Autologous nerve anastomosis versus human amniotic membrane anastomosis A rheological comparison following simulated sciatic nerve injury*☆

Guangyao Liu, Qiao Zhang, Yan Jin, Zhongli Gao

1Department of Orthopedics, China-Japan Friendship Hospital, Jilin University, Changchun 130031, Jilin Province, China

2Department of Ophthalmology, Second Hospital of Jilin University, Changchun 130041, Jilin Province, China

INTRODUCTION

Currently, clinical treatments for long-segment peripheral nerve injury commonly employ autologous nerve grafting; however is autologous nerve anastomosis the only effective therapeutic approach? This question is particularly relevant as autologous nerve anastomosis can easily lead to loss of nerve function in the donor area.

A number of studies have shown that human amniotic membrane anastomosis can inhibit fibrosis, prevent the formation of new blood vessels in scars, promote inflammatory cell apoptosis, and provide the appropriate milieu for axonal growth in the central nervous system[1-2].Human amniotic basilar membrane is remarkably strong and forms a support matrix containing laminin, fibronectin, type IV collagen, a large number of heparan sulfate proteoglycans and other types of proteoglycans, as well as neurotrophic factors[3-18].

The proportion of collagen fibers and elastic components present in the amniotic membrane effect its elasticity and plasticity[19].Collagen fibers are strong, yet highly flexible, so the amniotic membrane has a combination of strength and rigidity.Furthermore, elastic fibers are highly resilient;thus the amniotic membrane can extend under a load.Type II collagen in the extracellular matrix can help the amniotic membrane maintain a certain degree of elasticity and tensile strength[20].These biological characteristics enable the amniotic membrane to serve as a potential substrate for the repair of peripheral nerve damage.

No matter which approach is used—autologous nerve anastomosis or allogeneic human amniotic membrane anastomosis—the treatment for sciatic nerve injury should involve a consideration of the stress relaxation and creep characteristics of the sciatic nerve after transplantation, as well as attention to anastomotic tension and biocompatibility.

We speculate that the stress relaxation and creep characteristics of human amniotic membrane are similar to that of human sciatic nerve, based on previous physiological and morphological studies[3-17].Biomechanical studies of the amniotic membrane have solely focused on the simple tension method[21], which provides only a limited assessment of the mechanical behavior of the specimen.Both human amniotic membrane and sciatic nerve are viscoelastic tissues, showing stress relaxation and creep characteristics that help them to adapt to their particular physiological functions.In this study, we examine the stress relaxation and creep properties of the injured sciatic nerve following autologous nerve and human amniotic membrane anastomoses.

RESULTS

Stress relaxation of the sciatic nerve after autologous nerve and human amniotic membrane anastomoses

One hundred sets of data in the 7 200-second stress relaxation experiments of sciatic nerve specimens after autologous nerve and human amniotic membrane anastomoses were collected at 18 different time points for computer curve fitting; and because the stress decreased rapidly within 600 seconds and then subsequently diminished slowly, the time points for collecting data were 0, 60, 120, 180, 300, 420, 600, 900, 1 200, 1 500, 1 800, 2 400, 3 000, 3 600, 4 800, 5 400, 6 600,and 7 200 seconds (Figure 1).

Figure 1 Stress relaxation curves of the sciatic nerve specimens after autologous nerve and human amniotic membrane anastomosis.The vertical axis indicates the stress.The stress decreased rapidly within the first 600 seconds and then slowly declined.The stress relaxation curve tended to be normal at 7 200 seconds, appearing to obey a logarithmic function.The duration of the experiment was 7 200 seconds, and 100 sets of stress relaxation data were collected.Data are expressed as mean±SD.There were significant differences in stress reduction between the two groups, as revealed by t-tests(P < 0.05).

As shown in Figure 1, the stress on the injured sciatic nerve in the autologous nerve anastomosis group was 0.400 MPa at 0 second and 0.315 MPa at 7 200 seconds;decreasing by 0.085 MPa during this period.In the human amniotic membrane anastomosis group, the stress was 0.400 MPa at 0 second and 0.270 MPa at 7 200 seconds; decreasing by 0.13 MPa during this period.The reduction in stress in the human amniotic membrane anastomosis group was greater than that in the autologous nerve anastomosis group (P< 0.05).The normalized stress relaxation function curves of sciatic nerve specimens from the autologous nerve anastomosis group and the human amniotic membrane anastomosis group are provided in Figure 2.As this figure shows, the 7 200-second stress relaxation function of the injured sciatic nerve in the autologous nerve anastomosis group was less than that in the human amniotic membrane anastomosis group (0.68vs.0.79,P< 0.05).

Figure 2 Normalized stress relaxation function curves for sciatic nerve specimens from the autologous nerve anastomosis and human amniotic membrane anastomosis groups.The vertical axis represents the normalized stress relaxation function and the abscissa denotes time.Data are expressed as mean±SD.The 7 200-second stress relaxation function in the human amniotic membrane anastomosis group is less than that in the autologous nerve anastomosis group (P < 0.05).

Establishment of normalized stress relaxation functions of sciatic nerve specimens in the autologous nerve anastomosis and human amniotic membrane anastomosis groups

As shown in Figure 1, the stress relaxation curves resemble a logarithmic function, so they were defined as follows:

Wherecanddare undetermined coefficients.

The experimental data were entered into formula (2) to calculate thecanddvalues for the two groups.Subsequently, the resultant values were entered into formula (1)to obtain the normalized stress relaxation functions for the two groups of specimens, as follows:

Autologous nerve anastomosis group:

Human amniotic membrane anastomosis group:

Creep characteristics of sciatic nerve specimens in the autologous nerve anastomosis and human amniotic membrane anastomosis groups

The 7 200-second creep test of sciatic nerve specimens from the two groups were recorded at 18 different time points, for a total of one hundred sets of data for curve fitting.Because the stress decreased rapidly within 600 seconds and then gradually, the time points for collecting data were 0, 60, 120, 180, 300, 420, 600, 900, 1 200,1 500, 1 800, 2 400, 3 000, 3 600, 4 800, 5 400, 6 600,and 7 200 seconds (Figure 3).

As shown in Figure 3, the strain on the injured sciatic nerve in the autologous nerve anastomosis group was 8.63% at 0 second and 12.32% at 7 200 seconds, having increased 3.69%.In the human amniotic membrane anastomosis group, the strain was 9.28% at 0 second and 14.00% at 7 200 seconds, having increased 4.72%.Thus,the increase in the human amniotic membrane anastomosis group was greater than that in the autologous nerve anastomosis group (P< 0.05).The normalized creep function curves of sciatic nerve specimens from the autologous nerve anastomosis and human amniotic membrane anastomosis groups are shown in Figure 4.The 7 200-second creep function value for the human amniotic membrane anastomosis group was greater than that for the autologous nerve anastomosis group (1.14vs.1.12,P< 0.05).

Figure 4 Normalized creep function curves for sciatic nerve specimens in the autologous nerve anastomosis and human amniotic membrane anastomosis groups.The vertical axis represents the normalized creep function and the abscissa denotes time.Data are expressed as mean ±SD.The creep function value at the 7 200-second time point for the human amniotic membrane anastomosis group is greater than that for the autologous nerve anastomosis group (P < 0.05).

Establishment of normalized creep functions forsciatic nerve specimens from the autologous nerve anastomosis and human amniotic membrane anastomosis groups

As shown in Figure 3, the creep curves exhibited an exponential pattern, so the function was formulated as follows:

Two sets of data from creep experiments were entered into formula (4) to determine theaandbvalues for the two groups of specimens; then the resulting values were introduced into formula (3) to obtain normalized creep functions as follows:Autologous nerve anastomosis group:

Human amniotic membrane anastomosis group:

DISCUSSION

Stress relaxation tests demonstrated that stress reduction at the 7 200-second time point in the autologous nerve anastomosis group was significantly less than that in the human amniotic membrane anastomosis group.In conditions exhibiting similar constant tensile strain, reduced stress relaxation can help to lessen anastomotic tension and benefit the repair and anastomosis of injured nerves.

Creep tests revealed that the increase in strain at the 7 200-second time point in the allogeneic human amniotic membrane anastomosis group was significantly greater than that in the autologous nerve anastomosis group.Creep is the adaptive reaction of the tissue in response to tension (stress) and is important for determining the critical tension points for nerve damage and anastomotic rupture.Under protracted continuous physiological stress, good strain properties of the anastomosed nerve or other tissue can reduce the probability of anastomotic fracture, reduce anastomotic tension, and improve the chances for successful nerve repair.

A load beyond physical limits does not necessarily cause injury, because there is a range between the physical limit and the critical point for damage that plays an important role in the anastomosis of injured nerve.For nerve transection injury with minor damage,we can free and extend the nerve to bring the ends closer together to facilitate suturing[22].Under constant stress, nerve anastomosis exhibits creep (elongation)which is highly conducive to nerve anastomosis.

Our experimental data are unique due to differences in patient age and health status.In addition, complex clinical conditions may affect the type of stress on human amniotic membrane, such as the size, direction, time, frequency, and type of force.This study has some limitations because the elongated specimens were only tested by the creep and stress relaxation tests.The viscoelastic testing of biological soft tissues is influenced by many factors, so we should undertake an integrated and comprehensive analysis prior to clinical application.Further studies are required to conclusively determine creep characteristics under various stress conditions, and to more fully assess stress relaxation properties under several different strains, following human amniotic membrane transplantation.

MATERIALS AND METHODS

Design

A comparative study ofin vitroexperiments.

Time and setting

Experiments were performed from August to September 2009 in the Mechanical Experimental Center of Jilin University, China.

Materials

Experimental samples were harvested from the bilateral sciatic nerves from the gluteus maximus muscle of six Chinese adult male corpses who died of acute head trauma (aged 22–28 years) and were provided by the Department of Anatomy, Jilin University School of Medicine, China.Human amniotic membrane was from the placenta of five pregnant women who underwent cesarean section at the China-Japan Friendship Hospital of Jilin University (China) and was obtained following maternal informed consent.The sciatic nerve samples were stored in a plastic bag placed in a –20°C freezer and human amniotic membrane was preserved in normal saline at 4°C immediately after removing.

Methods

Preparation of amniotic membrane tubes

Amniotic epithelial cells were removed using the ammonia digestion method[23].The amniotic membrane specimens were cut into 25 multi-layer long curly amniotic membrane tubes at a diameter of 8.8–11.4 mm and 10 mm length, according to methods reported by Uchidaet al[18].

Preparation of sciatic nerve specimens

In total, 12 sciatic nerve samples were thawed at room temperature after been stored in the freezer for 1 day,and then each sample was cut into four pieces with a scalpel, for a total of 48 specimens.These simulated sciatic nerve injury specimens were randomly divided into the autologous nerve anastomosis and human amniotic anastomosis groups.Each group contained 24 specimens and 12 each were used for stress relaxation and creep experiments.The sciatic nerve specimens were cut in the middle using a sterile plastic-handle scalpel (Huaian Uniecom Medical Supplies Co., Ltd.,Huaian City, Jiangsu Province, China) to produce a 10-mm injury.The mutilated specimens were anastomosed with autologous nerve or human amniotic membrane using 7-0 nylon thread (Qingdao Nike Medical Material Co., Ltd., Qingdao, Shandong Province, China).

Stress relaxation experiments

The length and diameter of specimens (length 17.6–18.8 mm, diameter 8.8–11.4 mm) were measured with an AG-10TA electronic universal testing machine (Shimadzu,Tokyo, Japan; supplementary Figure 1 online) and a JXD-2 reading microscope (Changchun Optical Instrument Factory, Changchun City, Jilin Province, China)according to previously described methods[24], and each sample was pre-treated.In this experiment, the temperature was maintained at 36.5±0.5°C, to simulate human body temperature.Two groups of specimens were clamped in a soft tissue experimental fixture(Mechanical Experimental Center of Jilin University,China) which was connected to a glass cylinder that contained saline solution (pH 7.40).The specimens were immersed in saline and exposed to strain at 50%/min.Under 0.4 MPa stress, the strains in the autologous nerve anastomosis group and the allogeneic human amniotic membrane anastomosis group were 8.63% and 9.28%, respectively.Thus, the strain was kept constant.The duration of the stress relaxation experiment was 7 200 seconds, and 100 sets of experimental data were collected.

Creep experiments

The specimen clamping protocol, experimental temperature, data collection, and sample pre-treatment for the creep test were the same as those for the stress relaxation experiments, but the rate of stress increase was 0.5 GPa/min for the creep experiments.When the strains in the autologous nerve anastomosis and human amniotic membrane anastomosis groups reached 8.63%and 9.28%, respectively, under 0.4 MPa stress, the stress should remain constant.

Statistical analysis

A normalized approach was applied to analyze the stress relaxation and creep data for all specimens to establish a normalized stress relaxation equation and a normalized creep equation.Data were analyzed using SPSS 11.0 software (Chicago, IL, USA) and expressed as mean ±SD.Two groups of data were compared with pairedt-tests, and differences withP< 0.05 were considered statistically significant.

Author contributions:Guangyao Liu was responsible for study concept and design, and writing of the manuscript.All authors contributed to integrate, analyze and process the experimental data.Qiao Zhang was responsible for statistical analysis.Guangyao Liu and Yan Jin supervised manuscript preparation.Zhongli Gao was in charge of the funds.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by the Key Clinical Project from the Ministry of Health in China, No.2007-353.

Ethical approval:This study was approved by the Ethics Committee, China-Japan Friendship Hospital of Jilin University in China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol.6, No.31, 2011 after selecting the “NRR Current Issue” button on the page.

[1]Kim HS, Sah WJ, Kim YJ, et al.Amniotic membrane, tear film,corneal, and aqueous levels of ofloxacin in rabbit eyes after amniotic membrane transplantation.Cornea.2001;20(6):628-634.

[2]Goto Y, Noguchi Y, Nomura A, et al.In vitro reconstitution of the tracheal epithelium.Am J Respir Cell Mol Biol.1999;20(2):312-318.

[3]Mohammad J, Shenaq J, Rabinovsky E, et al.Modulation of peripheral nerve regeneration: a tissue-engineering approach.The role of amnion tube nerve conduit across a 1-centimeter nerve gap.Plast Reconstr Surg.2000;105(2):660-666.

[4]Wolford LM, Rodrigues DB.Autogenous grafts/allografts/conduits for bridging peripheral trigeminal nerve gaps.Atlas Oral Maxillofac Surg Clin North Am.2011;19(1):91-107.

[5]Aubá C, Hontanilla B, Arcocha J, et al.Peripheral nerve regeneration through allografts compared with autografts in FK506-treated monkeys.J Neurosurg.2006;105(4):602-609.

[6]Fujioka M, Tasaki I, Kitamura R, et al.Cavernous nerve graft reconstruction using an autologous nerve guide to restore potency.BJU Int.2007;100(5):1107-1109.

[7]O'Neill AC, Randolph MA, Bujold KE, et al.Preparation and integration of human amnion nerve conduits using a light-activated technique.Plast Reconstr Surg.2009;124(2):428-437.

[8]Henry FP, Goyal NA, David WS, et al.Improving electrophysiologic and histologic outcomes by photochemically sealing amnion to the peripheral nerve repair site.Surgery.2009;145(3):313-321.

[9]Pan HC, Yang DY, Chiu YT, et al.Enhanced regeneration in injured sciatic nerve by human amniotic mesenchymal stem cell.J Clin Neurosci.2006;13(5):570-575.

[10]O'Neill AC, Winograd JM, Zeballos JL, et al.Microvascular anastomosis using a photochemical tissue bonding technique.Lasers Surg Med.2007;39(9):716-722.

[11]Korte N, Schenk HC, Grothe C, et al.Evaluation of periodic electrodiagnostic measurements to monitor motor recovery after different peripheral nerve lesions in the rat.Muscle Nerve.2011;44(1):63-73.

[12]Yoshii S, Oka M, Shima M, et al.Bridging a 30-mm nerve defect using collagen filaments.J Biomed Mater Res A.2003;67(2):467-474.

[13]Toba T, Nakamura T, Lynn AK, et al.Evaluation of peripheral nerve regeneration across an 80-mm gap using a polyglycolic acid (PGA)--collagen nerve conduit filled with laminin-soaked collagen sponge in dogs.Int J Artif Organs.2002;25(3):230-237.

[14]Kottler UB, Jünemann AG, Aigner T, et al.Comparative effects of TGF-beta 1 and TGF-beta 2 on extracellular matrix production,proliferation, migration, and collagen contraction of human Tenon's capsule fibroblasts in pseudoexfoliation and primary open-angle glaucoma.Exp Eye Res.2005;80(1):121-134.

[15]Ueta M, Kweon MN, Sano Y, et al.Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction.Clin Exp Immunol.2002;129(3):464-470.

[16]Ravishanker R, Bath AS, Roy R."Amnion Bank"--the use of long term glycerol preserved amniotic membranes in the management of superficial and superficial partial thickness burns.Burns.2003;29(4):369-374.

[17]O'Neill AC, Randolph MA, Bujold KE, et al.Photochemical sealing improves outcome following peripheral neurorrhaphy.J Surg Res.2009;151(1):33-39.

[18]Uchida S, Inanaga Y, Kobayashi M, et al.Neurotrophic function of conditioned medium from human amniotic epithelial cells.J Neurosci Res.2000;62(4):585-590.

[19]Malak TM, Ockleford CD, Bell SC, et al.Confocal immunofluorescence localization of collagen types I, III, IV, V and VI and their ultrastructural organization in term human fetal membranes.Placenta.1993;14(4):385-406.

[20]Manabe Y, Himeno N, Fukumoto M.Tensile strength and collagen content of amniotic membrane do not change after the second trimester or during delivery.Obstet Gynecol.1991;78(1):24-27.

[21]Nakamura T, Yoshitani M, Rigby H, et al.Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction.Invest Ophthalmol Vis Sci.2004;45(1):93-99.

[22]Yin YQ, Liu ZJ, He GH.Effect of the different tensions on the morphologic feature of the median nerve in the rabbit.Acta Anat Sin.1995;26(4):346-350.

[23]Ngeow WC, Atkins S, Morgan CR, et al.The effect of Mannose-6-Phosphate on recovery after sciatic nerve repair.Brain Res.2011;1394:40-48.

[24]Hou XH, Li XY, Yang SB, et al.Effects of brachial plexus injury anastomosis simulation on biomechanical properties of adult brachial plexus.Neural Regen Res.2010;5(6):471-475.