Original Article Rat sciatic nerve regeneration through type I collagen-derivedartificial tubular implants

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1 Int J Clin Exp Med 2016;9(7): /ISSN: /IJCEM Originl Article Rt scitic nerve regenertion through type I collgen-derivedrtificil tubulr implnts Rong-Bo Wu *, Zhen-Cho Go *, Fng-Yi Chen, Xing-Fei Zhu, Rui Ding, Xin-Cho Zhng, Rong-Mei Hung, Li Wng, Ji-Ming Shi Deprtment of Orthopedic Surgery, Jinshn Hospitl of Fudn University, Shnghi , Chin. * Equl contributors. Received December 21, 2015; Accepted April 3, 2016; Epub July 15, 2016; Published July 30, 2016 Abstrct: Objective: To investigte the efficcy of type I collgen-derived rtificil tubulr implnt on scitic nerve regenertion. Mterils nd Methods: Sprgue-Dwley rt scitic nerve injury model ws built by cutting 5.0 mm defect on the scitic nerve of the right thigh. Rts with scitic nerve injury were treted with type I collgen-derived rtificil tubulr implnts (Group A), utologous common crotid rtery implnts (Group B), or utologous scitic nerve implnts (Group C). Two control groups were lso included: Group D (mock injury group: sme surgery ws performed on the rts without genertion of nerve injury) nd Group E (norml rts without surgery). After tretment, ll rts were monitored for chnge in clw reflection, nerve conduction velocity, evoked potentil, ltency nd histologicl ssessment on week 2, 4, 8 nd 12. Results: Compring to Group B, Group A showed fster clw reflection recovery s well s higher nerve conduction velocity nd voltility nd shorter ltency time. Also, histologicl ssessment lso reveled tht Group A demonstrted less gstrocnemius wet weight loss thn Group B. However, Group C ws better thn Group A in ll the bove ssessments. Conclusion: Type I collgen-derived rtificil tubulr implnttion is fesible wy for scitic nerve regenertion, but its efficcy is still not s good s utologous nerve implnttion. Keywords: Scitic nerve regenertion, rtificil tubulr implnt, type I collgen Introduction Due to trum or deliberte surgicl resection, peripherl nerve injury, common clinicl compliction, represents mjor cuse of morbidity nd disbility worldwide. In generl, bout 2.8% of the trum ptients would suffer from peripherl nerve injuries nd mny of them would hve life-long disbility [1]. Usully, nerve grft implnttion is required to bridge the nerve defect. Autologous nerve grft hs been considered s the gold stndrd technique for tretment of nerve defect, which requires nerve utogrft hrvested from nother site of the sme ptient [2]. Although the utologous nerve grft implnttion often provides good outcome, there re some serious limittions for this method, including donor site morbidity, secondry deformities, tissue vilbility nd possible differences in tissue structure nd size [2-4]. Besides utologous grfts, llogrfts hve lso been tried in the nerve defect tretment. However, due to immunosuppression, the results hve been not stisficing [5-7]. Therefore, novel techniques for tretment on peripherl nerve injuries re still needed. Beside the conventionl utologous nd llogeneic nerve implnttion techniques, rtificil nerve implnts hve offered n lterntive option with promising results [8, 9]. Artificil nerve grfts, usully in the form of nerve tubes or conduits, cn be mde of mny types of mterils, including type I collgen, chitosn nd polyglycolic cid etc [10-13]. These types of rtificil implnts re mde of bioresorbble mterils tht cn be biodegrded in the body fter its temporry function s nerve scffolds [13]. Moreover, their unique structure could llow sufficient nutrition infiltrtion into the inner cvity of the implnts [11, 14]. Their chrcteristics in biocomptibility nd biodegrdtion render these rtificil nerve implnts very strong cndidtes for peripherl nerve regenertion.

2 Tble 1. Clw reflection recovery time fter surgery (n=12) Group Recovery initition time (Dy) In this study, using Sprgue-Dwley (SD) rts s model, we hve investigted the fesibility of type I collgen-derived rtificil implnt for scitic nerve regenertion. Mterils nd methods Ethicl sttement nd nimls All the protocols involving nimls were reviewed nd pproved by the institutionl ethic review bord nd performed in ccordnce with the Provincil Guidelines on Animl Experimenttion. Mle SD rts ( g) were purchsed from Shnghi s & p-shll ky lbortory niml co., LTD (Btch No ) nd hosted in the niml center of Shnghi City Public Helth Center with food nd wter provided. All rts were rndomly divided into 5 groups: Group A-E. Group A-C: rts with scitic nerve injury were treted with type I collgen-derived rtificil tubulr implnts (Group A), utologous common crotid rtery implnts (Group B), or utologous scitic nerve implnts (Group C). Group D (mock injury group: sme surgery ws performed on the rts without genertion of nerve injury) nd Group E (norml rts without surgery) ws served s controls. Surgicl procedure Totl recovery time (Dy) Averge recovery time (Dy) A ±3.67 B ±3.78 C ±3.49 b D NA NA NA E NA NA NA, P < 0.05 (vs. Group B); b, P < 0.05 (vs. Group A nd B); NA: Not pplicble. All nimls were nesthetized by 10% chlorl hydrte solution (300 mg/kg) before surgery. For genertion scitic nerve defect, skin incision nd underlying muscle splitting were mde in the right lterl thigh. A segment of scitic nerve (bout 20 mm) ws resected nd left 5.0 mm long defect following retrction of the nerve ends. Subsequently, the nerve defect ws bridged by type I collgen-derived rtificil implnt (Group A), utologous common crotid rtery implnts (Group B), or utologous scitic nerve implnts (Group C). For the genertion of mock injury (Group D), sme surgicl procedure ws performed to isolte the scitic nerve but without the introduction of nerve defect. Generl observtion After surgery nd corresponding tretments, ll rts were monitored for chnges in their posture nd git, clw reflection, food drop, toe swollenness nd skin ulcer. Motor Nerve Conduction Velocity (MNCV) mesurement The MNCV of the injury scitic nerve ws mesured on week 2, 4, 8 nd 12 using noninvsive procedure in the scitic posterior tibil conducting system in temperture controlled environment, s previously described [15]. In brief, rts were first nesthetized with 10% chlorl hydrte solution (300 mg/kg) nd then the right scitic nerve ws stimulted by single 0.2 ms supr mxil (8 V) pulses t the scitic notch nd then t the Achilles tendon using myoelectricity evoked potentil equipment (Dntec Keypoint). The evoked potentils nd proximl ltency were recorded. MNCV ws clculted by the following formul: MNCV (m/ s)=the distnce between the two electrodes (in meters)/trnsduction time (in seconds). Histologicl ssessments At week 2, 4, 8 nd 12, the dhesion of scitic nerve to the surrounding tissue ws monitored nd grded s follows: Grde 0, no pprent dhesion; Grde 1, mild dhesion (dhesion ws observed but could be esily seprted) nd Grde 2: severe dhesion (dhesion ws observed nd hrd to be seprted). At week 2, 4, 8 nd 12, gstrocnemius muscles from both sides were hrvested nd wet weight ws mesured. The gstrocnemius muscle weight loss ws clculted s the weight difference in percentge between the surgery side nd helthy side. After weighting, the gstrocnemius muscles were fixed by 10% formlin, embedded in prffin nd cut into 5μm slides. Then HE stining ws performed nd the sectionl res s well s the dimeter of the muscle cells were mesured Int J Clin Exp Med 2016;9(7):

3 ws observed in ll rts from Group A to C since dy 12 fter surgery nd no sttisticl difference ws monitored mong these three groups (dt not shown). The verge clw reflection recovery time for Group A, B nd C were 48.05, nd dys, respectively (Tble 1). These dt indicted tht rts received rtificil nerve conduits implnttion hd fster clw reflection recovery thn rts with utologous common crotid rtery implnttion, but slower thn rts with utologous nerve implnttion. Since rts in Group D nd E hd no scitic nerve injury, no bnormlities were observed. Electrophysiologicl ssessment Figure 1. MNCV of scitic nerves fter surgery. (A) MNCV, (B) Ltency nd (C) evoked potentil of rt scitic nerves fter surgery t week 2, 4, 8 nd 12 were mesured. Dt shown re men ± SD of three independent experiments (n=12). Sttisticl nlysis Mesurement dt were expressed s men ± SD in this study. Sttisticl comprisons were performed using One-wy ANOVA plus SNK post hoc. A P < 0.05 ws considered s sttisticlly significnt. All sttisticl nlyses were performed with SPSS17.0 (SPSS. Inc). Results Microscopic observtion Due to the scitic nerve defect, ll rts from Group A, B nd C exhibited prlysis symptoms including foot drop nd disppered clw reflection. Neurologicl mlnutrition of the skin At week 2, 4, 8 nd 12, the nerve conduction velocity of the surgicl repired scitic nerves were tested nd MNCV, ltency s well s evoked potentils were recorded. Rts from Group D nd E hd the fstest MNCV nd shortest ltency time nd no pprent difference ws observed between the two groups, indicting tht surgicl procedure hd little impct on the nerve function (Figure 1A nd 1B). Aside from Group D nd E, Group C hd the fstest MNCV nd shortest ltency durtion, followed by Group A nd then Group B (Figure 1A nd 1B). The evoked potentils of Group D nd E remined reltively constnt while the other three groups exhibited grdul increse over time. Among Group A, B nd C, Group C showed the highest evoked potentil nd Group A next while Group B ws the lowest of the three (Figure 1C). These dt implied tht scitic nerve function ws better restored in rts with rtificil nerve tubulr implnttion thn in utologous common crotid rtery implnttion. But still, utogrft possessed the best repir efficiency mong the three. Evlution of gstrocnemius muscle At week 2, 4, 8 nd 12, the wet weight, cell dimeter s well s sectionl re were mesured on gstrocnemius muscle on both sides of the thigh. As shown in Tble 2, Group D nd E showed no pprent weight loss in gstrocnemius muscle over time while the other three groups showed significntly loss since surgery. Of notice, rts from Group A, B nd C ll strted to gin weight on gstrocnemius muscle since week 4. However, Group C exhibited the fstest weight-gin, which ws followed by Group A nd then Group B. Similr results were observed Int J Clin Exp Med 2016;9(7):

4 Tble 2. Gstrocnemius muscle wet weight loss (n=12) Group Week 2 (%) Week 4 (%) Week 8 (%) Week 12 (%) A 43.65± ± ± ±2.48 B 41.39± ± ± ±2.62 C 44.82± ± ±2.79 b 71.96±2.81 b D 97.61±2.91 c 98.05±2.73 c 98.36±2.85 c 97.73±2.48 c E 99.37±2.74 c ±2.1 c 99.84±2.81 c ±2.4 c, P < 0.05 (vs. Group B); b, P < 0.05 (vs. Group A nd B); c, P < (vs. Group A, B nd C). Tble 3. Dimeter of gstrocnemius muscle cells (n=4) Group Week 4 (μm) Week 8 (μm) Week 12 (μm) A 28.23± ± ±1.79 B 29.19± ± ±1.63 C 31.39±1.54 b 25.45±1.27 b 22.39±1.22 b D 46.67±1.73 c 47.05±1.63 c 48.50±1.39 c E 47.02±1.62 c 49.62±1.63 c 49.97±1.47 c, P < 0.05 (vs. Group B); b, P < 0.05 (vs. Group A nd B); c, P < (vs. Group A, B nd C). Tble 4. Sectionl re of gstrocnemius muscle cells (n=4) Group Week 4 (μm 2 ) Week 8 (μm 2 ) Week 12 (μm 2 ) A ± ± ±22.68 B ± ± ±23.19 C ±25.34 b ±22.72 b ±22.58 b D ±21.53 c ±22.82 c ±26.68 c E ±31.86 c ±33.28 c ±31.84 c, P < 0.05 (vs. Group B); b, P < 0.05 (vs. Group A nd B); c, P < (vs. Group A, B nd C). when the muscle cell dimeter nd sectionl re of gstrocnemius muscle were determined (Tbles 3 nd 4). The regenerted nerves s well s tissue inflmmtion were determined by HE stining. As shown in Figure 2, Group D nd E hd the most nerve fiber cells nd these cells were rrnged in order without obvious infiltrtion of inflmmtion cells. Contrry to the bove two groups, the other three groups ll demonstrted certin degree of nerve fiber loss nd inflmmtion. Among the three groups, Group A hd more properly rrnged nerve fiber cells nd less inflmmtion cells thn Group B, which, however, still slightly inferior to Group C. Adhesion of regenerted nerve to surrounding tissue No dhesion ws observed in Group E since no surgicl procedure ws performed. On the other hnd, different degrees of dhesion were monitored in the other four groups including the mock surgery group (Group D). The dhesion rtes of Group A to D were 50.0%, 91.7%, 66.7% nd 41.7%, respectively (Tble 5). These dt indicted tht tretment with rtificil nerve tubulr implnts exhibited slightest dhesion of nerve to surrounding tissue (Supplementry). Discussion Autologous nerve implnttion hs been considered the primry therpy for peripherl nerve injury nd it exhibits bout 50% functionl nerve repir in ptients [16, 17]. However, it lso hs some limittions including llogrft vilbility, donor site morbidity, nd dhesion of nerve to surrounding tissue [18]. Consequently, considerble effort hs been mde to develop lterntive tretments nd the doption of rtificil nerve grfts using bioresorbble mterils hs been one of them. A big vriety of bioresorbble mterils hve been investigted for nerve tissue engineering pplictions, such s chitosn, lginte, polyglycolic cid, poly-3-hydroxybutyrte nd type I collgen [19-21]. In the current study, we exmined the fesibility of type I collgen-derived rtificil nerve tubulr implnts for nerve defect tretment. Our results hve showed tht the dhesion of nerve to surrounding tissue ws significntly decresed with use of this rtificil nerve conduit. However, the nerve defect repir efficcy of the novel rtificil conduit ws still not s good s utologous nerve implnts. Growth fctors re cpble of promote cell growth, prolifertion nd differentition. It hs been reported tht certin growth fctors, such s nerve growth fctor (NGF), insulin-like growth fctor (IGF-I) nd IGF-II, could promote nerve regenertion [22]. In ddition, longitudinl biomteril filments hve lso been used into rtificil nerve tubulr implnts to guide nerve regenertion [23, 24]. Although beyond the scope of the current study, it would be very interesting to investigte whether the combin Int J Clin Exp Med 2016;9(7):

5 Figure 2. HE stining of gstrocnemius muscles. At week 2, 4, 8 nd 12, gstrocnemius muscles from both sides were hrvested nd fixed by 10% formlin, embedded in prffin nd cut into 5 μm slides. Then HE stining ws performed nd the sectionl res s well s the dimeter of the muscle cells were mesured. Representtive results re shown. Tble 5. The dhesion grde of scitic nerve to the surrounding tissue (n=12) Group Grde 0 Grde 1 Grde 2 Averge dhesion (%) A B C D E tion of type I collgen-derived rtificil nerve implnts with growth fctors nd/or longitudinl biomteril filments could offer better results in nerve regenertion. moted the weight gin of the gstrocnemius muscle, but it ws still slower thn using utologous nerve implnts. Motor nerve defect could led to denervtion of trget muscle nd consequently result in muscle fiber decrese in size nd weight nd trophy. This sitution could be stopped if the muscle is reinnervted [10, 25, 26]. Therefore, mesurement of muscle weight loss nd size is n effective wy to determine nerve regenertion. In our study, the size nd weight of the gstrocnemius muscle ws mesured to represent scitic nerve regenertion efficiency. Our results showed tht rtificil nerve conduit pro- In conclusion, our findings in the current study reveled tht type I collgen-derived rtificil tubulr implnttion is fesible wy for scitic nerve regenertion, but its efficcy is still not s good s utologous nerve implnttion. Future improvements on this rtificil nerve conduit re required before its use in preclinicl nd clinicl studies. Acknowledgements We thnk the niml center in Fudn University ffilited Public Helth center for the ssistnce on niml experimenttion, thnk Fudn University ffilited Jinshn Hospitl for the ssistnce on lbortory experiments. Supported by Shnghi Jinshn Science & Technology Commission Grnt: Disclosure conflict of interest None Int J Clin Exp Med 2016;9(7):

6 Address correspondence to: Rong-Bo Wu nd Xin- Cho Zhng, Deprtment of Orthopedic Surgery, Jinshn Hospitl of Fudn University, Shnghi , Chin. E-mil: (RBW); (XCZ) References [1] Rochkind S. New Aspects in Nerve Regenertion. Glicin Medicl Journl 2015; 22: [2] Anesti K, Cine P. Peripherl nerve injuries. Plstic nd Aesthetic Reserch 2015; 2: 309. [3] Ducker TB, Hyes GJ. Peripherl nerve grfts: experimentl studies in the dog nd chimpnzee to define homogrft limittions. J Neurosurg 1970; 32: [4] Evns GR. Chllenges to nerve regenertion. Semin Surg Oncol 2000; 19: [5] Hu J, Zhu QT, Liu XL, Xu YB, Zhu JK. Repir of extended peripherl nerve lesions in rhesus monkeys using cellulr llogenic nerve grfts implnted with utologous mesenchyml stem cells. Exp Neurol 2007; 204: [6] Hu M, Xio H, Niu Y, Liu H, Zhng L. Long-Term Follow-Up of the Repir of the Multiple-Brnch Fcil Nerve Defect Using Acellulr Nerve Allogrft. J Orl nd Mxillofc Surg 2016; 74: 218.e1-218.e11. [7] Heth CA, Rutkowski GE. The development of biortificil nerve grfts for peripherl-nerve regenertion. Trends Biotechnol 1998; 16: [8] Fields RD, Le Beu JM, Longo FM, Ellismn MH. Nerve regenertion through rtificil tubulr implnts. Prog Neurobiol 1989; 33: [9] Ichihr S, Ind Y, Nkmur T. Artificil nerve tubes nd their ppliction for repir of peripherl nerve injury: n updte of current concepts. Injury 2008; 39: [10] Wng X, Hu W, Co Y, Yo J, Wu J, Gu X. Dog scitic nerve regenertion cross 30- mm defect bridged by chitosn/pga rtificil nerve grft. Brin 2005; 128: [11] Jing X, Lim SH, Mo HQ, Chew SY. Current pplictions nd future perspectives of rtificil nerve conduits. Exp Neurol 2010; 223: [12] Hdlock TA, Sundbck CA, Hunter DA, Vcnti JP, Cheney ML. A new rtificil nerve grft contining rolled Schwnn cell monolyers. Microsurgery 2001; 21: [13] Keeley RD, Nguyen KD, Stephnides MJ, Pdill J, Rosen JM. The rtificil nerve grft: comprison of blended elstomer-hydrogel with polyglycolic cid conduits. J Reconstr Microsurg 1991; 7: [14] Rosen JM, Pdill JA, Nguyen KD, Pdill MA, Sbelmn EE, Phm HN. Artificil nerve grft using collgen s n extrcellulr mtrix for nerve repir compred with sutured utogrft in rt model. Ann Plstic Surg 1990; 25: [15] Coppey LJ, Dvidson EP, Dunlp JA, Lund DD, Yorek MA. Slowing of motor nerve conduction velocity in streptozotocin-induced dibetic rts is preceded by impired vsodiltion in rterioles tht overlie the scitic nerve. Int J Dibetes Res 2000; 1: [16] Lee SK, Wolfe SW. Peripherl nerve injury nd repir. J Am Acd Orthop Surg 2000; 8: [17] Millesi H. Repprisl of nerve repir. Surg Clin North Am 1981; 61: [18] Moore AM, Ksukurthi R, Mgill CK, Frhdi HF, Borschel GH, Mckinnon SE. Limittions of conduits in peripherl nerve repirs. Hnd 2009; 4: [19] Itoh S, Suzuki M, Ymguchi I, Tkkud K, Kobyshi H, Shinomiy K, Tnk J. Development of nerve scffold using tendon chitosn tube. Artif Orgns 2003; 27: [20] Young R, Terenghi G, Wiberg M. Poly-3- hydroxybutyrte (PHB): resorbble conduit for long-gp repir in peripherl nerves. Br J Plstic Surg 2002; 55: [21] Hshimoto T, Suzuki Y, Kitd M, Ktok K, Wu S, Suzuki K, Endo K, Nishimur Y, Ide C. Peripherl nerve regenertion through lginte gel: nlysis of erly outgrowth nd lte increse in dimeter of regenerting xons. Exp Brin Res 2002; 146: [22] Lee AC, Yu VM, Lowe JB 3rd, Brenner MJ, Hunter DA, Mckinnon SE, Skiym-Elbert SE. Controlled relese of nerve growth fctor enhnces scitic nerve regenertion. Exp Neurol 2003; 184: [23] Ribeiro-Resende VT, Koenig B, Nichterwitz S, Oberhoffner S, Schlosshuer B. Strtegies for inducing the formtion of bnds of Büngner in peripherl nerve regenertion. Biomterils 2009; 30: [24] Hung YC, Hung YY. Biomterils nd strtegies for nerve regenertion. Artif Orgns 2006; 30: [25] Rossoll W, Kröning AK, Ohndorf UM, Steegborn C, Jblonk S, Sendtner M. Specific interction of Smn, the spinl musculr trophy determining gene product, with hnrnp-r nd gry-rbp/ hnrnp-q: role for Smn in RNA processing in motor xons? Hum Mol Genet 2002; 11: [26] Thoms PK, Clne DB. Motor nerve conduction velocity in peronel musculr trophy: evidence for genetic heterogeneity. J Neurol Neurosurg Psychitry 1974; 37: Int J Clin Exp Med 2016;9(7):

7 Supplementry Beside the conventionl utologous nd llogeneic nerve implnttion techniques, rtificil nerve implnts hve offered n lterntive option with promising results. Artificil nerve grfts, usully in the form of nerve tubes or conduits, cn be mde of mny types of mterils, including type I collgen, chitosn nd polyglycolic cid etc. These types of rtificil implnts re mde of bioresorbble mterils tht cn be biodegrded in the body fter its temporry function s nerve scffolds. Moreover, their unique structure could llow sufficient nutrition infiltrtion into the inner cvity of the implnts. Their chrcteristics in biocomptibility nd biodegrdtion render these rtificil nerve implnts very strong cndidtes for peripherl nerve regenertion. In this study, using Sprgue-Dwley (SD) rts s model, we hve investigted the fesibility of type I collgen-derived rtificil implnt for scitic nerve regenertion. 1