THE INFLUENCE OF NERVE CONDUITS DIAMETER IN MOTOR NERVE RECOVERY AFTER SEGMENTAL NERVE REPAIR GUILHERME GIUSTI, M.D.,1 RICHARD H. SHIN, M.D.,1 JOO-YUP LEE, M.D., ph.D.,1 TIAGO G. MATTAR, M.D.,1 ALLEN T. BISHOP, M.D.,1,2 and ALEXANDER Y. SHIN, M.D.1,2*
Many conduits have demonstrated potential to substitute nerve autografts; however, the influence of conduit inner diameter (ID) has never been studied as a separate parameter. This experimental study compared motor recovery after segmental nerve repair with two different ID collagen conduits: 1.5 and 2.0 mm. In addition, the conduits were analyzed in vitro to determine the variations of ID before and after hydration. Thirty rats were divided into three groups: 2.0 mm ID, 1.5 mm ID, and a control group autograft. After 12 weeks, the 1.5 mm ID group demonstrated significant increase in force (P < 0.0001) and weight (P < 0.0001) of the tibialis anterior muscle and better histomorphometry results of the peroneal nerve (P < 0.05) compared to 2.0 mm ID group; nevertheless, autograft results outperformed both conduits (P < 0.0001). Conduits ID were somewhat smaller than advertised, measuring 1.59 6 0.03 mm and 1.25 6 0.0 mm. Only the larger conduit showed a 6% increase in ID after hydration, changing to 1.69 6 0.02 mm. Although autografts perform best, an improvement in motor recovery can be achieved with collagen conduits when a better size match conduit is being used. Minimal changes in collaC 2014 Wiley Periodicals, Inc. Microsurgery 34:646–652, 2014. gen conduits ID can be expected after implantation. V
Functional
recovery of segmental nerve lesions is typically poor despite the regenerative capacity of axons.1 The use of nerve autograft has proven to be the better option to repair those lesions,2,3 however disadvantages such as donor site morbidity and limited supply continue to motivate researchers to look for an alternative. After the disappointing results with silicone conduits,4 a variety of bioabsorbable nerve conduits have been described to replace autograft nerves.5–10 An ideal nerve conduit must demonstrate comparable axon regeneration as well as similar target recovery, sensory, or motor, as autograft. Allografts, PLC (poly lactate caprolactone), and collagen conduits have proven similar recovery to autografts in experimental studies using a short nerve gap model in the rat sciatic nerve.11–14 In a case series, collagen conduits have also demonstrated good and excellent sensibility recovery in 75% of individuals with short digital nerves injuries.15 While focusing on gap length and conduit materials to evaluate nerve recovery, conduit diameter has been overlooked as a parameter that could influence nerve recovery. In most of these studies, conduit diameter is empirically chosen and the available options are limited. Despite advances in developing novel conduit materials, nerve growth factors, or biological matrixes to enhance axon growing,16–20 conduit diameter has not been consistently reported12,21,22 or controlled as a separate variable.13,23,24 Changes in conduit inner
diameter could alter concentration of growth factors or change mechanical support for growing axons what could interfere directly with functional recovery. The purpose of this study is to compare the recovery of motor function after replacing a segmental nerve defect in rats with collagen conduits of different inner diameters. Additionally, the conduits were analyzed in vitro to determine the variations of inner diameter before and after hydration. MATERIAL AND METHODS
After approval by the Institutional Animal Care and Use Committee (IACUC), 30 male Lewis rats weighting between 200 and 300 g were divided into three experimental groups of 10 animals each. Group I had a unilateral 10 mm sciatic nerve segment substituted by a 2.0 mm inner diameter (ID) collagen conduit (NeuroMatrixTM, Stryker Orthopedics, Kalamazoo, MI). In group II, the animals received a 1.5 mm ID collagen conduit to bridge the same gap (NeuraGenTM, Integra Life Sciences, Plainsboro, NJ). In group III, the same nerve segment was reversed and used as an autologous nerve graft. Each animal also served as its own control by examination of the opposite, non-transected sciatic nerve. Surgical Procedure
The rats were anesthetized with an intraperitoneal injection of 10 parts Ketamine (KetasetV, Fort Dodge Animal Health, Fort Dodge, IA) and one part XylazineV (Vettek, Bluesprings, MO) at a dosage of 1 mg/kg body weight. To maintain anesthesia, subsequent doses of ketamine were given intraperitoneally. Body temperature was maintained at 37 C with a heating pad. Five milliliters of lactated Ringer’s and 30 mg/kg of trimethoprim/sulfadiazine (Tribissen, Five Star Compounding Pharmacy, Clive, IA) were administered separately subcutaneously R
R
1
Microvascular Research Laboratory, Mayo Clinic, Rochester, MN 2 Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN. *Correspondence to: Alexander Y. Shin, MD, Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: [email protected] Received 1 October 2013; Revision accepted 5 August 2014; Accepted 8 August 2014 Grant sponsor: Stryker and Integra. Published online 28 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/micr.22312 Ó 2014 Wiley Periodicals, Inc.
Influence of Conduit Diameter in Segmental Nerve Repair
for hydration and infection prophylaxis, respectively. Following skin closure, 0.1 ml/kg of buprenorphine hydrochloride (BuprenexV Reckitt Benckiser Pharmaceuticals, Richmond, VA) was given subcutaneously for pain control. Postoperatively, the animals were kept warm in a heating pad until they were stable and 300 mg/kg of acetaminophen (Q-pap, Qualitest Pharmaceuticals, Huntsville, AL) were added to the feed water for two days. Once the rats were anesthetized, sterile technique was maintained throughout the procedure. The sciatic nerve of the experimental side was fully exposed from the inferior margin of the piriformis muscle to 5 mm past the bifurcation of the peroneal and distal tibial branch. A 10 mm segment of the sciatic nerve was excised ending 5 mm proximal to the tibial peroneal bifurcation by a sharp transection with a microsurgical scissor under an operating microscope (Zeiss OpMi6, Carl Zeiss Surgical GmbH, Oberkochen, Germany). In groups I and II, the nerve gaps were repaired with a 12 mm collagen nerve conduit using two 8-0 nylon epineurial interrupted sutures and filled with saline solution. In both cases, nerve ends were positioned and sutured to lie 1 mm within the conduits on each side resulting in a final 10 mm nerve gap. In group III, the excised nerve segment was cut and sutured with 10-0 Nylon in a reverse position functioning as a devascularized autograft. Fibrin glue was added to augment all nerve repairs and to prevent hematoma from entering the conduits. Muscle flaps were approximated with simple 4-0 Vicryl sutures. Subcutaneous and skin were closed with interrupted sutures of 4-0 Vicryl and 4-0 Nylon, respectively. R
Compound Muscle Action Potential Test
After 12 weeks, the animals were anesthetized and, using the same dorsal approach, the main sciatic trunk proximal to the graft was exposed. A miniature bipolar stimulating electrode (Harvard Apparatus, Holliston, MA) was clamped around the exposed sciatic nerve. Ground and collecting electrodes were placed in the adjacent tissue and the tibialis anterior (TA), respectively. Using a VikingQuest Portable EMG (Nicolet Biomedical, Madison WI) and VikingQuest software on PC, the maximal amplitude of compound muscle action potential (CMAP) was measured in the TA muscle. Stimulation duration was set at 0.02 ms and intensity was increased until a maximum CMAP signal was obtained. To measure the contralateral side, the bipolar electrode was clamped to the sciatic nerve in a similar location to the experimental side. Skin was re-approximated with suture upon completion of CMAP testing until force measurements were conducted. The maximal amplitude from both sides was recorded and results were expressed as a percentage of the non-operated side.
647
Maximum Isometric Tetanic Force Test
Following CMAP test, the maximum isometric tetanic muscle force of TA muscle was performed as described by Shin et al.25 Briefly, a bipolar stimulator (Grass SD9, Grass Instrument Co., Quincy, MA) was clamped around the peroneal branch using a miniature electrode. The TA tendon was released at its insertion and the muscle was freed from the surrounding tissue while preserving its neurovascular pedicle. The hind limb was secured to a testing board with Kirschner wires (Pfizer Howmedica, Rutherford, NJ) and the distal TA tendon was attached to a force transducer (MDB-2.5, Transducer Techniques, Temecula, CA) by a custom clamp. The clamp was positioned in a way to transmit the force horizontally to the transducer and the signal was processed and analyzed using LabVIEW software (National Instruments, Austin, TX). Four parameters were identified and optimized to obtain the maximum isometric tetanic force of the TA: muscle length, stimulus intensity, pulse duration, and pulse frequency.25 During testing, body temperature was maintained at 37 C with a heating pad and the muscle was kept moist with a heated saline drip. After completion of one side, the skin was sutured and results were obtained in the contralateral side. The maximal isometric tetanic force from the experimental side was expressed as a percentage of the contralateral side. Tissue Collection and Histomorphometry
After muscle force test, animals were sacrificed with an overdose of phenobarbital and the TA was carefully dissected and weighed. A segment of peroneal nerve bilaterally, distal to the repair site, was harvested and fixed in a 2% Trump’s solution (37% formaldehyde and 25% glutaraldehyde). Sciatic nerve samples from the autograft group were also harvested to measure the average diameter in normal nerves. Nerve samples were cut with 1 mm thickness and stained with toluidine blue. Histomorphometric analysis was performed using the Image ProPlus software (Media Cybernetics, Bethesda, MD) where nerve area, number of axons, total axon area, and total myelin area were measured in a semi automatic fashion. The results were stated as a percentage of the non-operated, contralateral peroneal nerve. The normal sciatic nerve samples from autograft group were used for calculation of average cross-section nerve area. In Vitro Collagen Conduits Hydration
Three conduits from each diameter size had a 1 mm segment in length hydrated in 5 ml of saline solution at room temperature. Changes in the inner area and conduit thickness were monitored before and after hydration at 5 min interval over a period of 40 min. Digital pictures of cross-sections of the conduits were taken using a 4.0 Microsurgery DOI 10.1002/micr
648
Giusti et al.
Figure 1. Mean and standard deviation of muscle force, muscle weight, and CMAP for all groups. The results were expressed as a percentage of the control side. *Indicates statistical significance at P < 0.05.
megapixels digital camera attached to a Nikon microscope (Nikon Instruments, Melville, NY). The ID and wall thickness were measured using Image ProPlus software (Media Cybernetics, Bethesda, MD) and expressed as mean 6 standard deviation. Statistical Analysis
The groups were compared with respect to CMAP, muscle force, muscle weight and histomorphometry parameters. All results were normalized to the contralateral side and one-way analysis of variance (ANOVA) followed by Bonferronni post test were used to determine any statistical difference among the groups. All values were reported as mean 6 standard deviation and P values less than 0.05 were considered significant.
side after 12 weeks. Group II recovered 27.1 6 11.6% and group III presented the greatest recovery of 55.1 6 7.3%. Group comparison demonstrated statistical significance between all groups (P < 0.0001) with autograft performing better followed by group II. All animals in groups II and III had some recordable contraction but 30% of the animals in group I did not present any muscle contraction after electrical nerve stimulation (Fig. 1). Muscle Weight
The normalized results of TA muscle weight were 22.5 6 8.9% in group I, 51.8 6 6.9% in group II, and 55.1 6 7.3% in group III. Group I results were significantly inferior (P < 0.0001) but no statistical difference was seen between groups II (2.0 mm ID) and III (autograft) (P > 0.05) (Fig. 1).
RESULTS
Some degradation of collagen conduits was observed, however, after 12 weeks the implants were still present and retained their tubular structure in all animals. No blood clots or significant obstructions were observed in the lumen of the conduits. Animal Weight
The mean and standard deviation of animal weight gain was 43.1 6 10.6 g for group I, 50.9 6 7.4 g for group II, and 46.0 6 13.0 for group III without any significant differences among the groups (P 5 0.29). CMAP
The percentage of recovery of the experimental to the contralateral side was 5.1 6 7.1% in group I, 21.2 6 10.1% in group II, and 54.5 6 63.0 in group III. Significant difference was observed only between groups I and III (P < 0.05) (Fig. 1) Maximum Isometric Tetanic Force
In group I, the percentage of TA muscle force recovery was 6.9 6 9.1% when compared to the controlateral Microsurgery DOI 10.1002/micr
Histomorphometry
The average area of an intact sciatic nerve was 0.94 6 0.08 mm. The numbers of axons at the peroneal nerve normalized to the contralateral control side were 11.7 6 9.9% for group I, 51.8 6 24.5% for group II, and 89.8 6 36.5% for group III. The normalized total axon area was 11.8 6 10.0% for group I, 49.0 6 22.8% for group II, and 82.3 6 28.6% for group III. In both parameters, significant differences were found between all groups (P < 0.05) with autograft performing the better followed by 1.5 mm collagen conduit. Peroneal nerve area was significantly different only between 2.0 mm ID conduits and autografts (P < 0.05). The normalized myelin area of 1.5 mm ID conduits and autografts were significantly superior to 2.0 mm ID conduits (P < 0.05) (Figs. 2 and 3). Changes After Conduit Hydration
The average ID and wall thickness of NeuroMatrix conduit (2.0 mm ID) before hydration were 1.59 6 0.03 mm and 0.29 6 0.05 mm, respectively. The average ID and wall thickness of NeuraGen conduit
Influence of Conduit Diameter in Segmental Nerve Repair
649
Figure 2. Mean and standard deviation of histomorphometry results of peroneal nerve for all groups. The results of nerve area, number of axons, axon area, and myelin area were expressed as a percentage of the control side. *Indicates statistical significance at P < 0.05.
Figure 3. Histological transverse sections of the peroneal nerve at 203. A: An example of the histologic result after repair with 2.0 mm ID collagen conduit. The nerve diameter was smaller compared to the other groups (B and C), number and quality of axons were also inferior; (B) shows a histologic slide after repair with 1.5 mm ID collagen conduit group and (C) with autograft. Nerve size was very similar between those but number and quality of axons were superior in the autograft repair group (C).
(1.5 mm ID) before hydration were 1.25 6 0.0 mm and 0.33 6 0.05 mm, respectively. Both conduits swelled rapidly increasing the wall thickness after 5 min. NeuroMatrix increased 18.4% and NeuraGen 16.1%. However, only the NeuroMatrix conduit (2.0 mm ID) showed changes in ID after hydration. It increased 6.0% from 1.59 6 0.03 mm to 1.69 6 0.02 mm in the first 5 min, remaining unchanged after that. No significant changes in ID were observed with NeuraGen conduits (1.5 mm ID) (Figs. 4 and 5). DISCUSSION
Collagen conduits are commercially available and have been used to repair short sensory nerves gaps for many years,15 however, conduit diameter has been chosen based on surgeons experience and the limited available options. Clinical results after peripheral nerve repair still disappointing in a majority of cases though. If adjusting one small factor such as conduit diameter could optimize nerve regeneration, it would be one simple step to improve patient function and life quality, which is surgeon’s primary goal. In this study, we were able to demonstrate that a relative small difference in the conduit diameter could significantly change nerve recovery. The 2.0 mm ID collagen conduits were less effective in
producing muscle recovery compared to 1.5 mm ID conduits in the used model. The promising performance of collagen conduits as nerve substitutes has been previously described in the literature. In a well controlled animal study, Waitayawinyu et al. compared the results of TA muscle force, weight, and axon counting distal to the nerve conduit at 15 weeks after replacing a segmental nerve defect in rats with either autograft, PGA (polyglycolic acid), or collagen conduit. No difference was seen between collagen and autograft in any parameter; however, the exact relationship between conduit diameter and recipient nerves was not reported.12 Similarly, Archibald et al. and Li et al. performed studies on recovery of short segmental nerve repair in primates with collagen conduits with promising results but no information about the conduit diameter that were used.21,22 In this study, motor recovery using autografts exceeded that seen with either collagen conduits. The effect of conduit diameter as a variable may have played a role in some previous studies. Shin et al. compared different commercial available nerve conduits in motor nerve recovery in rats including PGA, PLC, and collagen conduits and alluded to the effect of the diameter of the conduit on motor outcome. The diameter of the PGA conduits (2.3 mm ID) was bigger than PLC and Microsurgery DOI 10.1002/micr
650
Giusti et al.
collagen conduits (1.5 mm ID) and the results in the PGA group were statistically inferior to the other groups.13 Another study reported by Kemp et al. demonstrated a significant increment in histomorphometry parameters in the 1.5 mm ID collagen conduits compared to 2.0 mm silicone in a rat model.24 Considering that the recipient nerves were similar in both studies, the
Figure 4. Results of 1.5 and 2.0 ID collagen conduits ID and wall thickness during hydration for 40 min. Results were expressed as mean 6 standard deviation.
decreased regeneration observed across larger conduits may have been due to either conduit material or diameter. There are only two experimental studies in the literature comparing 1.5 mm ID collagen conduits to autografts with controversial results. In a gap of 14 mm, Whitlock et al. measured the gastrocnemius muscle weight, sciatic functional index, and number of axons in the distal portion of the conduit after 12 weeks recovery. They reported no difference between collagen conduit and autograft.11 On the other hand, using a smaller nerve gap of 10 mm but same recovery time, Shin et al. demonstrated a significant inferior performance of collagen conduits compared to autografts based on results of isometric tetanic tibialis anterior muscle force and weight, electrophysiology, and number of axons of the peroneal nerve.13 Although Whitlock et al. used traditional methods to analyze nerve regeneration, the lack of differences between groups does not necessarily means that the nerve reconstructions are similar.26,27 Their small group size and the poor reproducibility of sciatic function index (SFI) for segmental nerve injuries are important limitations of their study.28–30 Fewer studies using collagen conduits with 2.0 mm ID for either experimental or clinical purpose report mismatch between the conduit and the nerve diameters. Lohmeyer et al. report their clinical experience after collagen conduit implantation to repair digital nerve injuries. Excellent or good sensibility recovery was achieved in 9 out of 12 patients. Digital nerves are 2–3 times larger than the rat sciatic nerve and would provide a better match with the 2.0 mm ID conduits.15 In another study, Alluin et al. used similar conduits to bridge a peroneal nerve gap in rats. After 12 weeks, the weight of tibialis anterior muscle was significantly superior in the autograft group. The peroneal nerve is approximately half size of the sciatic nerve in rats what makes the 2.0 mm ID conduit diameter four times larger.31 In our study, the average diameter of the rat sciatic nerve measured approximately 1mm. The relation between the recipient nerve and conduit diameter appears to be an important
Figure 5. Conduits on the left side of the figures: NeuraGen conduit (1.5 mm ID) which measured 1.25 6 0.0 mm before hydration; conduits in the right side of the figures: NeuroMatrix conduit (2.0 mm ID) which measured 1.59 6 0.03 mm before hydration. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Microsurgery DOI 10.1002/micr
Influence of Conduit Diameter in Segmental Nerve Repair
aspect for nerve regeneration and variations in this parameter, which is not often reported, make study comparison difficult. The ID of collagen conduits clearly demonstrated to be an important parameter for nerve regeneration in this study and no report was found in the literature regarding the changes after implantation. In order to simulate implantation, the conduits were hydrated in normal saline solution at room temperature and analyzed regarding their ID and wall thickness. The ID of both conduits measured before hydration was 15% smaller than advertised and minimal change was seen after hydration. Although conduit diameters were smaller, the sciatic nerve stumps could still be sutured to the conduits since the average rat’s sciatic nerve diameter was smaller (0.94 mm). With the results from this study we can conclude that the reconstruction with collagen conduits with ID of 1.5mm (real diameter 1.25 mm after hydration) generates a better nerve motor recovery than the 2.0 ID conduits (real diameter of 1.69 mm after hydration) in this animal model. We can hypothesize a few reasons why a better match nerve/conduit could generate a better nerve recovery: (1) A decrease in conduit dead space could increase the concentration of growth factors released from the nerve stumps improving quality and quantity of regenerating axons; (2) A better mechanical support for growing axons with better size-matching nerve conduits could improve axonal direction and sustainability. Those points were not analyzed at this time and the exact cause of difference in motor recovery cannot be answered with this study alone and should be the object of future investigations. This study had a few limitations. The two different collagen conduits tested in the study belonged to different providers and, although they were made from similar material as described by the manufacture’s brochures (bovine collagen type I), the companies have their own proprietary methods of production. Testing conduits from the same provider would be ideal and should be recommended for future diameter comparison purposes. Potential causes for the poor results using larger conduits would be a possible increase in conduit dead space leading to a decrease in growth factor concentration released from the nerve ends or simply the poor mechanical support for growing axons. The lack of detailed analyses of the environment inside the conduits would be another limitation of the study that could help to clarify the cause of difference in motor recovery between the conduits. CONCLUSION
Reconstruction with autograft nerve was significantly superior to both collagen conduits and still is the best
651
option for motor recovery after segmental nerve reconstruction in the present model. When considering reconstruction with collagen conduits, improvement in motor recovery can be expected with a better size match nerve conduit. After implantation of the described collagen conduits, variations in conduit ID would probably be minimal.
REFERENCES 1. Lundborg G. Richard P. Bunge memorial lecture. Nerve injury and repair—A challenge to the plastic brain. J Peripher Nerv Syst 2003; 8:209–226. 2. Berger A, Millesi H. Nerve grafting. Clin Orthop Relat Res 1978; 133:49–55. 3. Giusti G, Willems WF, Kremer T, Friedrich PF, Bishop AT, Shin AY. Return of motor function after segmental nerve loss in a rat model: Comparison of autogenous nerve graft, collagen conduit, and processed allograft (AxoGen). J Bone Joint Surg 2012;94:410–417. 4. Merle M, Dellon AL, Campbell JN, Chang PS. Complications from silicon-polymer intubulation of nerves. Microsurgery 1989;10:130– 133. 5. Penna V, Wewetzer K, Munder B, Stark GB, Lang EM. The longterm functional recovery of repair of sciatic nerve transection with biogenic conduits. Microsurgery 2012;32:377–382. 6. Lundborg G. A 25-year perspective of peripheral nerve surgery: Evolving neuroscientific concepts and clinical significance. J Hand Surg [Am] 2000;25:391–414. 7. Weber RV, Mackinnon SE. Bridging the neural gap. Clin Plast Surg 2005;32:605–616, viii. 8. Meek MF, Coert JH. Clinical use of nerve conduits in peripheralnerve repair: Review of the literature. J Reconstr Microsurg 2002; 18:97–109. 9. Millesi H. Progress in peripheral nerve reconstruction. World J Surg 1990;14:733–747. 10. Pfister LA, Papaloizos M, Merkle HP, Gander B. Nerve conduits and growth factor delivery in peripheral nerve repair. J Peripher Nerv Syst 2007;12:65–82. 11. Whitlock EL, Tuffaha SH, Luciano JP, Yan Y, Hunter DA, Magill CK, Moore AM, Tong AY, Mackinnon SE, Borschel GH. Processed allografts and type I collagen conduits for repair of peripheral nerve gaps. Muscle Nerve 2009;39:787–799. 12. Waitayawinyu T, Parisi DM, Miller B, Luria S, Morton HJ, Chin SH, Trumble TE. A comparison of polyglycolic acid versus type 1 collagen bioabsorbable nerve conduits in a rat model: An alternative to autografting. J Hand Surg [Am] 2007;32:1521–1529. 13. Shin RH, Friedrich PF, Crum BA, Bishop AT, Shin AY. Treatment of a segmental nerve defect in the rat with use of bioabsorbable synthetic nerve conduits: a comparison of commercially available conduits. J Bone Joint Surg 2009;91:2194–2204. 14. Tang P, Kilic A, Konopka G, Regalbuto R, Akelina Y, Gardner T. Histologic and functional outcomes of nerve defects treated with acellular allograft versus cabled autograft in a rat model. Microsurgery 2013;33:460–467. 15. Lohmeyer JA, Siemers F, Machens HG, Mailander P. The clinical use of artificial nerve conduits for digital nerve repair: A prospective cohort study and literature review. J Reconstruc Microsurg 2009;25: 55–61. 16. Kempton LB, Gonzalez MH, Leven RM, Hughes WF, Beddow S, Santhiraj Y, Archibald SJ, El Hassan B, Shott S, Kerns JM. Assessment of axonal growth into collagen nerve guides containing VEGFtransfected stem cells in matrigel. Anat Rec (Hoboken) 2009;292: 214–224. 17. Dodla MC, Bellamkonda RV. Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps. Biomaterials 2008;29:33–46.
Microsurgery DOI 10.1002/micr
652
Giusti et al.
18. Marchesi C, Pluderi M, Colleoni F, Belicchi M, Meregalli M, Farini A, Parolini D, Draghi L, Fruguglietti ME, Gavina M, Porretti L, Cattaneo A, Battistelli M, Prelle A, Moggio M, Borsa S, Bello L, Spagnoli D, Gaini SM, Tanzi MC, Bresolin N, Grimoldi N, Torrente Y. Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection. Glia 2007;55:425–438. 19. Wells MR, Kraus K, Batter DK, Blunt DG, Weremowitz J, Lynch SE, Antoniades HN, Hansson HA. Gel matrix vehicles for growth factor application in nerve gap injuries repaired with tubes: A comparison of biomatrix, collagen, and methylcellulose. Exp Neurol 1997;146:395–402. 20. Xu X, Yee WC, Hwang PY, Yu H, Wan AC, Gao S, Boon KL, Mao HQ, Leong KW, Wang S. Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits. Biomaterials 2003;24:2405–2412. 21. Li ST, Archibald SJ, Krarup C, Madison RD. Peripheral nerve repair with collagen conduits. Clin Mater 1992;9:195–200. 22. Archibald SJ, Shefner J, Krarup C, Madison RD. Monkey median nerve repaired by nerve graft or collagen nerve guide tube. J Neurosci 1995;15(5 Part 2):4109–4123. 23. Moore AM, Kasukurthi R, Magill CK, Farhadi HF, Borschel GH, Mackinnon SE. Limitations of conduits in peripheral nerve repairs. Hand (NY) 2009;4:180–186.
Microsurgery DOI 10.1002/micr
24. Kemp SW, Syed S, Walsh SK, Zochodne DW, Midha R. Collagen nerve conduits promote enhanced axonal regeneration, schwann cell association, and neovascularization compared to silicone conduits. Tissue Eng Part A 2009;8:1975–1988. 25. Shin RH, Vathana T, Giessler GA, Friedrich PF, Bishop AT, Shin AY. Isometric tetanic force measurement method of the tibialis anterior in the rat. Microsurgery 2008;28:452–457. 26. Altman D, Bland JM. Confidence intervals illuminate absence of evidence. BMJ 2004;328:1016–1017. 27. Altman DG, Bland JM. Absence of evidence is not evidence of absence. BMJ 1995;311:485. 28. Shenaq JM, Shenaq SM, Spira M. Reliability of sciatic function index in assessing nerve regeneration across a 1 cm gap. Microsurgery 1989;10:214–219. 29. Weber RA, Proctor WH, Warner MR, Verheyden CN. Autotomy and the sciatic functional index. Microsurgery 1993;14:323–327. 30. Kanaya F, Firrell JC, Breidenbach WC. Sciatic function index, nerve conduction tests, muscle contraction, and axon morphometry as indicators of regeneration. Plastic and reconstructive surgery 1996;98: 1264–1271, discussion 1272–1264. 31. Alluin O, Wittmann C, Marqueste T, Chabas JF, Garcia S, Lavaut MN, Guinard D, Feron F, Decherchi P. Functional recovery after peripheral nerve injury and implantation of a collagen guide. Biomaterials 2009;30:363–373.
Many conduits have demonstrated potential to substitute nerve autografts; however, the influence of conduit inner diameter (ID) has never been studied as a separate parameter. This experimental study compared motor recovery after segmental nerve repair with two different ID collagen conduits: 1.5 and 2.0 mm. In addition, the conduits were analyzed in vitro to determine the variations of ID before and after hydration. Thirty rats were divided into three groups: 2.0 mm ID, 1.5 mm ID, and a control group autograft. After 12 weeks, the 1.5 mm ID group demonstrated significant increase in force (P < 0.0001) and weight (P < 0.0001) of the tibialis anterior muscle and better histomorphometry results of the peroneal nerve (P < 0.05) compared to 2.0 mm ID group; nevertheless, autograft results outperformed both conduits (P < 0.0001). Conduits ID were somewhat smaller than advertised, measuring 1.59 6 0.03 mm and 1.25 6 0.0 mm. Only the larger conduit showed a 6% increase in ID after hydration, changing to 1.69 6 0.02 mm. Although autografts perform best, an improvement in motor recovery can be achieved with collagen conduits when a better size match conduit is being used. Minimal changes in collaC 2014 Wiley Periodicals, Inc. Microsurgery 34:646–652, 2014. gen conduits ID can be expected after implantation. V
Functional
recovery of segmental nerve lesions is typically poor despite the regenerative capacity of axons.1 The use of nerve autograft has proven to be the better option to repair those lesions,2,3 however disadvantages such as donor site morbidity and limited supply continue to motivate researchers to look for an alternative. After the disappointing results with silicone conduits,4 a variety of bioabsorbable nerve conduits have been described to replace autograft nerves.5–10 An ideal nerve conduit must demonstrate comparable axon regeneration as well as similar target recovery, sensory, or motor, as autograft. Allografts, PLC (poly lactate caprolactone), and collagen conduits have proven similar recovery to autografts in experimental studies using a short nerve gap model in the rat sciatic nerve.11–14 In a case series, collagen conduits have also demonstrated good and excellent sensibility recovery in 75% of individuals with short digital nerves injuries.15 While focusing on gap length and conduit materials to evaluate nerve recovery, conduit diameter has been overlooked as a parameter that could influence nerve recovery. In most of these studies, conduit diameter is empirically chosen and the available options are limited. Despite advances in developing novel conduit materials, nerve growth factors, or biological matrixes to enhance axon growing,16–20 conduit diameter has not been consistently reported12,21,22 or controlled as a separate variable.13,23,24 Changes in conduit inner
diameter could alter concentration of growth factors or change mechanical support for growing axons what could interfere directly with functional recovery. The purpose of this study is to compare the recovery of motor function after replacing a segmental nerve defect in rats with collagen conduits of different inner diameters. Additionally, the conduits were analyzed in vitro to determine the variations of inner diameter before and after hydration. MATERIAL AND METHODS
After approval by the Institutional Animal Care and Use Committee (IACUC), 30 male Lewis rats weighting between 200 and 300 g were divided into three experimental groups of 10 animals each. Group I had a unilateral 10 mm sciatic nerve segment substituted by a 2.0 mm inner diameter (ID) collagen conduit (NeuroMatrixTM, Stryker Orthopedics, Kalamazoo, MI). In group II, the animals received a 1.5 mm ID collagen conduit to bridge the same gap (NeuraGenTM, Integra Life Sciences, Plainsboro, NJ). In group III, the same nerve segment was reversed and used as an autologous nerve graft. Each animal also served as its own control by examination of the opposite, non-transected sciatic nerve. Surgical Procedure
The rats were anesthetized with an intraperitoneal injection of 10 parts Ketamine (KetasetV, Fort Dodge Animal Health, Fort Dodge, IA) and one part XylazineV (Vettek, Bluesprings, MO) at a dosage of 1 mg/kg body weight. To maintain anesthesia, subsequent doses of ketamine were given intraperitoneally. Body temperature was maintained at 37 C with a heating pad. Five milliliters of lactated Ringer’s and 30 mg/kg of trimethoprim/sulfadiazine (Tribissen, Five Star Compounding Pharmacy, Clive, IA) were administered separately subcutaneously R
R
1
Microvascular Research Laboratory, Mayo Clinic, Rochester, MN 2 Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN. *Correspondence to: Alexander Y. Shin, MD, Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: [email protected] Received 1 October 2013; Revision accepted 5 August 2014; Accepted 8 August 2014 Grant sponsor: Stryker and Integra. Published online 28 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/micr.22312 Ó 2014 Wiley Periodicals, Inc.
Influence of Conduit Diameter in Segmental Nerve Repair
for hydration and infection prophylaxis, respectively. Following skin closure, 0.1 ml/kg of buprenorphine hydrochloride (BuprenexV Reckitt Benckiser Pharmaceuticals, Richmond, VA) was given subcutaneously for pain control. Postoperatively, the animals were kept warm in a heating pad until they were stable and 300 mg/kg of acetaminophen (Q-pap, Qualitest Pharmaceuticals, Huntsville, AL) were added to the feed water for two days. Once the rats were anesthetized, sterile technique was maintained throughout the procedure. The sciatic nerve of the experimental side was fully exposed from the inferior margin of the piriformis muscle to 5 mm past the bifurcation of the peroneal and distal tibial branch. A 10 mm segment of the sciatic nerve was excised ending 5 mm proximal to the tibial peroneal bifurcation by a sharp transection with a microsurgical scissor under an operating microscope (Zeiss OpMi6, Carl Zeiss Surgical GmbH, Oberkochen, Germany). In groups I and II, the nerve gaps were repaired with a 12 mm collagen nerve conduit using two 8-0 nylon epineurial interrupted sutures and filled with saline solution. In both cases, nerve ends were positioned and sutured to lie 1 mm within the conduits on each side resulting in a final 10 mm nerve gap. In group III, the excised nerve segment was cut and sutured with 10-0 Nylon in a reverse position functioning as a devascularized autograft. Fibrin glue was added to augment all nerve repairs and to prevent hematoma from entering the conduits. Muscle flaps were approximated with simple 4-0 Vicryl sutures. Subcutaneous and skin were closed with interrupted sutures of 4-0 Vicryl and 4-0 Nylon, respectively. R
Compound Muscle Action Potential Test
After 12 weeks, the animals were anesthetized and, using the same dorsal approach, the main sciatic trunk proximal to the graft was exposed. A miniature bipolar stimulating electrode (Harvard Apparatus, Holliston, MA) was clamped around the exposed sciatic nerve. Ground and collecting electrodes were placed in the adjacent tissue and the tibialis anterior (TA), respectively. Using a VikingQuest Portable EMG (Nicolet Biomedical, Madison WI) and VikingQuest software on PC, the maximal amplitude of compound muscle action potential (CMAP) was measured in the TA muscle. Stimulation duration was set at 0.02 ms and intensity was increased until a maximum CMAP signal was obtained. To measure the contralateral side, the bipolar electrode was clamped to the sciatic nerve in a similar location to the experimental side. Skin was re-approximated with suture upon completion of CMAP testing until force measurements were conducted. The maximal amplitude from both sides was recorded and results were expressed as a percentage of the non-operated side.
647
Maximum Isometric Tetanic Force Test
Following CMAP test, the maximum isometric tetanic muscle force of TA muscle was performed as described by Shin et al.25 Briefly, a bipolar stimulator (Grass SD9, Grass Instrument Co., Quincy, MA) was clamped around the peroneal branch using a miniature electrode. The TA tendon was released at its insertion and the muscle was freed from the surrounding tissue while preserving its neurovascular pedicle. The hind limb was secured to a testing board with Kirschner wires (Pfizer Howmedica, Rutherford, NJ) and the distal TA tendon was attached to a force transducer (MDB-2.5, Transducer Techniques, Temecula, CA) by a custom clamp. The clamp was positioned in a way to transmit the force horizontally to the transducer and the signal was processed and analyzed using LabVIEW software (National Instruments, Austin, TX). Four parameters were identified and optimized to obtain the maximum isometric tetanic force of the TA: muscle length, stimulus intensity, pulse duration, and pulse frequency.25 During testing, body temperature was maintained at 37 C with a heating pad and the muscle was kept moist with a heated saline drip. After completion of one side, the skin was sutured and results were obtained in the contralateral side. The maximal isometric tetanic force from the experimental side was expressed as a percentage of the contralateral side. Tissue Collection and Histomorphometry
After muscle force test, animals were sacrificed with an overdose of phenobarbital and the TA was carefully dissected and weighed. A segment of peroneal nerve bilaterally, distal to the repair site, was harvested and fixed in a 2% Trump’s solution (37% formaldehyde and 25% glutaraldehyde). Sciatic nerve samples from the autograft group were also harvested to measure the average diameter in normal nerves. Nerve samples were cut with 1 mm thickness and stained with toluidine blue. Histomorphometric analysis was performed using the Image ProPlus software (Media Cybernetics, Bethesda, MD) where nerve area, number of axons, total axon area, and total myelin area were measured in a semi automatic fashion. The results were stated as a percentage of the non-operated, contralateral peroneal nerve. The normal sciatic nerve samples from autograft group were used for calculation of average cross-section nerve area. In Vitro Collagen Conduits Hydration
Three conduits from each diameter size had a 1 mm segment in length hydrated in 5 ml of saline solution at room temperature. Changes in the inner area and conduit thickness were monitored before and after hydration at 5 min interval over a period of 40 min. Digital pictures of cross-sections of the conduits were taken using a 4.0 Microsurgery DOI 10.1002/micr
648
Giusti et al.
Figure 1. Mean and standard deviation of muscle force, muscle weight, and CMAP for all groups. The results were expressed as a percentage of the control side. *Indicates statistical significance at P < 0.05.
megapixels digital camera attached to a Nikon microscope (Nikon Instruments, Melville, NY). The ID and wall thickness were measured using Image ProPlus software (Media Cybernetics, Bethesda, MD) and expressed as mean 6 standard deviation. Statistical Analysis
The groups were compared with respect to CMAP, muscle force, muscle weight and histomorphometry parameters. All results were normalized to the contralateral side and one-way analysis of variance (ANOVA) followed by Bonferronni post test were used to determine any statistical difference among the groups. All values were reported as mean 6 standard deviation and P values less than 0.05 were considered significant.
side after 12 weeks. Group II recovered 27.1 6 11.6% and group III presented the greatest recovery of 55.1 6 7.3%. Group comparison demonstrated statistical significance between all groups (P < 0.0001) with autograft performing better followed by group II. All animals in groups II and III had some recordable contraction but 30% of the animals in group I did not present any muscle contraction after electrical nerve stimulation (Fig. 1). Muscle Weight
The normalized results of TA muscle weight were 22.5 6 8.9% in group I, 51.8 6 6.9% in group II, and 55.1 6 7.3% in group III. Group I results were significantly inferior (P < 0.0001) but no statistical difference was seen between groups II (2.0 mm ID) and III (autograft) (P > 0.05) (Fig. 1).
RESULTS
Some degradation of collagen conduits was observed, however, after 12 weeks the implants were still present and retained their tubular structure in all animals. No blood clots or significant obstructions were observed in the lumen of the conduits. Animal Weight
The mean and standard deviation of animal weight gain was 43.1 6 10.6 g for group I, 50.9 6 7.4 g for group II, and 46.0 6 13.0 for group III without any significant differences among the groups (P 5 0.29). CMAP
The percentage of recovery of the experimental to the contralateral side was 5.1 6 7.1% in group I, 21.2 6 10.1% in group II, and 54.5 6 63.0 in group III. Significant difference was observed only between groups I and III (P < 0.05) (Fig. 1) Maximum Isometric Tetanic Force
In group I, the percentage of TA muscle force recovery was 6.9 6 9.1% when compared to the controlateral Microsurgery DOI 10.1002/micr
Histomorphometry
The average area of an intact sciatic nerve was 0.94 6 0.08 mm. The numbers of axons at the peroneal nerve normalized to the contralateral control side were 11.7 6 9.9% for group I, 51.8 6 24.5% for group II, and 89.8 6 36.5% for group III. The normalized total axon area was 11.8 6 10.0% for group I, 49.0 6 22.8% for group II, and 82.3 6 28.6% for group III. In both parameters, significant differences were found between all groups (P < 0.05) with autograft performing the better followed by 1.5 mm collagen conduit. Peroneal nerve area was significantly different only between 2.0 mm ID conduits and autografts (P < 0.05). The normalized myelin area of 1.5 mm ID conduits and autografts were significantly superior to 2.0 mm ID conduits (P < 0.05) (Figs. 2 and 3). Changes After Conduit Hydration
The average ID and wall thickness of NeuroMatrix conduit (2.0 mm ID) before hydration were 1.59 6 0.03 mm and 0.29 6 0.05 mm, respectively. The average ID and wall thickness of NeuraGen conduit
Influence of Conduit Diameter in Segmental Nerve Repair
649
Figure 2. Mean and standard deviation of histomorphometry results of peroneal nerve for all groups. The results of nerve area, number of axons, axon area, and myelin area were expressed as a percentage of the control side. *Indicates statistical significance at P < 0.05.
Figure 3. Histological transverse sections of the peroneal nerve at 203. A: An example of the histologic result after repair with 2.0 mm ID collagen conduit. The nerve diameter was smaller compared to the other groups (B and C), number and quality of axons were also inferior; (B) shows a histologic slide after repair with 1.5 mm ID collagen conduit group and (C) with autograft. Nerve size was very similar between those but number and quality of axons were superior in the autograft repair group (C).
(1.5 mm ID) before hydration were 1.25 6 0.0 mm and 0.33 6 0.05 mm, respectively. Both conduits swelled rapidly increasing the wall thickness after 5 min. NeuroMatrix increased 18.4% and NeuraGen 16.1%. However, only the NeuroMatrix conduit (2.0 mm ID) showed changes in ID after hydration. It increased 6.0% from 1.59 6 0.03 mm to 1.69 6 0.02 mm in the first 5 min, remaining unchanged after that. No significant changes in ID were observed with NeuraGen conduits (1.5 mm ID) (Figs. 4 and 5). DISCUSSION
Collagen conduits are commercially available and have been used to repair short sensory nerves gaps for many years,15 however, conduit diameter has been chosen based on surgeons experience and the limited available options. Clinical results after peripheral nerve repair still disappointing in a majority of cases though. If adjusting one small factor such as conduit diameter could optimize nerve regeneration, it would be one simple step to improve patient function and life quality, which is surgeon’s primary goal. In this study, we were able to demonstrate that a relative small difference in the conduit diameter could significantly change nerve recovery. The 2.0 mm ID collagen conduits were less effective in
producing muscle recovery compared to 1.5 mm ID conduits in the used model. The promising performance of collagen conduits as nerve substitutes has been previously described in the literature. In a well controlled animal study, Waitayawinyu et al. compared the results of TA muscle force, weight, and axon counting distal to the nerve conduit at 15 weeks after replacing a segmental nerve defect in rats with either autograft, PGA (polyglycolic acid), or collagen conduit. No difference was seen between collagen and autograft in any parameter; however, the exact relationship between conduit diameter and recipient nerves was not reported.12 Similarly, Archibald et al. and Li et al. performed studies on recovery of short segmental nerve repair in primates with collagen conduits with promising results but no information about the conduit diameter that were used.21,22 In this study, motor recovery using autografts exceeded that seen with either collagen conduits. The effect of conduit diameter as a variable may have played a role in some previous studies. Shin et al. compared different commercial available nerve conduits in motor nerve recovery in rats including PGA, PLC, and collagen conduits and alluded to the effect of the diameter of the conduit on motor outcome. The diameter of the PGA conduits (2.3 mm ID) was bigger than PLC and Microsurgery DOI 10.1002/micr
650
Giusti et al.
collagen conduits (1.5 mm ID) and the results in the PGA group were statistically inferior to the other groups.13 Another study reported by Kemp et al. demonstrated a significant increment in histomorphometry parameters in the 1.5 mm ID collagen conduits compared to 2.0 mm silicone in a rat model.24 Considering that the recipient nerves were similar in both studies, the
Figure 4. Results of 1.5 and 2.0 ID collagen conduits ID and wall thickness during hydration for 40 min. Results were expressed as mean 6 standard deviation.
decreased regeneration observed across larger conduits may have been due to either conduit material or diameter. There are only two experimental studies in the literature comparing 1.5 mm ID collagen conduits to autografts with controversial results. In a gap of 14 mm, Whitlock et al. measured the gastrocnemius muscle weight, sciatic functional index, and number of axons in the distal portion of the conduit after 12 weeks recovery. They reported no difference between collagen conduit and autograft.11 On the other hand, using a smaller nerve gap of 10 mm but same recovery time, Shin et al. demonstrated a significant inferior performance of collagen conduits compared to autografts based on results of isometric tetanic tibialis anterior muscle force and weight, electrophysiology, and number of axons of the peroneal nerve.13 Although Whitlock et al. used traditional methods to analyze nerve regeneration, the lack of differences between groups does not necessarily means that the nerve reconstructions are similar.26,27 Their small group size and the poor reproducibility of sciatic function index (SFI) for segmental nerve injuries are important limitations of their study.28–30 Fewer studies using collagen conduits with 2.0 mm ID for either experimental or clinical purpose report mismatch between the conduit and the nerve diameters. Lohmeyer et al. report their clinical experience after collagen conduit implantation to repair digital nerve injuries. Excellent or good sensibility recovery was achieved in 9 out of 12 patients. Digital nerves are 2–3 times larger than the rat sciatic nerve and would provide a better match with the 2.0 mm ID conduits.15 In another study, Alluin et al. used similar conduits to bridge a peroneal nerve gap in rats. After 12 weeks, the weight of tibialis anterior muscle was significantly superior in the autograft group. The peroneal nerve is approximately half size of the sciatic nerve in rats what makes the 2.0 mm ID conduit diameter four times larger.31 In our study, the average diameter of the rat sciatic nerve measured approximately 1mm. The relation between the recipient nerve and conduit diameter appears to be an important
Figure 5. Conduits on the left side of the figures: NeuraGen conduit (1.5 mm ID) which measured 1.25 6 0.0 mm before hydration; conduits in the right side of the figures: NeuroMatrix conduit (2.0 mm ID) which measured 1.59 6 0.03 mm before hydration. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Microsurgery DOI 10.1002/micr
Influence of Conduit Diameter in Segmental Nerve Repair
aspect for nerve regeneration and variations in this parameter, which is not often reported, make study comparison difficult. The ID of collagen conduits clearly demonstrated to be an important parameter for nerve regeneration in this study and no report was found in the literature regarding the changes after implantation. In order to simulate implantation, the conduits were hydrated in normal saline solution at room temperature and analyzed regarding their ID and wall thickness. The ID of both conduits measured before hydration was 15% smaller than advertised and minimal change was seen after hydration. Although conduit diameters were smaller, the sciatic nerve stumps could still be sutured to the conduits since the average rat’s sciatic nerve diameter was smaller (0.94 mm). With the results from this study we can conclude that the reconstruction with collagen conduits with ID of 1.5mm (real diameter 1.25 mm after hydration) generates a better nerve motor recovery than the 2.0 ID conduits (real diameter of 1.69 mm after hydration) in this animal model. We can hypothesize a few reasons why a better match nerve/conduit could generate a better nerve recovery: (1) A decrease in conduit dead space could increase the concentration of growth factors released from the nerve stumps improving quality and quantity of regenerating axons; (2) A better mechanical support for growing axons with better size-matching nerve conduits could improve axonal direction and sustainability. Those points were not analyzed at this time and the exact cause of difference in motor recovery cannot be answered with this study alone and should be the object of future investigations. This study had a few limitations. The two different collagen conduits tested in the study belonged to different providers and, although they were made from similar material as described by the manufacture’s brochures (bovine collagen type I), the companies have their own proprietary methods of production. Testing conduits from the same provider would be ideal and should be recommended for future diameter comparison purposes. Potential causes for the poor results using larger conduits would be a possible increase in conduit dead space leading to a decrease in growth factor concentration released from the nerve ends or simply the poor mechanical support for growing axons. The lack of detailed analyses of the environment inside the conduits would be another limitation of the study that could help to clarify the cause of difference in motor recovery between the conduits. CONCLUSION
Reconstruction with autograft nerve was significantly superior to both collagen conduits and still is the best
651
option for motor recovery after segmental nerve reconstruction in the present model. When considering reconstruction with collagen conduits, improvement in motor recovery can be expected with a better size match nerve conduit. After implantation of the described collagen conduits, variations in conduit ID would probably be minimal.
REFERENCES 1. Lundborg G. Richard P. Bunge memorial lecture. Nerve injury and repair—A challenge to the plastic brain. J Peripher Nerv Syst 2003; 8:209–226. 2. Berger A, Millesi H. Nerve grafting. Clin Orthop Relat Res 1978; 133:49–55. 3. Giusti G, Willems WF, Kremer T, Friedrich PF, Bishop AT, Shin AY. Return of motor function after segmental nerve loss in a rat model: Comparison of autogenous nerve graft, collagen conduit, and processed allograft (AxoGen). J Bone Joint Surg 2012;94:410–417. 4. Merle M, Dellon AL, Campbell JN, Chang PS. Complications from silicon-polymer intubulation of nerves. Microsurgery 1989;10:130– 133. 5. Penna V, Wewetzer K, Munder B, Stark GB, Lang EM. The longterm functional recovery of repair of sciatic nerve transection with biogenic conduits. Microsurgery 2012;32:377–382. 6. Lundborg G. A 25-year perspective of peripheral nerve surgery: Evolving neuroscientific concepts and clinical significance. J Hand Surg [Am] 2000;25:391–414. 7. Weber RV, Mackinnon SE. Bridging the neural gap. Clin Plast Surg 2005;32:605–616, viii. 8. Meek MF, Coert JH. Clinical use of nerve conduits in peripheralnerve repair: Review of the literature. J Reconstr Microsurg 2002; 18:97–109. 9. Millesi H. Progress in peripheral nerve reconstruction. World J Surg 1990;14:733–747. 10. Pfister LA, Papaloizos M, Merkle HP, Gander B. Nerve conduits and growth factor delivery in peripheral nerve repair. J Peripher Nerv Syst 2007;12:65–82. 11. Whitlock EL, Tuffaha SH, Luciano JP, Yan Y, Hunter DA, Magill CK, Moore AM, Tong AY, Mackinnon SE, Borschel GH. Processed allografts and type I collagen conduits for repair of peripheral nerve gaps. Muscle Nerve 2009;39:787–799. 12. Waitayawinyu T, Parisi DM, Miller B, Luria S, Morton HJ, Chin SH, Trumble TE. A comparison of polyglycolic acid versus type 1 collagen bioabsorbable nerve conduits in a rat model: An alternative to autografting. J Hand Surg [Am] 2007;32:1521–1529. 13. Shin RH, Friedrich PF, Crum BA, Bishop AT, Shin AY. Treatment of a segmental nerve defect in the rat with use of bioabsorbable synthetic nerve conduits: a comparison of commercially available conduits. J Bone Joint Surg 2009;91:2194–2204. 14. Tang P, Kilic A, Konopka G, Regalbuto R, Akelina Y, Gardner T. Histologic and functional outcomes of nerve defects treated with acellular allograft versus cabled autograft in a rat model. Microsurgery 2013;33:460–467. 15. Lohmeyer JA, Siemers F, Machens HG, Mailander P. The clinical use of artificial nerve conduits for digital nerve repair: A prospective cohort study and literature review. J Reconstruc Microsurg 2009;25: 55–61. 16. Kempton LB, Gonzalez MH, Leven RM, Hughes WF, Beddow S, Santhiraj Y, Archibald SJ, El Hassan B, Shott S, Kerns JM. Assessment of axonal growth into collagen nerve guides containing VEGFtransfected stem cells in matrigel. Anat Rec (Hoboken) 2009;292: 214–224. 17. Dodla MC, Bellamkonda RV. Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps. Biomaterials 2008;29:33–46.
Microsurgery DOI 10.1002/micr
652
Giusti et al.
18. Marchesi C, Pluderi M, Colleoni F, Belicchi M, Meregalli M, Farini A, Parolini D, Draghi L, Fruguglietti ME, Gavina M, Porretti L, Cattaneo A, Battistelli M, Prelle A, Moggio M, Borsa S, Bello L, Spagnoli D, Gaini SM, Tanzi MC, Bresolin N, Grimoldi N, Torrente Y. Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection. Glia 2007;55:425–438. 19. Wells MR, Kraus K, Batter DK, Blunt DG, Weremowitz J, Lynch SE, Antoniades HN, Hansson HA. Gel matrix vehicles for growth factor application in nerve gap injuries repaired with tubes: A comparison of biomatrix, collagen, and methylcellulose. Exp Neurol 1997;146:395–402. 20. Xu X, Yee WC, Hwang PY, Yu H, Wan AC, Gao S, Boon KL, Mao HQ, Leong KW, Wang S. Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits. Biomaterials 2003;24:2405–2412. 21. Li ST, Archibald SJ, Krarup C, Madison RD. Peripheral nerve repair with collagen conduits. Clin Mater 1992;9:195–200. 22. Archibald SJ, Shefner J, Krarup C, Madison RD. Monkey median nerve repaired by nerve graft or collagen nerve guide tube. J Neurosci 1995;15(5 Part 2):4109–4123. 23. Moore AM, Kasukurthi R, Magill CK, Farhadi HF, Borschel GH, Mackinnon SE. Limitations of conduits in peripheral nerve repairs. Hand (NY) 2009;4:180–186.
Microsurgery DOI 10.1002/micr
24. Kemp SW, Syed S, Walsh SK, Zochodne DW, Midha R. Collagen nerve conduits promote enhanced axonal regeneration, schwann cell association, and neovascularization compared to silicone conduits. Tissue Eng Part A 2009;8:1975–1988. 25. Shin RH, Vathana T, Giessler GA, Friedrich PF, Bishop AT, Shin AY. Isometric tetanic force measurement method of the tibialis anterior in the rat. Microsurgery 2008;28:452–457. 26. Altman D, Bland JM. Confidence intervals illuminate absence of evidence. BMJ 2004;328:1016–1017. 27. Altman DG, Bland JM. Absence of evidence is not evidence of absence. BMJ 1995;311:485. 28. Shenaq JM, Shenaq SM, Spira M. Reliability of sciatic function index in assessing nerve regeneration across a 1 cm gap. Microsurgery 1989;10:214–219. 29. Weber RA, Proctor WH, Warner MR, Verheyden CN. Autotomy and the sciatic functional index. Microsurgery 1993;14:323–327. 30. Kanaya F, Firrell JC, Breidenbach WC. Sciatic function index, nerve conduction tests, muscle contraction, and axon morphometry as indicators of regeneration. Plastic and reconstructive surgery 1996;98: 1264–1271, discussion 1272–1264. 31. Alluin O, Wittmann C, Marqueste T, Chabas JF, Garcia S, Lavaut MN, Guinard D, Feron F, Decherchi P. Functional recovery after peripheral nerve injury and implantation of a collagen guide. Biomaterials 2009;30:363–373.
