An experimental study of the effect of autologous platelet rich plasma on the healing process of tendon in donkeys

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1 Proceedings of the 5 th Scientific Conference, College of Veterinry Medicine, University of Mosul An experimentl study of the effect of utologous pltelet rich plsm on the heling process of tendon in donkeys A. Kh. Aljoory Deprtment of Surgery nd Theriogenology, College of Veterinry Medicine, University of Mosul, Mosul, Irq. Astrct The im of this study ws to evlute the regenertive effect of pltelets rich plsm PRP on tendon heling in donkeys. Eight dult donkeys from oth sexes were used. The ge rnge from 2-4 yers nd weighting kg. The nimls divided into two groups control nd tretment group ech group consists of four nimls. The donkeys in oth groups were given deep nrcosis with 10% chlorl hydrte t dose of 5 g 50 kg of ody weight I.V fter the premediction with 2% xylzine t dose of 1.1mg/kg of ody weight I.V. The superficil flexor digitl tendon of forelim ws severed trnsversely nd sutured y unnell-myer technique using stinless steel suture nd the lim ws totlly immoilized with plster of pris. In the second group donkeys underwent the sme procedures with topicl ppliction of pltelets rich plsm on the site of sectioned tendon. At 30 post opertive dys the results reveled tht there ws noticele improvement of the functionl ctivity of the injured tendon treted with pltelets rich plsm PRP. Histologiclly there ws grnultion tissue t the site of heling of sectioned tendon. We conclude tht pltelets rich plsm led to functionl improvement of the injured tendon. Keywords: Tendon, Heling, Pltelet rich plsm, Donkey. Aville online t - -, ) %. ( / ) % ( /

2 Proceedings of the 5 th Scientific Conference, College of Veterinry Medicine, University of Mosul Introduction The process of tendon repir ws descried with ccurcy s fr ck s the seventeenth century (1). Flexor tendon wounds re serious ecuse of the weight ering function of this structure, Further more distl lim wounds usully involve dditionl soft tissue structures such s joint cpsules, tendon sheths nd neurovsculr undles which complictes tretment nd heling nd negtively ffect the prognosis (2). Rehilittion following tendon injury nd repir is long process. In cse of flexor tendon injuries, return to norml function is not llowed until t lest eight to twelve weeks fter repir (3). The pltelet rich plsm (PRP) is n utologous product tht concentrte high numer of pltelets in smll volume of plsm (4). Its iocomptile nd iodegrdle properties prevent the PRP from inducing foreign ody rections, tissue necrosis, or extensive firosis (5). Reports in the literture differ out the intensity nd durtion of lood centrifugtion, the numer of pltelets present in the PRP, nd the use of thromin or other fctors to ctivte PRP (4-6). PRP cn e used s heling sustnce for different kinds of tissue injury cusing less post-surgicl pin (7,8). During pltelet degrnultion mny iologic ctive sustnces re relesed which prticipte in the primry homeostsis nd help the following reprtion nd regenertion of the soft nd hrd tissues. The most importnt sustnces re: serotonin, ctecholmine, firinogen, fctor V, fctor VIII, thromoxn A2, clcium etc. Mny growth fctor re lso very importnt: Pltelet-derived growth fctor (PDGF); Trnsforming growth fctor- (TGF-); Vsculr endothelil growth fctor (VEGF); Interleucin-1(IL-1); Bsic Firolst Growth Fctor (BFGF); Insulin like Growth Fctor (IGF). In the pltelet rich plsm there re lso smll quntities of immune cells s well s plsm (9-11). The im of present study ws to evlute the effect of pltelet rich plsm on the heling process of the injured tendon. Mterils nd methods Animls Eight dult donkeys weighing etween 150 nd 180 kg nd ging etween 2 nd 4 yers were used in the current study. The nimls were kept in the frm of the College of Veterinry Medicine, University of Mosul. The nimls divided into two groups of four nimls in ech group the control group underwent surgicl procedure in which the superficil flexor digitl tendon of the forelim ws sectioned nd repired surgiclly y unnell-myer technique using stinless suture mteril nd ws totlly immoilized with plster of Pris. In the second group the sme procedure performed with locl ppliction of pltelet rich plsm on the cut surfce of the tendon. Postopertive oservtions were dily performed during the first week nd weekly therefter.pltelets rich Plsm preprtion. Four lood smples from four nimls were collected. A 100 ml of lood ws drwn under strict septic condition from the jugulr vein the lood ws spirted with 21 G needle. A 50 ml sized syringe preloded with 7 ml of nticogulnt citrte dextrose solution ACD ws used to void cogultion. Ech lood smple ws centrifuged 2400 prm for 10 minute t 5 ºC y cooled centrifuge resulting in three following lyers. The inferior lyer composed red lood cells, the intermedite lyer composed of white lood cells nd the superior lyer mde of plsm, the plsm lyer ws centrifuged for nother 15 minute t 3400 prm in order to otin two prt: the upper prt which is pltelet poor plsm PPP nd the lower prt ws the pltelet rich plsm PRP. Then the PRP ws spirted with nother pipette nd plced in sterile tue. The PRP ws thus prepred for ctivtion y 10% clcium chloride CCl2 which inhiit the lood thinning effect of ACD. After ctivtion, PRP turned into gel like solution tht is redy for use (12). Anesthesi Animls were premedicted with 2% Xylzine t dose of 1.1mg kg of ody weight intrvenously then 10% chlorl hydrte t dose of 5 g 50 kg of ody weight intrvenously ws given s deep nrcosis (13). Surgicl procedure Under septic condition longitudinl skin incision ws mde directly over the superficil flexor digitl tendon nd then complete trnsverse incision ws mde with surgicl lde through the midwy of the tendon. This incision ws immeditely sutured with unnell-myer technique using stinless steel suture (figure1) then the sectioned tendon ws llocted to receive either pltelet rich plsm or control tretment. Totl immoiliztion of the ffected lim ws mde y plster of pris which extended from crpl joint to the coffin joint for two weeks. Figure 1: Drwing showing unnell-myer technique utilized for end to end nstomosis of tendon (14). 82

3 Proceedings of the 5 th Scientific Conference, College of Veterinry Medicine, University of Mosul Gross pthologicl study nd iopsy collection Histopthologicl exmintion of the specimens were done 30 dys fter surgery. Histologicl specimens were stined with hemtoxylin nd eosin. Results Clinicl oservtion The ility of the nimls in the tretment group to use the operted leg is etter thn the nimls in the control group in which the niml exert gret efforts in order to stnd. The nimls in the control group tend to remin recument most of the time nd stnding occurred fter severl trils s compred to those of the tretment group. The git of the nimls of the oth group ws oserved cliniclly two weeks fter opertion. The tretment groups showed significnt improvement in comprison with control group nimls. And the tretment with PRP ws considered successful when the nimls return to the sme pre-injury movement without relpse of the injury in the follow-time. While the control nimls suffered from lmeness for longer period when compred with tretment nimls. Gross finding: There were mcroscopic differences etween the groups of nimls t four weeks fter opertion the skin repir site ws showed incresed grnultion tissue especilly in the tretment group. The repir site ws hypertrophic nd semi trnslucent. The cross sectioned re of tendon ws incresed in dimeter in oth groups. Histopthologicl results of the control group reveled skin wound heling through firoplsi nd epitheliztion t 30 postopertive dys. Heling of sectioned tendon ws occurred y prolifertion of collgen fiers nd firolsts "firoplsi", ssocited with ngioplsi in intertendineous gp which ws filled with mture grnultion tissue tht rrnged in prllel fshions to long xis of the tendon. This grnultion tissue cn recognized from originl tendon tissue nd there is severe infiltrtion of mononucler inflmmtory cells in the heling tendon tissue s well s edem (figure 2 nd 3). In the tretment group heling of tendon occurred in similr pttern tht mentioned ove histopthologiclly only there ws evidence for polymorphonucler inflmmtory cells (inflmmtory nodules) in the heling tissue within the intertendineous gp (figure 4 nd 5). Figure 2: Photomicrogrph of heling tendon t 30 dys fter tenotomy in group 1, showed presence of grnultion tissue consisting from newly cpillries () nd collgen fier (). H& E t 40 X. Figure 3: Photomicrogrph of heling tendon t 30 dys fter tenotomy in group 1, showed mture grnultion tissue within intertendineous gp (rrow). (H & E 40 X). Figure 4: Photomicrogrph of heling tendon t 30 dys fter tenotomy in group 2 treted within PRP fter tenotomy, showed the presence of collgen fier (), newly cpillries (), (H & E 40 X). 83 Figure 2 photomicrogrph of heling

4 Proceedings of the 5 th Scientific Conference, College of Veterinry Medicine, University of Mosul cells in the tretment group ws due to PDGF which relesed s result of pltelets degrnultion nd this fctor mplified the inflmmtory responses with incresed wound influx neutrophils (18). In oth groups there ws infiltrtion of inflmmtory cells ecuse it is thought tht the inflmmtory responses were mrkedly diminished t six weeks nd were miniml t twelve weeks (19). In conclusion the heling of tendon seem to e complicted process nd the PRP hd positive effect on functionl improvement of injured tendon. It is recommended tht the durtion of the studies in the future should e extended for more thn twelve weeks. Figure 5: Photomicrogrph of heling tendon t 30 dys fter tenotomy in group 2 treted within PRP fter tenotomy, showed the presence of mture grnultion tissue () with inflmmtory nodules (), (H & E 40 X). Discussion A chemicl sustnce 10% clcium chloride CCl2 ws used for the ctivtion of the PRP nd this could void the use of heterologous lood components such s ovine thromin s ctivtor which cn cuse immune rections nd ssocited with development of ntiodies which hd occsionlly led to life-thretening cogulopthies, nd in turn, led to negtive results tht could e flsely scried to PRP (14). PRP is identicl in sustrte to the lood clot tht form in every wound nd therefore it cnnot support cteril growth in ddition to tht PRP hs ph of 6.5 to 6.7 where s mture lood clot hs ph of 7.0 to 7.2 nd therefore my even inhiit cteril growth s other cidic solutions do (15). The sectioned tendon ws sutured with stinless steel suture mteril ecuse it is iologiclly inert to decrese tissue rection. The injured lim ws totlly immoilized postopertively in oth groups to prevent further extension which my negtively ffect heling process. The clinicl improvement of the git of the nimls in the tretment group during the first two weeks fter opertion my relted to the powerful nd long-term nlgesic nd nti-inflmmtory effect of the PRP due to presence of importnt mount of trnsforming growth fctor TGF-B nd ngiogenic VEGF nd prolifertive nd nolic PDGF, IGF growth fctors which hve significnt importnce in the wound repir process (16). Histologiclly there ws prolifertion of collgen fiers nd firoplsi, ngioplsi with mture grnultion tissue due to the effect of the pltelet derived growth fctors upon the prolifertion phse nd lso these fctors enhnce ngiogenesis, firolst prolifertion vi ctivtion of their mitotic ctivity (17,18). Histopthologiclly there were no differences t repir site in oth groups ecuse of short period of the study. The presence of polymorphonucler Acknowledgement Author wish to thnk the College of Veterinry Medicine, University of Mosul for support nd fcilities. Assistnt Prof. Dr. Intisr Al-kenny kindly contriuted with this knowledge nd performed creful histopthologicl exmintions. Apprecition is lso extended to Prof. Dr. Hfez I. Alsdy for scientific consulttion. References 1. The History of Tendons Suture In Historicl Medicine. Med J Ree. Cited y Wrenn RN, Goldner JL, Mrkee JL. An experimentl study of the effect of cortisone on the heling process nd tensile strength of tendon.j Bone Joint Surg.Am.1954;36: Auer JA, Stick JA, Equine surgery. 2 nd ed.w.b Sunders Compny, Phildelphi p. 3. Boyer MI, Stricklnd JW, Engles D, Schr K, Leversedge FJ. Flexor tendon repir nd rehilittion : stte of the rt in Instr curse lec. 2003;52: Mrx RE. Pltelet-Rich Plsm: evidence to support its use. J Orl Mxillofc Surg. 2004;62: Dijkstr-Tiekstr MJ, Vn der Schoot CE, Pietersz RN, Reesink HW. White lood cell frgments in pltelet concentrtes prepred y the pltelet-rich plsm or uffy-cot methods. Vox Sng. 2005;88: Efeoglu C, Akcy YD, Erturk S. A modified method for prepring pltelet-rich plsm: n experimentl study. J Orl Mxillofc Surg. 2004;62: Pierce GF, Mustoe TA, Altrock BW, Deuel TF, Thomson A. Role of pltelet-derived growth fctor in wound heling. J Cell Biochem. 1991;45: Simon D, Mneul S, Geeth V, Nik BR. Potentil for osseous regenertion of pltelet-rich plsm comprtive study in mndiulr third molr sockets. Indin J Dent Res. 2004; 15: Cochrn DL, Rouse CA, Lynch SE,Grves DR. Effect of plteletderived growth fctor isoform on relese from neontl mouse clvrine Bone 1993;14(1): Mrx RE. Pltelet-rich plsm (PRP): wht is PRP nd wht is not PRP? Implnt Dent. 2001;10(4): Petrungro PS. Using pltelet-rich plsm to ccelerte soft tissue mturtion in esthetic periodontl surgery.compend Contin Educ Dent. 2001;22(9): ,734, Gimeno FL, Gtto S, Ferro J, Croxtto JO, Gllo JE. Preprtion of pltelet-rich plsm s tissue dhesive for experimentl trnsplnttion in rits Throm J.2006;4:18. 84

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