sign in sign up
<<
Home , Honey

The Effects of Different Types of Honey on Tensile Strength Evaluation of Tissue Healing

M.Z. Rozaini, BSca A.B.Z. Zuki, PhD, DVMa M. Noordin, PhD, MSc, DVMa Y. Norimah, PhDb A. Nazrul Hakim, PhD, DVMc aFaculty of Veterinary , University Putra Malaysia, Selangor Darul Ehsan, Malaysia bMalaysian Institute for Nuclear Technology Research (MINT) Bangi, Selangor Darul Ehsan, Malaysia cFaculty of Medicine and Health Sciences, University Putra Malaysia, Selangor Darul Ehsan, Malaysia

KEY WORDS: honey, tensile strength, can improved healing with regards burn wound healing to the tensile strength property.

ABSTRACT INTRODUCTION The effects of different types of honey on One of the most crucial phases in dermal the tensile strength of burn wound tissue wound healing is the progressive increase in healing were evaluated in 105 male biomechanical strength of the tissue result- Sprague-Dawley rats. The rats were random- ing from the formation and turnover of gran- ly divided into 7 groups of 15 rats each. ulation tissue.1 The mechanical properties of Rats were anesthetized and burn wounds the are mainly attributed to the function were created using cylindrical aluminum of the dermis in relation to the structure of templates heated in a waterbath for 3 hours collagen and elastic fiber networks.2 at a constant temperature of 85˚C. Honey Experimental and clinical trials designed harvested from different vegetation (0.5 mL) to influence wound strength or healing rates was applied to wounds of rats in 5 of the have been conducted with mixed success. groups approximately 30 minutes after the Cytokines or growth factors applied systemi- skin was burned. A positive control group cally, topically or more recently, by gene was treated with silver sulfadiazine cream transfer are some such examples.3 Honey has and one group served as untreated controls. also been shown to accelerate wound healing The rats were euthanized 3, 7, 14, 21, and by augmenting the rate of collagen synthesis 28 days after were created. Test areas when applied topically and administered sys- from the skin were removed in strips and a temically.4 In 1993, Suguna and his col- universal testing machine was used to meas- leagues demonstrated that honey accelerated ure the tensile strength. In general, the ten- the synthesis and maturation of collagen, thus sile strength values increased with time. resulting in increased tensile strength of the Tensile strength of skin treated with Manuka wound healing skin.5 Tensile strength has honey was highest throughout the study commonly been associated with the organiza- except on Day 21. The present study showed tion, content, and physical properties of the that topical application of honey on burn collagen fibril network.6 According to

290 Vol. 2, No. 4, 2004 • Intern J Appl Res Vet Med Jimenez and Rampy, 7 tensile strength is one and location of the burn was marked of the necessary parameters for determining between the last ribs and the horizontal line the pharmacologic effects of potential wound of the sacroiliac joints. The rat was posi- healing agents. tioned in sternal recumbency, and a burn was Consequently, the present study was inflicted on the dorsum of the body between designed to correlate the tensile properties the last thoracic vertebra and the first of the burned skin with the healing process. sacrum by placing the heated and moistened The effects of various types of honey on the template at right angles perpendicular to the tensile strength property of the burn wound dorsum of the rat on the pre-marked location tissue healing were evaluated in the study. for 5 seconds using an analogue stopwatch. Minimal and constant pressure was applied MATERIALS AND METHODS to ensure a perfect contact between the tem- Animals plate surface and the skin. The shaved skin One hundred-five male Sprague-Dawley was smooth ensuring sufficient contact and rats weighing 250 to 300 g each were used uniform pressure over the entire lesion. in this study. The rats were kept in the ani- Treatments mal unit at least 1 week prior to initiation of The rats were randomly divided into seven the study. The rats were given commercial groups of 15 animals each. Four selected pellet and water ad libitum throughout the monofloral honeys which pro- study. The protocol was approved by the duced by kept under different planta- Faculty’s Ethics Committee. tion were used in this study.Their floral sources were from Melaleuca spp. (Gelam) Skin Preparation trees, Cocos Nucifera spp. (Kelapa) trees, Rats were anesthetized with an IM injection Ananas Comosus spp. (Nenas) trees and of ketamine (50 mg/kg) and xylazine (5 spp. () trees.The mg/kg). Under anesthesia, the back and flank honeys were named according to their floral of both sides of the body were shaved. sources.All the honeys were supplied by the Following this procedure, rats were returned Department of Agriculture, Malaysia.The to their cages for 24 hours to allow any edema Manuka honey was supplied by ZHR caused by the shaving procedure to recede. Technologies (Sdn. Bhd., Petaling Jaya, Thermal Source Selangor, Malaysia), while silver sulphadi- A method described by Kaufman et al,8 was azine (SSD) cream was purchased from the used with modification. Cylindrical alu- Hospital Kuala Lumpur.All of the honeys minum templates (2.5 cm diameter ? 3 cm and the SSD cream were irradiated at length, a handle measuring 24 cm, and total 25kGy using a sinagama machine (Model weight 400 g) were heated in a waterbath at JS8900; Nordion International Inc., ) a constant temperature of 85˚C for 3 hours for sterilization purposes. prior to inflicting burn areas on the skin of the rats. Five templates were heated simulta- Mode of Treatment neously and used alternately and then were For all the treatment groups except in con- returned to the waterbath to ensure mainte- trol group, the different type of honey in nance of the desired temperature of the tem- quantities of approximately 0.5 mL was plate surface. Approximately 5 minutes applied topically onto the surface of each elapsed between each use of a template. burn wound.A thin layer of SSD cream was Burn Lesions applied on the burn wound in the positive Rats were again anesthetized with an IM control group.The first treatment was injection of ketamine (50 mg/kg) and applied approximately half an hour after xylazine (5 mg/kg). The anesthetized rat was burn infliction. The treatment groups restrained and stretched on a metal stage, received topical application twice a day until

Intern J Appl Res Vet Med • Vol. 2, No. 4, 2004 291 Table 1. Tensile Strength Measurement (MPa) of Healing Wounds* Experimental Days Post Injury Groups 3 7 14 21 28 Control 0.81±0.36a,x 1.35±0.52a,xy 1.47±0.20a,xy 1.97±0.32a,y 2.08±0.70a,y Ananas Comosus 0.95±0.43ab,x 1.25±0.26a,x 2.40±1.03a,xy 2.61±0.67a,xy 3.95±1.89a,y (Nenas) honey Melaleuca 0.93±0.14ab,x 1.55±0.68a,x 1.71±0.26a,xy 2.47±0.31a,yz 2.80±0.49a,z (Gelam) honey Durio Zibethinus 1.28±0.31ab,x 1.61±0.65a,x 2.19±1.29a,x 2.93±1.05a,x 3.56±3.98a,x (Durian) honey Cocos Nucifera 1.12±0.46ab,x 1.24±0.04a,x 1.43±0.91a,x 2.37±1.34a,x 2.54±0.65a,x (Kelapa) honey Manuka honey 1.53±0.24b,x 1.74±0.20a,x 2.73±0.46a,y 2.81±0.31a,y 4.68±0.39a,z SSD 0.86±0.04a,x 1.22±0.33a,x 1.67±1.01a,xy 1.79±0.28a,xy 2.54±0.37a,y *Data of tensile strength (MPa) expressed in mean ± S.D. a-bValues within the same column with different superscript is significant at 0.05 level (P<0.05) due to treatment group. x-zValues within the same row with different superscript is significant at 0.05 level (P<0.05) due to days of post injury. they were euthanized. The negative control being 30 mm.The Instron machine was set group received identical burn and environ- to a constant crosshead speed of 100 mental exposure but no treatment given. mm/min and a data collection speed of 100 Preparation of Strips data points per second.The program was The rats were serially euthanized at 3, 7, 14, started and allowed to run until the sample 21 and 28 days post burned.After euthana- tore.As the specimen elongated, the resist- sia, the skin samples were cut into 6.0 cm x ance of the specimen increased and detected 1.0 cm strips parallel to the long axis of the by a load cell.This load value (Force) was body by using perplex template and sharp recorded by the instrument.The elongation blades. The strips were then placed in a con- of the specimen was continued until a rup- tainer soaked with saline solution to prevent ture of the specimen was observed. drying of the skin. Statistical Analysis Tensile Strength Measurement The results were analyzed statistically using The Instron mechanical testing machine two-way and one-way ANOVA methods to (Model 4301, Instron Corporation, Canton, identify the differences between experimen- Mass) was used to apply tensile stresses on tal groups within the period of study. The the samples. Instructions provided for cali- data were considered significant at P<0.05. brating and balancing the Instron machine prior to each trial were used, that is, the load RESULTS and gauge length were set to zero before The results of the tensile strength measure- each trial was performed. The tensile ment is shown in Table 1 and expressed graph- machine has fixed or essentially stationary ically in Figure 1. In general, tensile strength member, carrying one grip and a movable values increased with time in all the experi- member carrying a second grip. Statistical mental groups.Tensile strength of the animals calculations were performed automatically treated with Manuka honey demonstrated the and presented on a visual display and hard highest value throughout the study except at copy printout at the end of the test. day 21 post burned, and followed by Durio The strips of skin were held into place Zibethinus (Durian) and Ananas Comosus on the machine with pneumatic clamps.The (Nenas)-treated groups as opposed to other samples were positioned vertically into top experimental groups. However, the difference clamps first and then the bottom clamp, between all experimental groups was statisti- with the distance between the two clamps cally not significant except at day 3 post injury

292 Vol. 2, No. 4, 2004 • Intern J Appl Res Vet Med Figure 1. Tensile strength of healing burn wounds for all treatment groups. where the tensile strength of Manuka-treated to break the wound, is an important measure wounds was significantly higher (P<0.05) since it reflects the subdermal organization (1.5393 MPa) compared to untreated control of the collagen fibers in the newly deposited group (0.8177 MPa) and SSD-treated group collage.7 Tensile strength indicates how (0.8623 MPa) with the untreated control group much the repaired tissue resists to break being the lowest value. under tension and may indicate in part the Topical application of Manuka honey on quality of the repaired tissue.9 the burns wound demonstrated a significant Collagen is one of the major components increase in wound tensile strength at day 14 that is mainly responsible for the mechanical (2.7333 MPa) and day 28 (4.6860 MPa) properties of the skin.10 The net amount of post burned as compared to other groups. wound collagen deposition depends on colla- The increment of tensile strength value of gen turnover and is a reflection of collagen the burn wound tissue for all the groups synthesis minus collagen breakdown.3 The from day 3 post-burned to day 28 post- changes in the diameter of collagen fibrils burned was significantly different except in have also been related to mechanical Durio Zibethinus (Durian) and Cocos strength of the skin. Apparently thick colla- Nucifera (Kelapa)-treated groups. gen fibrils can resist greater tensile strength as opposed to thin ones.11 Once the skin is DISCUSSION injured, the normal collagen will be replaced One of the most important factors in the by scar collagen and the connective tissue healing of wounds is the stimulation of will not regain the original highly organized wound strength.7 Wound strength is deter- structure of collagen.Thus, the healing skin mined by the amount and quality of newly is weaker and results in lower tensile synthesized and deposited collagen, as well strength as opposed to the normal skin.12 as degradation of preformed collagen.3 In this study, the initial phase of wound Tensile strength, which demonstrates the healing was characterized by low tensile force per unit of cross-sectional area needed strength, which resulted from the severing of

Intern J Appl Res Vet Med • Vol. 2, No. 4, 2004 293 collagen bundles and fibrils at the wound increased as the collagen reorganized and site. Heat denaturation, such as in thermal matured into larger bundles and thus increased burn, destroys the helical structure of the the strength of the wound.6 Subsequently, an collagen molecule and if the temperature is increase in the extent of intermolecular cova- sufficiently high, cleaves the Schiff-base lent cross-linking within a fibril and establish- bond thus solubilizing a fraction of intact ment of collagen-ground substance interaction collagen as a high molecular weight gela- within the wound, such as proteoglycan or gly- tin.13 Newly formed collagen does not pos- coprotein, may be additional factors responsi- sess any substantial mechanical strength ble for greater tensile strength.6 The increase in until cross-links of the Schiff-base type have tensile strength in the Manuka, Durio been formed between collagen molecules Zibethinus (Durian) and Ananas Comosus and this process is catalyzed by lysyl-oxi- (Nenas) honey-treated wounds might be due to dase.11 Although the mechanical strength of the increase in collagen concentration per unit soft connective tissues is mediated by the area and stabilization of the fibrils.5 collagen, there are no substantial amounts of Maturation of collagen fibrils resulted in sta- collagen present in a wound until after 2 to 3 ble cross links between several chains, and 14 days of healing. This is due to the fibroblast these cross links are responsible for the gain in proliferation and collagen synthesis, which strength. In addition, honey was claimed to 7 usually begins 2 to 3 days post wounding. contain high levels of glycine, methionine, A slow increase in wound tensile strength arginine, and proline, which play definite roles corresponds to the increase in fibroblasts, in collagen formation and deposition.17 which begin to produce immature collagen According to Jimenez and Rampy7 sub- during the proliferative phase of wound heal- stantial amounts of collagen undergo fibril 7 ing. Fibroblasts play an important role in pro- maturation in an intricate cross-linking sys- ducing the collagen necessary to restore the tem. Collagen intermolecular cross-links 15 tensile strength of wounded skin. Suguna et were essential for providing the stability and 4 al have proved that honey enhanced collagen tensile strength of the skin. Cross-linking synthesis, particularly by stimulating the formation took place as the triple helical col- activity of fibroblasts due to the pres- lagen molecules lined up and began to form 16 ent in honeys. Al-Jady et al have demonstrat- fibrils and then fibres.12 Cross-linking was ed that honey treatment significantly influenced by many factors and the cross- increased cell proliferation in the newly linking pattern may, therefore, reflect the formed granulation tissues, which reflected structural status of the collagen fibrils.18 The the high activity of fibroblasia. Honey con- increased in tensile strength of the 3 differ- tains sugars, amino , minerals, , ent types of honey-treated wounds namely and low level of , which Manuka, Durio Zibethinus (Durian) and help in enhancing cell proliferation, as well as Ananas Comosus (Nenas) honeys may be in stimulating fibroblast proliferation.16 Since due to the increased in collagen maturation fibroblasts are necessary for collagen produc- and crosslinking. Since wounds treated with tion, it may be inferred that application of these 3 types of honey showed greater tensile Manuka, Durio Zibethinus (Durian) and strength, it may be inferred that it not only Ananas Comosus (Nenas) honeys could play increases collagen synthesis per cell, but also a role in increasing the amount of granulation aids in the crosslinking of the . The tissue, by attracting fibroblasts to the wound gain in tensile strength may also be related site and facilitating and accelerating the to collagen interaction with other macro- wound healing process, thus augmenting the molecules, such as proteoglycan or glycopro- tensile strength. tein, or possibly by a slower process of In the latter stage of healing also known as cross-linking between scar tissue collagen the remodeling phase, the tensile strength and the mature collagen of the skin adjacent

294 Vol. 2, No. 4, 2004 • Intern J Appl Res Vet Med to the wound.19 Honey also has been proven cal strength. Since wounds treated with to enhance the synthesis of glycosaminogly- Durio Zibethinus (Durian) honey and cans.16 Glycosaminoglycans are one of the Ananas Comosus (Nenas) honey showed major components synthesized by fibroblasts greater tensile strength compared to wounds in the wound area, as a provisional matrix on treated with two other honeys (Melaleuca which collagen fibers are embedded.20 [Gelam] and Cocos Nucifera [Kelapa]), it Honey could be an important cellular may inferred that the former honey groups mediator responsible for the initiation and increased collagen concentration and stabi- acceleration of wound healing and may lization of the fibers. However, in the future, enhance the healing of burn wounds. Based further studies need to be carried out to con- on our findings, Manuka honey and the two firm it. honeys, namely Durio Zibethinus (Durian) and Ananas Comosus (Nenas), may be REFERENCES potent stimulators of wound healing, as 1. Paul G, Tarlton JF, Purslow PP, et al. demonstrated by increased mechanical Biomechanical and biochemical study of a stan- dardized wound healing model. Int J Biochem strength. Manuka Honey derived from the Cell Biol. 1997;29:211–220. manuka tree (), a 2. Daly CH. Biomechanical properties of dermis. J native of New Zealand is currently one of Invest Dermatol. 1982;79:17–20. 21 the honeys approved for therapeutic use. 3. Witte MB, Thornton FJ, Kiyama T, et al. Manuka is thought to have additional thera- Metalloproteinase inhibitors and wound healing: peutic properties, which are derived from A novel enhancer of wound strength. Surgery. 1998;124:464–470. the floral source.21 Manuka honey has a uniquely high level of herbal antibacterial 4. Suguna L, Chandrakasan G, Thomas-Joseph K. Influence of honey on collagen dur- component and is particularly effective ing wound healing in rats. J Clin Biochem Nutr. against some of the important wound-infect- 1992;13:7–12. ing bacteria.The increased in tensile strength 5. Suguna L, Chandrakasan G, Ramamoorthy U, of honey (Manuka, Durio Zibethinus Joseph KT. Influence of honey on biochemical and biophysical parameters of wounds in rats. J [Durian] and Ananas Comosus [Nenas]) Clin Biochem Nutr. 1993;14:91–99. treated wounds could reflect not only the 6. Dombi GW, Haut RC, Sullivan WG. Correlation increased collagen content but also accelera- of high-speed tensile strength with collagen con- tion of collagen maturation resulting from tent in control and lathyritic rat skin. J Surg Res. the cross-linking of the collagen deposited 1993;54:21–28. by the fibroblasts. In addition, the pH of 7. Jimenez PA, Rampy MA. Keratinocytes Growth Factor-2 accelerates wound healing in incisional honey was claimed to create and maintain wounds. J Surg Res. 1999;81:238–242. 21 optimal conditions for fibroblast activity. 8. Kaufman T, Lusthaus SN, Sagher U, Wexler MR. The recovery of tensile strength of heal- Deep partial skin thickness burns: a reproducible ing skin related to several factors, including animal model to study burn wound healing. healing time and the nutritional status of the Burns. 1990;16(1):13–16. healing tissues.14 Conditions such as scurvy 9. Rashed AN, Afifi FU, Disi AM. Simple evalua- tion of the wound healing activity of a crude and lathyrism may impair the synthesis or extract of Portulaca oleracea L. (growing in stability of fibrillar collagen and thus have a Jordan) in Mus musculus JVI-1. J Ethnopharma- detrimental effect on wound strength.22 col. 2003;88:131–136. Honey contains a wide range of amino 10. Oxlund H, Andreassen TT. The roles of hyaluron- ic , collagen and elastin in the mechanical acids, vitamins, and trace elements, in addi- properties of connective tissues. J Anat. 1980; tion to large qualities of readily assimilable 131(4):611–620. 4 sugars, which help stimulate tissue growth. 11. Jorgensen PH, Bang C, Andreassen TT, The present study shows that topical Jorgensen KD. Growth hormone influences colla- gen deposition and mechanical strength of intact application of honey on burn wounds can rat skin. A dose-response study. Acta Endocrino- improve healing through increased mechani- logica (Copenh). 1989;120:767–772.

Intern J Appl Res Vet Med • Vol. 2, No. 4, 2004 295 12. Diegelmann RF. Collagen Metabolism. Wounds. in buffaloes. Indian J Anim Sci. 2001;13(5):177–182. 1992;62(6):521–523. 13. Quirinia A, Viidik A. The influence of age on the 18. Wulf HC, Sandby-Moller J, Kobayasi T, healing of normal and ischemic incisional skin Gniadecki R. Skin aging and natural photopro- wounds. Mech Ageing Develop.1991;58:221–232. tection. Micron. 2004;35(3):185–191. 14. Lu WW, Ip WY, Jing WM, Holmes AD, Chow 19. Forrest L. Jackson DS. Intermolecular cross-link- SP. Biomechanical properties of thin skin flap ing of collagen in human and guinea pig scar tis- after basic fibroblast growth factor (bFGF) sue. Biochimica Biophysica Acta. administration. Brit J Plast Surg. 1971;229:681–689. 2000;53:225–229. 20. Chitra P, Sajithlal GB, Chandrakasan G. 15. Jorgensen PH, Andreassen TT. Influence of Influence of aloe vera on the healing of dermal biosynthetic human growth hormone on biome- wounds in diabetic rats. J Ethnopaharmacol. chanical properties of rat skin incisional wounds. 1998;59:195–201. Acta Chir Scand. 1988;154:623. 21. Lusby PE, Coombes A, Wilkinson JM. Honey: A 16. Al-jady AM, Kamaruddin MY, Jamal AM, potent agent for wound healing. J Wound Ostomy Mohd-Yassim MY. Biochemical study on the Continence Nurses Soc. 2002;29:295–300. efficacy of Malaysian Honey on inflicted 22. Scott PG, Chambers M, Johnson BW, Williams wounds: an animal model. Medical J Islamic HT. Experimental wound healing: increased break- Acad Sci. 2000;13(3):125–132. ing strength and collagen synthetic activity in 17. Gupta SK, Singh H, Vardhney AC, Prakash P. abdominal fascial wounds healing with secondary Therapeutic efficacy of honey in infected wounds closure of the skin. Br J Surg. 1985;72:777–779.

296 Vol. 2, No. 4, 2004 • Intern J Appl Res Vet Med