EXPERIMENTAL AND THERAPEUTIC MEDICINE 6: 1507-1511, 2013 Celecoxib effectively inhibits the formtion of joint dhesions FENGFENG LI 1*, BIN HE 2*, SHEN LIU 1 nd CUNYI FAN 1 1 Deprtment of Orthopedics, Sixth Affilited People's Hospitl of Shnghi Jiotong University, Shnghi 200233; 2 Deprtment of Orthopedics, Second Affilited Hospitl of Nnjing Medicl College, Nnjing 210011, P.R. Chin Received My 31, 2013; Accepted September 25, 2013 DOI: 10.3892/etm.2013.1336 Abstrct. The present study investigted the effectiveness of celecoxib in preventing the formtion of joint dhesions. Rbbit models of joint dhesion were creted nd the rbbits in two tretment groups were orlly dministered celecoxib or ibuprofen (s positive control) for 30 dys. Rbbits in the control group did not receive ny tretment. Following the 30 dy experimentl period, the inhibitory effects of celecoxib nd ibuprofen on the formtion of joint dhesion were ssessed using number of methods, including the study of mcroscopic ppernce, histology nd contrcture ngle. Thick fibrous dhesions developed in the knees of the rbbits in the control group. By contrst, few dhesions were observed in the two tretment groups, nd those observed were soft, wek nd esily stretched. Fewer dhesions were observed in the rbbits treted with celecoxib thn in the rbbits in the other groups. The dhesion scores nd contrcture ngles in the celecoxib (P<0.001) nd ibuprofen (0.001<P<0.0025) groups were significntly lower thn those of the control group. Moreover, the dhesion scores nd contrcture ngles were significntly lower in the celecoxib group thn in the ibuprofen group (0.025<P<0.05). Histologiclly, the dhesion tissues in the two tretment groups, prticulrly in the celecoxib group, were loose nd thin with sprse fiber formtion. The cell densities in the two tretment groups, of which the ibuprofen group hd higher cell densities (0.025<P<0.05), were significntly lower thn those in the control group (celecoxib group, P<0.001; ibuprofen group, 0.001<P<0.0025). These results indicted tht celecoxib effectively inhibited the formtion of joint dhesions nd therefore my provide novel nd potent pproch for their prevention. Correspondence to: Dr Cunyi Fn, Deprtment of Orthopedics, Sixth Affilited People's Hospitl of Shnghi Jiotong University, 600 Yishn Rod, Shnghi 200233, P.R. Chin E mil: fengmle@163.com * Contributed eqully Key words: celecoxib, ibuprofen, nonselective cyclooxygense 2 enzyme inhibitor, selective cyclooxygense 2 enzyme inhibitor, joint dhesion Introduction The formtion of fibrous joint dhesions following surgery or trum severely restricts functionl recovery nd is mjor problem in the field of orthopedics. The efficcy of certin phrmceuticl gents in the prevention of dhesion formtion, following locl dministrtion, hs been exmined (1 4). These ttempts hve chieved limited success nd thus dhesion formtion remins n unsolved problem. A number of selective nd nonselective cyclooxygense (COX) 2 enzyme inhibitors hve been shown to reduce dhesion formtion vi inhibition of inflmmtory responses involving polymorphonucler leukocytes, mcrophges, fibrin, fibroblsts nd new blood vessel formtion. However, s result of their limited effects, COX 2 inhibitors hve rrely been pplied cliniclly (5 11). Celecoxib, selective COX 2 inhibitor, hs previously been shown to produce mximl reduction in intr bdominl dhesion formtion compred with ibuprofen nd other nonselective COX 2 inhibitors (12,13). In ddition to its COX 2 inhibitory effects, nd unlike other COX enzyme inhibitors, celecoxib lso possesses ntingiogenic nd ntifibroblstic properties (7,14). These dditionl properties my contribute to the greter efficiency of celecoxib in reducing dhesion formtion compred with nonselective COX 2 enzyme inhibitors. Therefore, celecoxib my provide promising therpy for joint dhesions, s ll types of dhesions re considered to rise through the sme mechnism. The im of the present study ws to demonstrte mrked inhibition of joint dhesion formtion by orl dministrtion of celecoxib. A rbbit model tht ccurtely mimicked the fetures of humn joint dhesion ws creted nd the inhibitory effects of celecoxib on the formtion of dhesions were ssessed in this model. The effects were investigted from number of spects, including joint flexion contrcture ngle, mcroscopic ppernce, histology nd collgen content. To provide positive comprison, ibuprofen ws lso dministered in this study due to its mximl ntidhesion effect mong the nonselective COX 2 inhibitors (13,15). Mterils nd methods Animl model. A totl of 60 femle New Zelnd White rbbits (weight, 2.5 3.0 kg; ge, 3 4 months) were purchsed from the Shnghi Lbortory Animl Center (Chinese Acdemy of Sciences, Shnghi, Chin). All experimentl procedures were pproved by the Reserch Ethics Committees
1508 LI et l: CELECOXIB INHIBITS JOINT ADHESION FORMATION Figure 1. Cretion of the niml model nd mesurement of the flexion contrcture ngle. (A) Removl of n osteochondrl portion of the condyle. (B) Immobiliztion of the knee joint with Kirschner wire. (C) Extension of the knee joint by pulling the suture with tension of 4.9 N while the left thigh ws firmly fixed to the tble. The tension ws produced using dynmometer. (D) Rdiogrphy ws performed to determine the contrcture ngle. A 1 0 suture ws tied to the left nkle nd the direction of the suture ws mintined perpendiculr to the tibil xis., flexion contrcture ngle. of the Sixth Affilited People's Hospitl of Shnghi Jiotong University (Shnghi, Chin). The rbbits were rndomly ssigned to one of the following three groups (n=20 per group): Celecoxib, ibuprofen nd control groups. Using intrvenous pentobrbitl sodium (Dinihon, Osk, Jpn) to chieve nesthesi, the left knee joint ws prepred for surgery under septic conditions. Following skin incision, the knee ws opened using lterl prptellr pproch, nd the medil nd lterl sides of the femorl condyle were exposed. A prtil cpsulotomy nd synovectomy were performed using n osteotome nd n osteochondrl portion of the condyle (~5x10 mm) ws removed to ensure the underlying cncellous bone ws exposed (Fig. 1A). Ech limb tht ws subjected to surgery underwent knee joint immobiliztion t 140 of flexion with Kirschner wire (Zimmer, Shnghi, Chin) for 30 dys (Fig. 1B). Rbbits were housed in individul cges in which motion ws not limited. They hd ccess to food nd wter d libitum. Drug tretment. Following recovery from nesthesi, ll rbbits were dministered prophylctic cefzolin nd buprenorphine (Shnghi Phrm, Shnghi, Chin) for pin mngement s required. In ddition, the rbbits in the two tretment groups received orl celecoxib (2.86 mg/kg twice dy; Pfizer, Shnghi, Chin) or ibuprofen (3.81 mg/kg three times dy; GSK, Shnghi, Chin), commencing on the dy of surgery, for 30 dys. The rbbits in the control group did not receive orl celecoxib or ibuprofen. Mcroscopic evlution. Thirty dys subsequent to surgery, eight rbbits in ech group were scrificed by intrvenous dministrtion of n overdose of pentobrbitl sodium (Dinihon) nd the Kirschner wires were removed. The knee joint of ech rbbit ws exposed vi prptellr skin incision nd held t 140 of flexion. The presence nd severity of intr rticulr dhesions were semiquntittively ssessed by blinded observers using severity score scle of 0 4 (16) s follows: Grde 0, no dhesions; grde 1, filmy wek dhesions, esily exfolited by light preprtion with forceps; grde 2, mild dhesions, esily exfolited by moderte preprtion with forceps; grde 3, modertely dense dhesions, which my be prtly exfolited by strong preprtion with forceps; grde 4, dense fibrous dhesions, which my not be exfolited vi strong preprtion with forceps. Histologicl evlution of dhesion tissues. Following mcroscopic evlution of the dhesions, intr rticulr dhesion tissues were hrvested from the knee nd fixed in 10% neutrl buffered formlin for one week. Subsequently, the tissues were embedded in prffin nd 6 µm sections were prepred. The sections were mounted on silne coted slides nd stined with hemtoxylin nd eosin (H&E). The sections were then observed under microscope (VnoxAH 2; Olympus, Tokyo, Jpn), nd fibrosis, predominnt cells nd cell densities were evluted. Imge Pro Plus (Medi Cybernetics, Silver Spring, MD, USA) ws used to nlyze cell numbers. Mesurement of the flexion contrcture ngle. Following mcroscopic nd histologicl evlutions, the nimls remining in ech group were lso scrificed vi dministrtion of n overdose of pentobrbitl sodium (Dinihon), nd were immeditely subjected to biomechnicl evlutions. Following removl of the Kirschner wire, thick silk thred
EXPERIMENTAL AND THERAPEUTIC MEDICINE 6: 1507-1511, 2013 1509 Tble I. Adhesion scores of the rbbits in the control nd tretment groups. Group Number Adhesion scores P vlue P vlue b Control 8 3.6±0.4 NA NA Ibuprofen 8 1.9±0.3 0.001<P<0.0025 NA Celecoxib 8 1.4±0.3 P<0.001 0.025<P<0.05 Compred with the control group; b Compred with the ibuprofen group. All nimls in ech group were used for this evlution. Dt re presented s the men ± stndrd devition. Vlues of P<0.05 re considered to indicte sttisticlly significnt difference. NA, not pplicble. A B C D E F Figure 2. Photogrphs nd histologicl nlyses of dhesions in knee joints. Compred with the dhesions in (A nd D) the control group nd (B nd E) the ibuprofen group, the dhesions in (C nd F) the celecoxib group were reduced in number nd volume. Sections were stined with hemtoxylin nd eosin (originl mgnifiction, x100). ws hooked onto the lower left leg, 10 cm distl from the knee, nd n extension of 5.5 N ws pplied perpendiculr to the tibi by pulling the thred with dynmometer (Fig. 1C). The niml ws lid on the X ry tble on its left side nd the flexion contrcture ngle ws determined on lterl view rdiogrph of the left knee (Fig. 1D, ngle ). All mesurements were mde within 15 min of scrifice. Sttisticl nlysis. Sttisticl nlyses of the differences mong the groups were performed using one wy nlysis of vrince (ANOVA) nd post hoc Student Newmn Keuls test. The dt re presented s the men ± stndrd devition (SD). P<0.05 ws considered to indicte sttisticlly significnt difference. All sttisticl nlyses were conducted using SPSS 11.0 (SPSS, Inc., Chicgo, IL, USA). Results In generl, the rbbits dpted to Kirschner wire immobiliztion well during the 30 dys of the experimentl period. However, of the 60 rbbits used in this study, one niml in the celecoxib group died on dy 7 of unknown cuses nd two nimls suffered from wound infection (one ech from the ibuprofen nd control groups). In ddition, the fixtion of two nimls becme loose in the celecoxib nd ibuprofen groups, respectively. These seven nimls mentioned were excluded from the study. All other nimls gined weight nd ppered helthy, with no signs of impired wound heling. At 30 dys following surgery, thick fibrous dhesions hd developed in the knees of the rbbits in the control group (Fig. 2A). By contrst, the dhesions in the two tretment groups were few, soft, wek nd esily stretched (Fig. 2B nd C). Furthermore, fewer dhesions were observed in the rbbits treted with celecoxib compred with those treted with ibuprofen (Fig. 2C). Accordingly, the dhesion scores in the ibuprofen (0.001<P<0.0025) nd celecoxib (P<0.001) groups were significntly lower thn those in the control group (Tble I). Moreover, the dhesion scores were significntly lower in the celecoxib group thn those in the ibuprofen group (Tble I; 0.025<P<0.05). Histologiclly, the dhesion tissues in the control group were dense, thick nd fibrous (Fig. 2D), while those in the ibuprofen group nd prticulrly in the celecoxib group were loose nd thin with sprse fiber formtion (Fig. 2E nd F). In ll three groups, the predominnt cells were considered to be fibroblsts nd few inflmmtory cells were observed. The cell
1510 LI et l: CELECOXIB INHIBITS JOINT ADHESION FORMATION Tble II. Cell densities of the rbbits in the control nd tretment groups. Group Number Totl cells (per hpf) P vlue P vlue b Control 8 229±19 NA NA Ibuprofen 8 158±13 0.001<P<0.0025 NA Celecoxib 8 124±9 P<0.001 0.025<P<0.05 Compred with the control group; b Compred with the ibuprofen group. All nimls in ech group were used for this evlution. Dt re presented s the men ± stndrd devition. Vlues of P<0.05 re considered to indicte sttisticlly significnt difference. hpf, high power field; NA, not pplicble. Tble III. Contrcture ngles of the rbbits in the control nd tretment groups. Group Number Contrcture ngles ( ) P vlue P vlue b Control 11 120.0±11.2 NA NA Ibuprofen 9 44.2±4.4 0.001<P<0.0025 NA Celecoxib 9 26.4±3.4 P<0.001 0.025<P<0.05 Compred with the control group; b Compred with the ibuprofen group. All nimls in ech group were used for this evlution. Dt re presented s the men ± stndrd devition. Vlues of P<0.05 re considered to indicte sttisticlly significnt difference. NA, not pplicble. densities in the celecoxib (rnge, 117 130; men, 124; P<0.001) nd the ibuprofen (rnge, 151 165; men, 158; 0.001<P<0.0025) groups were significntly lower thn those in the control group (rnge, 220 239; men, 229; Tble II). Moreover, the cell densities were lower in the celecoxib group thn those in the ibuprofen group (Tble II; 0.025<P<0.05). As exhibited in Tble III, the flexion contrcture ngles in the control group rnged between 112 nd 125 (men, 120 ). Despite the differences between the two tretment groups, the contrcture ngles in the celecoxib group (rnge, 21 32 ; men, 26.4 ; P<0.001) nd the ibuprofen group (rnge, 40 48 ; men, 44.2 ; 0.001<P<0.0025) were significntly lower thn those in the control group. Moreover, the contrcture ngles were significntly lower in the celecoxib group thn in the ibuprofen group (0.025<P<0.05). Discussion Adhesion development is process tht involves tissue fibrosis, with inflmmtory responses involving polymorphonucler leukocytes, mcrophges, fibrin, fibroblsts nd new blood vessel formtion. The fibroblsts in dhesions express COX 2 enzymes, while those in non dhesion bering res do not (17). Consequently, certin selective nd nonselective COX 2 enzyme inhibitors my, to n extent, inhibit dhesion formtion. In ddition, the formtion of ll types of dhesions is prtly dependent on ngiogenesis (18,19), nd there is experimentl evidence indicting tht COX 2 induced prostglndins my modulte fibroblst growth fctor nd vsculr endothelil growth fctor induced ngiogenesis (20). Thus, the COX 2 inhibitor, celecoxib, my selectively inhibit the ngiogenesis ssocited with newly forming dhesions vi COX 2 bsed mechnism (8), thereby producing reduction in intr bdominl dhesion formtion (13). In view tht the sme mechnism underlies the formtion of different dhesion types, celecoxib my represent promising therpy for joint dhesions. In the present study, the inhibitory effects of celecoxib on the formtion of joint dhesions were demonstrted in vivo from different spects, including dhesion score, histology, collgen content nd joint contrcture ngle. The results of the histologicl nd biochemicl nlyses of the dhesion tissues were closely correlted with those of the mcroscopic nd biomechnicl evlutions. For exmple, collgen content is one of the key fctors determining the mechnicl strength of repir tissues (21). The collgen type rtio is nother determinnt of tissue strength, nd higher proportion of type III collgen is ssumed to decrese the strength by reducing the fibril dimeter, which is closely correlted with the strength of connective tissues (22,23). Thus, the histologicl nd biochemicl ltertions in the dhesion tissues my serve to improve joint contrcture. In the present study, cliniclly pplicble dosing regimen of the tested drugs ws used. Furthermore, celecoxib ws dministered vi orogstric feeding on dily bsis for 30 dys, thereby effectively simulting clinicl setting. Using n orl dily dosing schedule, it ws demonstrted tht ibuprofen nd celecoxib reduced the formtion of joint dhesions. However, the inhibitory effect of ibuprofen on dhesion formtion ws not s extensive s tht of celecoxib. Furthermore, the nonselective COX 2 inhibitor, ibuprofen, hs the disdvntge of cusing hrmful side effects ssocited with COX 1 inhibition, including gstrointestinl nd renl complictions, s well s bleeding, s result of inhibition of pltelet ggregtion. Furthermore, it ppers tht long term dministrtion nd high doses of COX 2 inhibitors increse the risk of dverse crdiovsculr events (24 26). However, ptients t risk of joint dhesion formtion my represent new cohort suitble for celecoxib tretment due to the potentilly low risk to benefit
EXPERIMENTAL AND THERAPEUTIC MEDICINE 6: 1507-1511, 2013 1511 rtio. Firstly, it ppered tht long term celecoxib dministrtion ws not required to prevent dhesion formtion, since joint dhesions re formed within 30 dys, nd 30 dy tretment led to long term dhesion prevention in the present study. Secondly, routine doses of celecoxib re likely to be required for efficcy in humns, thereby minimizing the incresed risk of crdiovsculr events ssocited with the high doses used for treting diseses such s rheumtoid rthritis. Furthermore, celecoxib hs lower toxicity profile thn other COX 2 inhibitors (27). An niml model tht ccurtely mimics the fetures of humn joint dhesions is essentil for the preclinicl evlution of the efficcy nd sfety of this form of drug therpy. An intr rticulr dhesion model hs been developed in rbbits nd is currently being used to evlute severl methods for inhibiting the formtion of joint dhesions (1,28). In the present study, similr type of intr rticulr dhesion model in rbbits ws used to evlute the effects of celecoxib for the tretment of joint dhesions. The high incidence rte (100%) of joint dhesion formtion in the control group indicted tht the experimentl surgicl procedure used in the present study ws pproprite for this type of study. In ddition, the dhesions developed in the rticulr cvity of this model; therefore, the dhesion tissues were of sufficient volume to enble biochemicl nlyses without contmintion by other tissues. The present study demonstrted tht orl delivery of celecoxib meliorted joint dhesion formtion effectively nd sfely in rbbit model. The study provides novel nd promising strtegy tht my serve s n lterntive tretment for the prevention of joint dhesion formtion. Further studies involving lrger nimls re required to support this hypothesis. In ddition, clinicl trils re required to confirm the beneficil properties of celecoxib in reducing joint dhesions. Acknowledgements This work ws supported finncilly by the Nturl Science Foundtion of Chin (GSCX0818005). References 1. Brunelli G, Longinotti C, Bertzzo C, et l: Adhesion reduction fter knee surgery in rbbit model by Hyloglide, hyluronn derivtive gel. J Orthop Res 23: 1377 1382, 2005. 2. Fukui N, Tshiro T, Hirok H, et l: Adhesion formtion cn be reduced by the suppression of trnsforming growth fctor betl ctivity. J Orthop Res 18: 212 219, 2000. 3. Fukui N, Nkjim K, Tshiro T, et l: Neutrliztion of fibroblst growth fctor 2 reduces intrrticulr dhesions. Clin Orthop Relt Res 383: 250 258, 2001. 4. Hyshi M, Sekiy H, Tktoku K, et l: Experimentl model of knee contrcture in extension: its prevention using sheet mde from hyluronic cid nd crboxymethylcellulose. Knee Surg Sports Trumtol Arthrosc 12: 545 551, 2004. 5. He Y, Revel M nd Loty B: A quntittive model of post lminectomy scr formtion. Effects of nonsteroidl nti inflmmtory drug. Spine (Phil P 1976) 20: 557 563, discussion 579 580, 1995. 6. 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