CORONARY BAND AUTOGRAFTING IN DONKEYS: CLINICAL AND HISTOLOGICAL EVALUATION OF HEALING QUALITY OF HOOF WALL DEFECTS

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1 Bulgrin Journl of Veterinry Medicine, 2015, 18, No 2, ISSN ; DOI: /jvm.811 Originl rticle CORONARY BAND AUTOGRAFTING IN DONKEYS: CLINICAL AND HISTOLOGICAL EVALUATION OF HEALING QUALITY OF HOOF WALL DEFECTS M. DARADKA, W. ALHAMAD & Z. B. ISMAIL Deprtment of Veterinry Clinicl Sciences, Fculty of Veterinry Medicine, Jordn University of Science nd Technology, Irid, Jordn Summry Drdk, M., W. Alhmd & Z. B. Ismil, Coronry nd utogrfting in donkeys: clinicl nd histologicl evlution of heling qulity of hoof wll defects. Bulg. J. Vet. Med., 18, No 2, Twelve helthy, dult donkeys of oth sexes ged etween 2 nd 4 yers were used to evlute the fesiility of using coronry nd utogrfts to id in the heling of deep derml hoof wll injuries. Wound heling nd grft tke were monitored visully nd evluted histologiclly t different time points. Epiderml cells of the coronry ppille survived the process of hoof wll stripping. Almost ll res of the sl memrne were intct nd ttched to the corium t the site of hoof wll stripping. Grfted sl cells were le to produce new genertions of cells (kertinocytes or kertin producing cells) which mtured nd kertinised, resulting in the production of new strtum medium. Inspite of the fct tht the new hoof wll ws rough nd irregulr in shpe, it ws functionl nd donkeys were no longer lme t 28 dys post surgery. Key words: utogrft, coronry nd, donkey, hoof wll INTRODUCTION The hoof is modified portion of the epiderml skin (Kiner, 1989; Drdk, 2000; Furst & Lischer, 2006); it origintes from the coronry nd. Hoof nd hoof wll injuries re common in equine prctice nd their mngement remins chllenging nd frustrting for oth veterinrins nd owners. Hoof wll crcks, hoof kertom, nd coronry nd vulsion need long term nd expensive tretment with gurded prognosis (Prks, 1999; Stshk, 2002; Stshk & Christine, 2008). Injuries to the coronry germinl lyer result in hoof wll defects of the hrd kertinised strtum medium (Dreiner et l., 2003; Celeste & Szoke, 2005). The defect moves distlly long with the direction of hoof wll growth (Dreiner et l., 2003; Celeste & Szoke, 2005). Injuries to the hoof wll hel y replcement of the defect y new hoof wll tht grows from the intct coronry nd (Dollr, 1993; Prks, 1999).

2 M. Drdk, W. Alhmd & Z. B. Ismil The response of strtum germintivum of the coronry nd differs ccording to the extent of the injury. For exmple hoof wll injuries involving the derml portion of the hoof, strtum germintivum produces scr horn rther thn prolifertion of sl cells (Dreiner et l., 2003; Celeste & Szoke, 2005). However, in injuries involving the epiderml lyer, strtum germintivum reserves its prolifertive potentil (Pollitt, 1998; Drdk, 2000). Therefore, in order to prevent scr tissue formtion, we propose coronry nd utogrft for the tretment of coronry nd injuries in donkeys s model for use in horses. Hoof wll striping is well known surgicl procedure in equine prctice nd frequently used to fcilitte the repir of crcks nd to remove foreign odies nd tumors eneth the hoof wll (Dreiner et l., 2003; Celeste & Szoke, 2005). The lrge deficit in hoof wll tht results from wll strip surgery hels remrkly well (Pollitt, 1998; Drdk, 2000). In equine prctice s well s in the interested scientific community, it is elieved tht coronry nd injuries re unsuitle for vrious reconstructive surgicl procedures tht might hve een helpful in other tissues. Hence, the primry ojective of this study ws to evlute the fesiility of using coronry nd utogrfts to id in the heling of deep derml hoof wll injuries in donkeys s model for horses. MATERIALS AND METHODS The experimentl protocol used in this study ws reviewed nd pproved y the Institutionl Animl Use nd Cre Committee t Jordn University of Science nd Technology (JUST-ACUC). Twelve helthy, dult donkeys of oth sexes, etween 2 nd 4 yers of ge nd 100 nd 150 kg ody weight were used in the study. All donkeys were free of lmeness nd hoof normlities. The donkeys were housed t the Veterinry Helth Center of the Fculty of Veterinry Medicine in fenced yrd with shded re ville. Animls were divided rndomly into 2 groups: Group 1 (n=10) underwent hoof wll stripping nd utogrfting of the coronry nd. Group 2 (n=2) underwent hoof wll striping nd coronry nd resection without grfting nd served s control. Hoof wll striping nd grft collection Prior to surgery, ll nimls were cliniclly exmined. Premediction ws otined y intrvenous injection of xylzine (Xylject, Adwi, Egypt) t 1.1 mg per kg Fig. 1. Left: donor nd recipient site with the dorsl hoof wll stripped nd redy for grfting. Right: Coronry nd utogrft prepred for grfting. BJVM, 18, No 2 141

3 Coronry nd utogrfting in donkeys: clinicl nd histologicl evlution of heling qulity of... of ody weight. Anesthesi ws then induced y intrvenous injection of 10% thiopentl sodium (Thiotl, Vetoquinol, Cnd) t 10 mg per kg ody weight. The niml ws then plced on lterl recumency nd the distl forelims nd hooves were clipped nd prepred for septic surgery. Hoof wll striping ws performed on the middle dorsl spect of oth forelims s descried previously (Drdk, 2000). Ech of the stripped hooves cted s donor nd recipient site. Of the stripped prt of the hoof, rectngulr full-thickness portion (10 mm wide 25 mm long) of the coronry epithelium including the coronry corium nd perioplic corium up to the lmellr corium ws otined (Fig. 1). This portion of tissue ws incised nd peeled off deep to the dermis in preprtion for trnsplnttion in the opposite hoof. Ech tissue ws trnsferred to leled Petri dish contining 10 ml norml sline nd 1 ml of 10% gentmicin witing for grfting. Tissue grfting The recipient site ws kept moist until the grft ws redy for trnsplnttion. Fullthickness coronry utogrfts were hndled gently nd sutured to the recipient site of the ipsilterl hoof using four simple interrupted sutures using 2/0 silk. Numerous simple interrupted sutures were mde in order to ring full contct of the grfted tissue to the recipient site. The surgicl site ws then ndged using sterile non-dherent dressing (Non-dherent dressing, Johnson nd Johnson, USA). In control nimls, hoof wll stripping nd resection of coronry nd without grfting ws crried nd left to hel y second intension. Wound dressing ws chnged every 48 hours for 7 dys. Periopertive ntiiotics using 2.2 mg per kg ody weight gentmicin nd 200,000 IU per kg ody weight of potssium penicillin twice dily intrvenously for 5 dys. Heling ws monitored dily, nd photogrphs of the heling site were tken weekly throughout the experimentl period (90 dys). Histopthology To otin tissues from different ges of grft trnsplnttion, 2 nimls were euthnised t 14, 28, 60 nd 90 dys fter the opertion. Control nimls were euthnised y the 90 th dy. Euthnsi ws performed using intrvenous injection of pentoritl sodium t 100 mg/kg (Beuthnsi-D; Schering-Plough Animl Helth, NJ, USA). Immeditely fter euthnsi, the middle dorsl hoof wll, coronry nd lmellr epidermis nd corium were tken using the method of Pollitt (1996). Collected tissues were trnsferred to 10% uffered neutrl formlin solution nd processed for routine histologicl exmintion. Sections of 5 µm thickness were cut nd stined using H & E stin nd Periodic cid Schiff (PAS) specil stin. RESULTS The coronry ppille epiderml cells survived in the grfting ed. Cells in the grfted tissues ppered to proliferte nd kertinise producing new kertinocytes immeditely fter grfting. The tuules nd intertuulr horn were prominent s erly s 28 dys post-opertively (Fig. 2 nd 3). Tle 1 descries the gross nd histologicl chrcteristics of the grfted tissues s it ppered t ech exmintion time point. At 7 dys old coronry ppille utogrft, progressive growth of grnultion tissue nd revsculristion could e detected (Fig. 2). At 14 dys old grft, 142 BJVM, 18, No 2

4 M. Drdk, W. Alhmd & Z. B. Ismil Tle 1. Gross nd histologic description of grfted tissue t the recipient site t different time points post-opertively Post opertive dy Gross description 7 Progressive growth of helthy grnultion tissue 14 Prt of superficil lyer of grfted tissue ws desqumted, while the deep lyer ws still intct 28 Beginning of kertinistion of oth coronry nd lmellr epiderml sl cells 60 Thickness of kertinised lyer ws proportionl to kertinistion process progression, the thicker the kertinised lyer, the more the kertinistion 90 The coronry kertinised lyer ecme level with the djcent norml hoof wll prt of the superficil lyer of grfted tissue ws desqumted, while the deep lyer ws still intct. At 28 dys old grft, the eginning of kertinistion of oth coronry nd lmellr epiderml sl cells could e detected (Fig. 3). At 60 dys old grft, the thickness of oth coronry nd lmellr kertinised lyer incresed to 2 mm (Fig. 4). Thickness of kertinised lyer ws proportionl to kertinistion process progression, the thicker the kertinised lyer, the more the kertinistion. The more the time pssed, the hrder the hoof ecme. At 90 dys old grft, the thickness of oth coronry nd lmellr kertinised lyer incresed to 4 mm (Fig. 5). The coronry kertinised lyer ecme level with the djcent norml hoof wll. The hoof wll ecme hrder thn efore. Coronry nd trnsplnt did not pper to lter neither the process of kertinistion nor the process of pigmenttion. Histologic description Prolifertion of epiderml sl cells of coronry ppille Grnultion tissue formtion ws evident nd new lood cpillries were formed t the site of grft tke High pigmenttion of the proliferting cells of coronry ppille Kertinocytes egn to lose their nuclei dding to strtum corneum of the hoof wll. Collgen fiers were more prominent Epiderml sl cells of coronry ppille proliferted nd produced new kertinocytes dding to the hoof wll strtum medium. Kertinistion process incresed with time Norml kertinocytes nd norml pigmented horn were produced. The rte of hoof c Fig. 2. A 7 dys old coronry ppille utogrft. There re different sizes of derml coronry ppille nd proliferted nd cornified coronry epiderml sl cells (strtum medium)., : derml coronry ppille, c: intertuulr horn, d: coronry cushion. Trnsverse section, H & E, Br=50 µm. d BJVM, 18, No 2 143

5 Coronry nd utogrfting in donkeys: clinicl nd histologicl evlution of heling qulity of... c Fig. 3. A 28 dys old coronry ppille utogrft. Notice the mount of pigmenttion of the proliferting sl cells. : derml coronry ppill, : sl cells, c: intertuulr horn. Trnsverse section, H&E, Br=50 µm. d c Fig. 4. A 60 dys old coronry ppille utogrft showing heled scr t the site of grft tke.,, c: compressed vsculture, d: condensed firolst. Trnsverse section, H&E, Br=50 µm. wll production fter grfting ws similr to the rte of hoof wll production in control nimls (1 cm/month). No complictions ssocited with the procedure were oserved. DISCUSSION The hoof is modified portion of the epiderml skin; thus heling of coronry nd grfts cn e compred with tht of skin grft. Concerning hoof wll injuries 144 Fig. 5. A 90 dys old coronry ppille utogrft showing derml coronry ppill, coronry epiderml sl cells proliferted nd cornified to produce new hoof wll (strtum medium). : derml coronry ppill, : dense connective tissue of coronry cushion. Longitudinl section, H&E, Br=100 µm. in the equine species, it is the strtum germintivum, not sl cells who proliferte in response to deep hoof wll injuries involving hoof horn nd dermis to produce scr tissue (Prks, 1999; Stshk, 2002; Dreiner et l., 2003; Celeste & Szoke, 2005; Furst & Lischer, 2006; Stshk & Christine, 2008). On the other hnd, strtum germintivum reserves its prolifertive potentil for the heling of epiderml injuries (Pollitt, 1998; Drdk, 2000). In this study, we were le to demonstrte tht coronry nd grft is well incorported in the recipient ed, proliferted nd produced ner norml shpe nd functionl new hoof. In medicl literture, grfting of epiderml ppendges is limited to hir follicles nd corne of the eye (IJzermns, 2001). Nevertheless, grfting of hrd thick kertinised tissues such s the hoof wll is not esy nd hs never reported efore in horses. In donkeys, hoof wll stripping ws performed sfely nd successfully. Survived grfted coronry ppillr epiderml cells kept their ility to proliferte nd kertinise in order to produce new hoof BJVM, 18, No 2

6 M. Drdk, W. Alhmd & Z. B. Ismil wll, in wy similr to tht occuring in hoof wll strip. This grees with Drdk (2000), who reported the process of prolifertion nd kertinistion of oth coronry nd lmellr sl cells in stripped hoof wll in horses. Immoilistion of the ffected hoof using csted mteril hs een proposed s essentil for heling of hoof injuries (Prks, 1999; Stshk, 2002; Dreiner et l., 2003; Celeste & Szoke, 2005; Furst & Lischer, 2006; Stshk & Christine, 2008). In this study, degree of immoilistion ws chieved y suturing the grfted coronry ppille to the recipient ed using numerous simple interrupted sutures. In ddition, the donkeys were given limited exercise to limit their movement during the post-opertive period. Despite of the limited motion provided, grfted tissues heled stisfctorily. Immoilistion using pressure ndges seemed to e sufficient s ws suggested previously to protect the grfting site (Lin et l., 1985). Degree of contrction of grfted tissue is positively proportionl to myofirolsts production ut negtively proportionl to the mount of dermis included in the grft (split-thickness or full-thickness) (Vn de Berg & Rudolph, 1993). In this study, coronry nd (dense connective tissue) contriuted to the high numer of mesenchyml cells, which could trnsform to myofirolsts nd cused grft contrction. Contrction ws ovious since coronry nd grfted tissue ws splitthickness grft rther thn full-thickness compred to skin ut grft tke ws successful. These findings re in greement with those otined y Vn de Berg & Rudolph (1993). Grft tke ws successful. Helthy grnultion tissue ws seen grossly s erly s 3 dys following grft trnsplnttion. Microscopiclly, chnnels connecting lood cpillries etween derml prt of recipient re nd grfted tissue could e detected s erly s 7 dys post-opertively. Firolst prolifertion could e detected t the sme time. This grees with Stshk (2002). Desqumtion of prt of superficil lyer of grfted tissue t 14 dys old coronry ppille utogrft ws expected, which grees with Pecock s sttement (1984). Coronry ppillr epiderml cells produced tuules nd intertuulr horn in the sme direction of norml hoof wll tuules (verticl to ground surfce) ut were not prllel to ech other (due to grft contrction). The newly formed hoof wll pssed over stripped lmellr tissue in proximo-distl direction. A degree of deformity ws oserved which eventully will dispper in fully grown hoof wll. These findings re in greement with the findings of Lech (1980), Budrs et l. (1980) nd Drdk (2000). Histologiclly, norml coronry ppille were seen in mture hoof wll grft tissue. In trnsverse sections, the sl cells orgnised in the sme rchitecture of the norml hoof wll. The sl cells proliferted nd kertinised to produce the ulk of the hoof wll (strtum medium). The growth rte of the grfted hoof wll ws 1 cm/month which ws similr to tht in norml hoof wll (Budrs et l., 1980; Drdk 2000). The process of prolifertion nd kertinistion ws proportionl to time. At 90 dys old coronry ppille utogrft, hoof wll thickness ws 4 mm which is close to the norml thickness of the hoof wll. This grees with the study of horse hoof kertin y Bertrm & Gosline (1987) nd hoof wll growth y Budrs et l. (1980) nd Lech (1980). BJVM, 18, No 2 145

7 Coronry nd utogrfting in donkeys: clinicl nd histologicl evlution of heling qulity of... CONCLUSIONS The primry ojective of this study ws to evlute the fesiility of using coronry nd utogrfts to id in the heling of deep derml hoof wll injuries. In equine prctice s well s in the interested scientific community, it ws elieved tht coronry nd injuries were unsuitle for vrious reconstructive surgicl procedures tht might hve een helpful in other tissues. Although the reserchers in this project do not recommend t this stge using utogrfts s tretment of hoof wll injuries, the ide is novel. Our intent ws to prove the vlidity nd viility of coronry nd grfting using utogrfts s preliminry study. In the future, the vlidity nd viility of using llogrfts re wrrnted. This will prove lifesving procedure for mny horses tht might e otherwise lost ecuse of devstting hoof wll injuries. REFERENCES Bertrm, J. E. A. & J. M. Gosline, Functionl design of horse hoof kertin, the modultion of mechnicl properties through hydrtion effects. Journl of Experimentl Biology, 130, Budrs, K. D., R. L. Hullinger & W. O. Sck, Light nd electron microscopy of kertiniztion in the lminr epidermis of the equine hoof with reference to lminitis. Americn Journl of Veterinry Reserch, 50, Celeste, C. J. & M. O. Szoke, Mngement of equine hoof injuries. Veterinry Clinics of North Americ Equine Prctice, 21, Dreiner, R. M., W. A. Moyer & G. K. Crter, Trum to the sole nd wll. In: Dignosis nd Mngement of Lmeness in the Horse. 1 st edn, W. B. Sunders, USA, pp Drdk, M., The equine hoof, growth, repir nd dimensions. PhD Thesis. The University of Queenslnd, School of Veterinry Medicine, Austrli. Dollr, J. A. W., The protective structures of the foot. In: A Hndook of Horse-Shoeing. Centur Forge Ltd., Burlington, pp Furst, A. E. & C. J. Lischer, Equine Surgery. 3 rd edn, W. B. Sunders, Phildelphi, pp IJzermns, J. N. M., Trnsplnttion Surgery, Springer, London. Kiner, R. A., Clinicl ntomy of the equine foot. Veterinry Clinics of North Americ Equine Prctice, 5, Lech, D. H., The structure nd function of the equine hoof wll. PhD Thesis. Deprtment of Veterinry Antomy, University of Ssktchewn, Ssktoon, Ssktchewn, Cnd. Lin, S. D., C. S. Li, M. F. Hou & C. C. Yng, Amnion overly meshed skin utogrft. Interntionl Society for Burn Injuries, 11, Prks, A. H., Equine foot wounds: Generl principles of heling nd tretment. Proceedings of the Americn Assocition of Equine Prctice, 45, Pecock, E. E., Wound Repir, 3 rd edn, W. B. Sunders Compny. Pollitt, C. C., Bsement memrne pthology, feture of cute lminitis. Equine Veterinry Journl, 28, Pollitt, C. C., The ntomy nd physiology of the equine hoof wll. Equine Veterinry Eduction, 10, Stshk, T. A., The foot. In: Adm s Lmeness in Horses. 5 th edn, Lippincott, Willims nd Wilkins, Phildelphi, pp Stshk, T. S. & L. T. Christine, Equine wound mngement, 2 nd edn, Wiley-Blckwell, USA Vn de Berg, J. S. & R. Rudolph, Immunohistochemistry of fironectin nd ctin in ungrfted wounds nd wounds co- 146 BJVM, 18, No 2

8 M. Drdk, W. Alhmd & Z. B. Ismil vered with full- nd split-thickness skin grfts. Plstic nd Reconstructive Surgery, 91, Pper received ; ccepted for puliction Correspondence: Mous Drdk Deprtment of Clinicl Veterinry Sciences, Fculty of Veterinry Medicine, Jordn University of Science nd Technology, Irid, P.O.Box 3030 Jordn. Phone: ext Fx: E-mil: drdk@just.edu.jo BJVM, 18, No 2 147