Köpeklerde Deneysel Diyafizer Radius Kırıklarında dl-alfa-tokoferol-asetat'ın Kırık İyileşmesi Üzerine Etkisi

Bu deneysel çalışma köpeklerde radiusun diyafizer kırıklarında, kırık iyileşmesi üzerine E vitamininin (dl-alfa-tokoferol asetat) etkisini araştırmak amacıyla gerçekleştirdi.

Onsekiz adet, erişkin (ortalama 3 yaşında), melez dişi köpekler iki eşit gruba ayrıldılar. Radiusun diyafizinde kırık oluşturulduktan sonra fiksasyon plaka ile gerçekleştirildi. Operasyondan sonra birinci gruptaki (deneme grubu) köpeklere dl-alfa-tokoferol asetat (20 mg/kg/gün) intramusküler olarak bir hafta süre ile enjekte edildi. İkinci gruptaki köpekler kontrol grubu olarak izlemeye alındılar. Çalışmanın sonucu operasyondan sonra 15, 30 ve 60. günlerde klinik ve radyolojik olarak değerlendirildi. Her muayene gününde her gruptan 3 olgunun doku örnekleri alındı ve bu köpekler serbest bırakıldı.

Dl-alfa-tokoferol asetat'ın erken dönemde (ilk 15 günde) daha etkili olması kırık bölgesinde oluşan serbest oksijen radikalleri üzerine antioksidan etki göstermesine bağlandı. Ancak bu farkın uzun dönemde (30 günden sonra) kapandığı saptandı. Sonuç olarak kırık oluştuktan hemen sonra (erken dönemde) bir hafta süreyle verilen dl-alfa-tokoferol asetat'ın köpeklerde kırık iyileşmesi üzerinde olumlu etkisinin olduğu kanısına varıldı.

This experiment was conducted to determine the effect of vitamin E (dl-alpha-tocopherol acetate) on healing of radial diaphysis fracture in dogs.

Eighteen adult (mean 3 -y-old), mongrel female dogs were divided into two equal groups. Fixation was performed with bone plate after fracture constituted on radial diaphysis. Postoperatively, dl-alpha-tocopherol acetate (20 mg/kg/day) was injected intramuscularly to the first group (treatment group) for one week. The second group was kept as control group. The result of the study was evaluated clinical and radiographically on postoperative days of 15th, 30th and 60th. At each examination time tissue samples of three cases of each group were harvested and these cases were freed.

Dl-alpha-tocopherol acetate being more effective in early stages (first fifteen days) was evaluated due to its antioxidant effect on free oxygen radicals in the fracture region. Consequently, it was understood that dl-alpha-tocopherol acetate given for a week had a positive effect on the fracture healing in dogs in case of applying immediately (in early stages).

Introduction

Free oxygen radicals are reactive chemical species with an unpaired electron that are produced through a variety of physiological and pathological processes[1-3]. Toxic levels of free oxygen radicals are observed in inflammation, wound healing, ischemia-reperfusion, and ionized radiation[4-6]. Oxygen radicals may also directly damage cell membrane, apparently through the peroxidation of structurally important polyansatured fatty acids within the phospholipid structure of the membrane itself[7]. Lipid peroxidation have recently been shown to play a role in bone metabolism especially in osteoclast activation and resorption activity[8,9]. Free radicals are also found to be cytotoxic to osteoblast cells[9]. Antioxidants inhibit lipid peroxidation by means of blocking of peroxidation or scavenging reactive oxygen species[10,11].

Vitamin E is a natural biological antioxidant, which prevents peroxides from accumulating and protects cells from damaging effects of free radicals[2,12]. Vitamin E also ensures the stability and integrity of biological membranes[13,14]. It has been demonstrated that vitamin E protects against cellular lipid peroxidation in cartilage to sustain normal bone growth and modelling[14,15], and results from animal experiments argue for an osteo-protective effect of vitamin E[16,17]. Therefore, the aim of this study was to determine the effect of vitamin E (alpha tocopherol acetate) administration on the healing processes of radial diaphyseal fracture clinically, radiologically and histopathologically in dogs.

Material and Method

Eighteen adult mongrel female clinically healthy dogs aged between 2 and 4-y-old (mean 3-y-old) were used in this study. The dogs were provided by Elazig Municipality, Turkey. The dogs were treated against antiparasite and vaccined. The animals were housed freely in three separate rooms, all cases were left free walk in the room and had free access to water and standard feed throughout follow up periods.

Preoperatively the dogs were left hungry for 12 hours. General anaesthesia was induced in animals by intramusculer administration of a combination of xylazine HCl 2 mg/kg (Rompun, Bayer, 23.32 mg/ml), and ketamine HCl 15 mg/kg (Ketalar, Parke-Davis, 50 mg/ml). Dogs were divided into two equal groups. Radial diaphysis was opened appropriately to surgical rules[18]. Diaphyseal fracture was performed on radial diaphysis by a Gigli saw. Fixation of fractures was performed by means of a bone plate and screws. Operation wound was closed appropriately to routine surgical procedures. Postoperatively, 20 mg/kg/day dl-alpha-tocopherol acetate (Evigen ampul, Aksu Farma, 2 ml x 5, 300 mg/ml dl-alpha-tocopherol acetate) was injected intramuscularly to treatment group for one week. The second group was kept as control group. The fractured limbs were supported with a bandage reinforced by plastic casts, and the bandages were kept for a month by renewing every week. Postoperatively, 3 ml penicilline and streptomycine (Strepto-veticilline, Eczacıbaşı, procaine penicilline G 15 000 000 IU, crystalise penicilline G 500 000 IU and streptomycine sulphate 2000 mg/10 ml) were administered parenterally for 5 days. Neither restriction in walking nor bandage were applied to the cases after 30^th days.

Clinical and radiographical evaluations were carried out at the 15^th, 30^th and 60^th days after operation. Clinical examinations were performed evaluating by grading (if present) of lameness, utilizing the classification system (Table I). At the end of 15^th, 30^th and 60^th day, three dogs in each group were reoperated under general anaesthesia to harvest a piece of bone including fracture region for histopathological examinations. The samples were placed in 10% neutral buffered formalin immediately after reoperation. The section was fixed in 10% neutral buffered formalin for 5 days and decalcifed in an decalcification solution (15 ml HNO₃+10 ml 10% neutral buffered formalin + 85 ml distilled water). The samples were cut into 5 mm sections after complete decalcification and stained with hematoxylin and eosin (H&E) and examined by light microscopy. In the histopathological examinations of the entire lenght of longitudinal bone sections, bleeding in the fracture area, osteoblastic activity, fibroblast proliferation, cartilage production, encondral ossification, bone marrow formation and bone union parameters were assessed and the differences between the groups were investigated [19, 20].

The dogs were released following taking tissue samples at the time points mentioned before.

Number of the cases examined at 15^th, 30^th and 60^th days are shown in Table II.

Results

The results were evaluated under three sections as clinical, radiological, and histopathological findings.

Clinical examinations showed the presence of moderate lameness in all cases at the 15^th day. At the end of the 30th day slight lameness was observed in two cases in control group, but no lameness in the other cases. All cases were free of lameness at the end of follow up period.

At the 15^th day, radiological examinations showed that callus formation began in all groups except two cases of control group. At the 30^th day, union was completed in all cases of the treatment group, whereas it was incompleted in 4 cases in control group. At the 60^th day, union was completed in all cases (Figure 1, 2), except only one case of control group.

In the histopathological findings, the cases in the treatment group were seen to be better regarding condroid production and osteoblastic activity on the fifteenth day. Callus tissue was determined in the medulla and periosteum of the fracture region in treatment group; however, callus tissue was observed only periosteum of fracture region in control group. Callus tissue decreased in the 30^th day according to the 15^th day, and abundant bone tissue was observed in control group. Whereas bone tissue maturated in the most of areas in treatment group. At the 60^th day, new bone tissue was determined in control group. There was bone union in the fracture ends, but a small focus of callus tissue continued in the fracture line. Bone tissue maturated completely in the treatment group. Bone marrow formation was better in the treatment group at the 60^th day (Figure 3).

Discussion

Free radicals play a role in a variety of diseases and it has become apparent that pathogenesis of many diseases can be lightened by means of understanding cell sources of free radicals and defence mechanism against free radicals. Free radicals are originated from activated phagocytes, antineoplastic agents, irradiation, addictive drugs, stress, otooxidation of small molecules, enzymes, proteins, electron transport systems of mitocondrium, membran of plasma and conditions of oxidative stress[1,2,4,7]. Free radicals effect metabolism of cells through the damaging effects on the metabolism of protein, DNA, carbonhydrate, lipids, enzymes and other molecule groups[5,10,11,21,22].

The protective effect of Vitamin E (alpha tocopherol) might be attributed to a structural effect, however, and there is an evidence from experiments upon cell cultures that the presence of vitamin E can affect the types of fatty acids that become incorporated into membrane lipids[22]. Keskin et al.[20] and Göktürk[23] investigated the effect of alpha tocopherol on the healing of bone in rabbits and rats respectively. In this study, it was to investigate the effect of alpha tocopherol on the healing of bone fracture in dogs.

Vasoconstruction and temporary ischemic period are developed in fracture site when a bone fractured, an arterial vasodilatation and reperfusion in fracture sites are then observed. Polymorphonuclear leucocytes, macrophages and mast cells are migrated to fracture sites in the first 5 days of fracture. This phase is important for fracture healing. It is believed that free oxygen radicals produced through the activation of polymorphonuclear leucocytes damage granulation tissue and retard wound healing[6,20,23-25]. Negative effects of free oxygen radicals on the fracture healing were reported[8,15,17,26]. Norazlina et al.[9], reported that Vitamin E deficiency may cause loss of bone calcium in growing female rats. Hodis[27] has mentioned that for normal antioxidant effect 400 IU vitamin E per day and for maximum antioxidant effect 800 IU per day should be administered 1000 IU per day is consider as megadose[27]. Some other authors[28,29] have suggested a prophylactic dose of 1000 IU per day for 3-4 months to decrease coronary health problems. Keskin et al.[20] and Durak et al.[24] have administered 20 mg/kg/day alpha-tocopherol in rabbits. For that reason, in this study 20 mg/kg/day dl-alpha-tocopherol acetate was injected intramuscularly to treatment group for one week as the first 5 days of fracture is important for fracture healing[2,20,23,24].

A role of free radicals has been proposed in the toxicity of numerous chemicals and in the pathogenesis of many diseases[30]. An extensive list of disorders in which free radicals are implicated is still growing, at least in part because these reactive molecules can produce most of the tissue changes that have been identified during a variety of injurious processes[3]. Some substances defined as antioxidants are used to either prevent formation of or to scavenge free oxygen radicals and their damages. Dl-alpha tocopherol is most active antioxidant among the tocopherols. Vitamin E prevents oxidation of other molecules by means of easily being oxidated[31-33]. Free radicals levels were not analized in this study because the aim of this study was to investigate the effect of vitamin E administration on the healing of fracture clinically, radiologically and histopathologically.

Vitamin E deficiency would increase lipid peroxidation. It has been shown that lipid peroxidation enhance bone resorption by directly activating osteoclasts[8,9,34,35]. Avitabile et al.[36] reported that an association between low activity of antioxidant systems and demineralization of bone, consequent upon enhanced free radical levels. Yee and Ima-Nirwana[37] reported that exposure to an oxidizing agent, ferric nitrilotriacetate, reduced bone calcium content, and that this was prevented by vitamin E supplementation. Therefore, it is suggested that the vitamin E deficiency increased free radical activity, thus enhancing bone resorption and demineralization, which was seen as significantly low bone calcium content. Cohen and Meyer[38] found that vitamin E and selenium deficiency predispose rabbit bones to osteomalacia and decreased the biomechanical strength of the bones. However, vitamin E supplementation was protective against bone loss due to rotational stress in rats[38]. Sergeev et al.[39,40], found that rats with a vitamin E deficiency had decreased absorption of calcium through the intestines and kidneys, as well as decreased deposition of calcium in bones. Similar to these results, results of current study showed that it is clear that vitamin E plays a role in normal bone mineralization, either by its antioxidant effects or by increasing calcium availability for bone deposition.

The clinic, radiographic and histopathologic findings of the present study shows that in order to prevent negative effects free oxygen radicals on osteogenesis the administration of 20 mg/kg/day dl-alpha-tocopherol, a high antioxidant feature appeared to have a usefull effect on early healing processes (first fifteen days) of osteogenesis in experimentally induced fracture dog model.

References

1. Akkuş I. Serbest Radikaller ve Fizyopatolojik Etkileri. Konya: Mimoza Yayınları, 1995.
2. Durmuş AS and Ünsaldı E. Free oxygen radicals, antioxidants and bone healing. Doğu Anadolu Bölgesi Araştırmaları Dergisi 2005; 3(3): 20-27.
3. Kehrer JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 1993; 23(1): 21-48.
4. Defraigne JO, Pincemail J, Franssen C, et al. In vivo free radical production after cross-clamping and reperfusion of the renal artery in the rabbit. Cardiovasc Surg 1993; 1: 343-349.
5. Serin E, Yılmaz E, Yılmaz S, et al. Free oxygen radicals at the site of ischemia-reperfusion damage (Experimental study on rats). Artroplasti Artroskopik Cerrahi 1998; 9(1): 36-39.
6. Seyama A. The role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal ischemia/reperfusion injury. Jpn J Surg 1993; 23: 1060.
7. Bulkley GB. The role of oxygen free radicals in human disease processes. Surgery 1983; 94(3): 407-411.
8. Garrett IR, Boyce BF, Oreffo ROC, et al. Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J Clin Invest 1990; 85: 632-639.
9. Norazlina M, Ima-Nirwana S, Gapor MTA, et al. Tocotrienols are needed for normal bone calcifcation in growing female rats. Asia Pacific J Clin Nutr 2002; 11(3): 194-199.
10. McCord JM. Oxygen-derived free radicals and tissue injury. N Engl J Med 1985; 312(3): 159.
11. McCord JM. Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed Proc 1987; 46(7): 2402.
12. Lucy JA and Dingle JT. Fat-soluble vitamins in biological membranes. Nature 1964; 204: 156-160.
13. Maiorano G, Manchisi A, Salvatori G. et al. Influence of multiple injections of vitamin E on intramuscular collagen and bone characteristics in suckling lambs. J Anim Sci 1999; 77: 2452-2457.
14. Mendez JA, Aguilar MR, Abraham GA, et al. New acrylic bone cements conjugated to vitamin E: curing parameters, properties, and biocompatibility. J Biomed Mater Res 2002; 62: 299-307.
15. Xu H, Watkins BA and Seifert MF. Vitamin E stimulates trabecular bone formation and alters epiphyseal cartilage. Calcif Tissue Int 1995; 57: 293-300.
16. Adam O. Effects of vitamin E on immune and inflammatory responses in rheumatic diseases. Fett/Lipid 1997; 99: 70-73.
17. Seifert MF and Watkins BA. Role of dietary lipid and antioxidants in bone metabolism. Nutr Res 1997; 17(7): 1209-1228.
18. Piermattei DL and Greeley RG. An Atlas of Surgical Approaches to the Bones of the Dog and Cat. Philadelphia: WB Saunders Company, 1979.
19. Gurley AM and Roth SI. Bone. In: S.S. Sternberg (Editor). Histology for Pathologists. New York: Raven Press, 1992: 61-69.
20. Keskin D, Karsan O, Ezirmik N, et al. Tavşanlarda kırık iyileşmesi üzerine alfa-tokoferolün etkisi. Artroplasti-Artroskopik Cerrahi 1999; 10(2): 207-210.
21. Winrow VR, Winyard PG, Morris CJ, et al. Free radicals in inflammation: second messengers and mediators of tissue destruction. Br Med Bulletin 1993; 49(3): 506-522.
22. Halliwell B and Gutteridge JMC. Free Radicals in Biology and Medicine. Second Ed. Oxford: Clarendon Press, 1996.
23. Göktürk E. Sıçanlarda serbest oksijen radikallerinin kırık iyileşmesine etkisi. Acta Orthop Traumatol Turc 1997; 31: 353-356.
24. Durak K, Bilgen OF, Kaleli T, et al. Antioxidant effect of alfa-tocopherol on fracture haematoma in rabbit. J Int Med Res 1996; 24: 419-424.
25. Engle WA, Yoder MC, Baurley JL, et al. Vitamin E decreases superoxide anion production by polymorphonuclear leucocytes. Pediatric Res 1998; 23: 245-248.
26. Koveshnikov VG and Pikaliuk VS. The prolipherative processes in the skeleton of white rats administered dipal experimentally and after antioxidant therapy with tocopherol. Morfologia 1993; 104: 34-39.
27. Hodis HN. Effect of alpha tocopherol in patients with coronary artery disease. The Journal of American Medicine Association 1996; 4(4): 196-201.
28. Abuja PM, Liebmann P, Hayn M et al. Antioxidant role of melatonin in lipid peroxidation of human LDL. FEBS Lett 1997; 18(2): 289-293.
29. Mosca L, Rubenfire M, Mandel C et al. Antioxidant nutrient supplementation reduces the susceptibility of low density lipoprotein to oxidation in patients with coronary artery disease. J Am Coll Cardiol 1997; 128(1): 97-105.
30. Turek JJ, Watkins BA, Schoenlein IA, et al. Oxidized lipid depresses canine growth, immune function, and bone formation. J Nutr Biochem 2003; 14: 24-31.
31. Liebler DC. The role of metabolism in the antioxidant function of vitamin E. Crit Rev Toxicol 1993; 23: 147-169.
32. Takenaka Y, Miki M, Yasuda H, et al. The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites. Arch. Biochem. Biophys 1991; 285: 344-350.
33. Traber MG and Packer L. Vitamin E. beyond antioxidant function. Am J Clin Nutr 1995; 62: 1501-1509.
34. Key LL, Ries WL, Taylor RG, et al. Oxygen-derived free radicals in osteoclasts: the specificity and location of the nitroblue tetrazolium reaction. Bone 1990; 11: 115-119.
35. Suda N. Role of free radicals in bone resorption. Kokubyo Gakkai Zasshi 1991; 58: 603-612.
36. Avitabile M, Rasa R, Campagna NE et al. Calcium release from the mineral matrix of the mandibular bone due to hydrogen peroxide exposure. Minerva Stomatol 1996; 45: 401-403.
37. Yee JK and Ima-Nirwana S. Palm vitamin E protects against ferricnitrilotriacetate-induced impairment of bone calcification. Asia Pacific J Pharmacol 1998; 13: 1-7.
38. Cohen ME and Meyer DM. Effects of dietary vitamin E supplementation and rotational stress on alveolar bone loss in rice rats. Arch Oral Biol 1993; 38: 601-606.
39. Sergeev IN, Arkhapchev IP and Spirichev VB. The role of vitamin E in metabolism and reception of vitamin D. Biokhimiia 1990; 55: 1989-1995.
40. Sergeev IN, Kha KP, Blazheevich NV et al. Effect of combined vitamin D and E deficiencies on calcium metabolism and bone tissue of the rat. Vopr Pitan 1987; 1: 39-43.