokines, coagulation, and fibrinolysis in peritoneal tissue repair. Eur J Surg. 165(11):10121019.
Knighton DR, Fiegel VD. 1993. Growth factors and comprehensive surgical care of diabetic wounds. Curr Opin Gen Surg. 3239.
Kowalewski K. 1996. Effect of anabolic steroids upon various fractions of tissue hydroxyproline in cortisone treated rats. Acta Endocrinol. 53(1):7378.
Kuhn CM. 2002. Anabolic steroids. Recent Prog Horm Res. 57:411434.
Kumara Swamy HM, Krishna V, Shankar murthy K, Abdul Rahiman B, Mankani KL, Mahadevan KM, Harish BG, Rajanaika H. 2007. Wound healing activity of embelin isolated from the ethanol extract of leaves of Embelia ribes Burm. J Ethnopharm. 109(3):529534.
Lee DM, Lee KH, Cheon YP, Chun T, Choi I. 2010. Effect of anabolic steroids of pork on proliferation and differentia-tion of myogenic satellite cell. Korean J Food Sci Ani Resour. 30(5):845850.
Miller S, Wolf RR. 2008. The dangers of weight loss in the elderly. J. Nutr Health Aging. 12(7):487491.
Nandi N, Mukheryer S, Chandra X. 1986. Role of anabolic steroid in clean surgical wounds. J Indian Med Assoc. 84(4):116118.
O’Kane S, Ferguson MW. 1997. Transforming growth factor bs and wound healing. Int J Biochem Cell Biol. 29(1): 6378.
Park JH, Choi SC, Lee JH, Lee DY. 2010. Recipient site preparation for epidermal grafting using carbon dioxide Silktouch laser. Clin Exp Dermatol. 35(4):443444. Pitkow H, Kess A, Kressler M, Johnson P. 1988. The effect of
anabolic steroids on wound healing in lower extreamly skeletal muscles of normal and diabetic rats. Med Sci Res. 16:993997.
Rajkumar VS, Shiwen X, Bostrom M, Leoni P, Muddle J, Ivarsson M, Gerdin B, Denton CP, Bou-Gharios G, Black CM, et al. 2006. Platelet-derived growth factor-beta receptor activation is essential for fibroblast and
pericyte recruitment during cutaneous wound healing. Am J Pathol. 169(6):22542265.
Roberts AB. 1995. Transforming growth factor-b: activity and efficacy in animal models of wound healing. Wound Repair Regen. 3(4):408418.
Ross EV, Grossman MC, Duke D, Grevelink JM. 1997. Long-term results after CO2 laser skin resurfacing: a comparison of scanned and pulsed systems. J Am Acad Dermatol. 37(5 Pt 1):709718.
Saito H. 1998. Anabolic agents in trauma and sepsis reflecting body mass and function. Nutrition. 17(6): 554556.
Sharath R, Harish BG, Krishna V, Sathyanarayana BN, Swamy HMK. 2009. Wound healing and protease inhibition activity of Bacosides-A, isolated from Bacopa monnieri wettest. Phytother Res. 24(8):12171222. Sibilia J. 2003. Corticosteroids and inflammation. Rev Prat.
53(5):495501.
Spungen AM, Rasul M, Koehler KM, Cytryn AS, Bauman WA. 1999. Effect of anabolic steroid therapy on healing of long-standing pressure ulcers: nine case reports in patients with spinal cord injury. Presented at the Amer-ican Spinal Injury Assocation meeting, Atlanta, GA. Stadelmann WK, Digenis AG, Tobin GR. 1998. Physiology
and healing dynamics of chronic cutaneous wounds. Am J Surg. 176(Suppl. 2A):26S38S.
Tennenbaum R, Skhlar G. 1970. Effect of anabolic steroids on the healing of experimental extraction wounds. Oral Med Oral Path. 30(6):824834.
Uehara M, Plank LD, Hill G. 1999. Components of energy expenditure in patients with severe sepsis and major trauma: a basis for clinical care. Crit Care Med. 27(7):12951302.
Woerman H, Strack R, de Boer H. 1992. Growth hormone
secretion and administration in catabolic patients
with emphasis on the critically ill patient. Neth J Med. 41(56):222244.
Wray CJ, Mammen JMV, Hasselgren P. 2002. Catabolic response to stress and potential benefits of nutrition support. Nutrition. 18(1112):971977.
Xu Z, Xu H, Ploplis VA, Castellino FJ. 2010. Factor VII deficiency impairs cutaneous wound healing in mice. Mol Med. 16(56):167176.
Zhang X. 2002. Anabolic action of insulin on skin wound protein is augmented by exogenous amino acids. Am J Physiol Endocrinol Metab. 282(6):E308E313.
Ziegler T, Wilmore D. 1994. Strategies for attenuating protein-catabolic responses in the critically ill. Am Rev Med. 45:459463.
