Photochemical Sealing Improves Outcome Following Peripheral Neurorrhaphy

Journal of Surgical Research 151, 33–39 (2009) doi:10.1016/j.jss.2008.01.025

Photochemical Sealing Improves Outcome Following Peripheral Neurorrhaphy Anne C. O’Neill, M.D.,* Mark A. Randolph, M.A.S.,*,1 Kenneth E. Bujold, B.S.,† Irene E. Kochevar, Ph.D.,† Robert W. Redmond, Ph.D.,† and Jonathan M. Winograd, M.D.* *Plastic Surgery Research Laboratory; †Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston Massachusetts Submitted for publication September 17, 2007

Key Words: peripheral nerve; neurorrhaphy; photochemical tissue bonding; nerve wrapping; human amniotic membrane.

Introduction. Peripheral nerve transection initiates a complex molecular response in the severed nerve endings, resulting in the release of neurotrophic and neurotropic factors that are central to axonal survival and regeneration. In this study we tested the hypothesis that sealing the neurorrhaphy site from the surrounding environment using a photochemically bonded nerve wrap would optimize the endoneural environment and enhance regeneration and nerve function recovery. Materials and methods. Adult rats underwent unilateral sciatic nerve transection and standard epineural nerve repair. The repair site was wrapped with amniotic membrane or autologous vein and then was either sealed using photochemical tissue bonding (PTB) or secured with sutures. Photochemical sealing without a wrap was also carried out. Functional recovery was assessed at 2-wk intervals using walking track analysis and nerve histomorphometry was assessed at 12 wk. Results. Treating nerves with PTB-sealed amnion significantly improved functional recovery and increased distal axon and fiber diameters and myelin thickness compared to nerves treated with standard neurorrhaphy alone. Direct PTB sealing of the repair site also improved function. Neither amnion secured with sutures nor vein wraps exhibited improved functional or histological recovery compared to standard neurorrhaphy. Conclusions. These results suggest that sealing the peripheral nerve repair site with amnion using a photochemical technique may lead to earlier restoration of neural homeostasis and consequent enhanced repair of nerve injury. © 2009 Elsevier Inc. All rights reserved.

INTRODUCTION

Photochemical tissue bonding (PTB) is a developing tissue repair technique that utilizes a photosensitizing dye and visible laser light to produce an immediate, water-tight seal between tissue surfaces. The dye, without additional proteins or polymers, is applied to the tissue surfaces, which are then brought into contact and irradiated with visible light. While the exact mechanism of action is yet to be elucidated, we believe that light activation of the dye initiates chemical reactions that form covalent bonds between proteins on the tissue surfaces. PTB differs fundamentally from earlier laser tissue welding techniques that used lasers to generate thermal energy, resulting in denaturation of tissue proteins [1]. During the cooling process, the denatured proteins anneal, thus welding adjacent tissues. Laser welding is ultimately limited by the collateral tissue damage produced by heat. In contrast, thermographic studies have demonstrated that, using light and dye parameters that bond the tissue, the temperature in PTB-treated tissues does not rise above 40°C. PTB has been used experimentally in a number of tissue repair models and has been shown to seal corneal incisions in vivo and ex vivo [2– 4], bond transected tendon to produce increased early repair strength [5], seal incisional and excisional skin wounds with healing equivalent to sutured closure [6], and adhere skin grafts [7]. We have also previously demonstrated that PTB can be successfully used in peripheral nerve repair with functional and histological results similar to those ob-

1

To whom correspondence and reprint requests should be addressed at Division of Plastic Surgery, Massachusetts General Hospital, WAC 435, 15 Parkman Street, Boston, MA 02114. E-mail: [email protected].

33

0022-4804/09 $34.00 © 2009 Elsevier Inc. All rights reserved.

34

JOURNAL OF SURGICAL RESEARCH: VOL. 151, NO. 1, JANUARY 2009

tained with conventional epineural sutures [8]. That study indicated that circumferential sealing of the nerve repair site resulted in excellent restoration of normal neural architecture. Our understanding of the molecular processes governing nerve regeneration has been greatly improved in recent years [9, 10], and this has fuelled a new approach to nerve repair. Following peripheral nerve injury, the normal intraneural homeostasis is lost [11]. Injury initiates a complex series of events involving the up-regulation of important growth factors from resident cells, e.g., Schwann cells, and from transient cells, e.g., macrophages, in the nerve stumps [12]. Many of these factors have now been well characterized and their specific roles in the regenerative process identified [9, 10]. The precisely timed fluctuation in the regulation of neurotrophic factors and their receptors is likely to play an essential role in the peripheral nervous system’s ability to regenerate. With conventional suture approximation, the repair site is not completely sealed and isolated from the surrounding environment, which potentially reduces the concentration of growth factors and their ability to promote survival, regeneration, and restoration of normal nerve function. Many investigators have attempted to manipulate the process by the addition of individual growth factors [10, 13, 14] but precise reenactment of kinetics of the changes in concentration of these factors is not practical. In an alternative approach, tubulization techniques have been described in a bid to confine natural neurotrophic factors at the repair site [15–19]. While the results of clinical trials have been promising, they have failed to demonstrate a clear advantage over conventional suturing and there is concern regarding the risk of nerve compression within the silicone tubes. In this study we attempted to contain the growth factors within the repaired nerve by sealing the repair site, thus optimizing the endoneural environment and exploiting the nerve’s inherent capacity for regeneration. The concept of nerve wrapping has been advocated both experimentally and clinically. Nerve wraps have been applied as a secondary surgical procedure in cases of chronic and recurrent perineural scarring. Autologous vein wraps have the advantage of being readily available and producing minimal donor site defect [20, 21] and have been shown to be safe and effective in the prevention of perineural scarring and adhesions [22, 23]. More recently successful nerve wrapping with human amnion has been described [24]. Human amniotic membrane induces minimal inflammatory or immunological response [25] and therefore does not contribute to the compression of the nerve. In this study, we combined PTB with a modified nerve-wrapping technique using autologous vein and human amniotic membrane to seal the peripheral nerve repair site. We tested whether this adjunct to

standard neurorrhaphy can improve peripheral nerve regeneration in an acute rat sciatic nerve injury model. MATERIALS AND METHODS Surgical Procedures The institutional Subcommittee on Research Animal Care at Massachusetts General Hospital approved all procedures in this study. Forty-eight male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 250 –350 g were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg; Abbott Laboratories, Chicago, IL). A left groin incision was made to expose the femoral vessels. Using an operating microscope (Codman, Randolph, MA), the femoral vein was carefully dissected from the surrounding tissues. A maximal portion of the vein was excised. The femoral vein was excised in all animals to overcome any impact this may have on subsequent walking track analysis. The right sciatic nerve was then exposed through a dorso-lateral muscle-splitting incision. Using the operating microscope, the nerve was dissected from the surrounding tissues and sharply transected with a scalpel blade at a point 1 cm distal to its exit from the pelvis. The nerve was repaired immediately with six 10-O nylon epineural sutures (Ethicon, Somerville, NJ). Animals were then randomized to one of six experimental groups (Table 1).

Group 1: Control Group (n ⴝ 8) These animals received only standard neurorrhaphy repair. The results from this group served as the standard against which the results of other treatments are compared.

Group 2: PTB (n ⴝ 8) Rose Bengal dye (Aldrich, Milwaukee, WI; 0.1% (w/v)) in phosphatebuffered saline was applied to the repair site and allowed to absorb for 1 min. Excess dye was removed and the repair site was exposed to green laser light at 532 nm from a Compass 415 continuous-wave frequencydoubled Nd/YAG laser (Coherent Inc., Santa Clara, CA) at an irradiance of 0.5 W/cm 2 for 1 min followed by 180° rotation and repeat irradiation on the opposite side. Laser parameters and dye concentration were determined in pilot ex vivo studies.

Group 3: Amnion Wrap with PTB (n ⴝ 8) A 1-cm 2 segment of human amniotic membrane was treated with 0.1% Rose Bengal. The dye was allowed to absorb for 1 min. The segment was then wrapped around the repair site, covering the epineural repair. This area was then exposed to green laser light using the parameters described for group 2.

TABLE 1 Functional Recovery at 12 wks Postoperatively Group 1 2 3 4 5 6

Sciatic function index (SFI) ⫾ SD

Standard neurorrhaphy (SN) SN ⫹ PTB SN ⫹ amnion wrap ⫹ PTB SN ⫹ amnion wrap ⫹ suture SN ⫹ vein wrap ⫹ PTB SN ⫹ vein wrap ⫹ suture Note. Values are mean ⫾ standard deviation. * P ⬍ 0.05 compared to group 1.

⫺70.8 ⫾ 4.2 ⫺60.8 ⫾ 6.8* ⫺55.7 ⫾ 3.2* ⫺68.6 ⫾ 5.0 ⫺71.6 ⫾ 6.1 ⫺73.8 ⫾ 7.7

O’NEILL ET AL.: PHOTOCHEMICAL SEALING IMPROVES OUTCOME

Group 4: Amnion Wrap with Sutures (n ⴝ 8) 2

A one-cm segment of amnion was treated with Rose Bengal as outlined for group 3 and wrapped around the repair site. It was secured to the epineurium using a single 10-O nylon suture proximally and distally.

Group 5: Autologous Vein Wrap with PTB (n ⴝ 8) The harvested femoral vein segment was irrigated with saline to remove any blood remnants. The vein segment was trimmed to 1 cm length and the adventitia was removed. Ex vivo pilot studies indicated that the external surface of the vein bonded more readily than the luminal surface using PTB; consequently, 0.1% Rose Bengal was applied to this surface. Dye was allowed to absorb for 1 min before the excess was removed. The segment was then wrapped around the repair site, covering the epineural repair. This area was then exposed to green laser light using the parameters described for group 2.

35

ness were made 5 mm proximal and 5 mm distal to the repair site. Sections were stained with 0.5% (w/v) toluidine blue for light microscopy. Detailed histomorphometrical analysis was carried out on six nerves from each experimental group. Nerve architecture was examined at 400⫻ magnification in a blinded manner. Five 400⫻ images were taken of each proximal and distal segment at sites evenly distributed within the nerve. The number of myelinated fibers in each image was manually counted using Microsoft Photo Editor Software (Microsoft Corp., Redmond, WA). The average number of fibers in a 400⫻ field was calculated for proximal and distal segments of each nerve sample. The total area of each nerve was determined using Metamorph Imaging Software v4.6 (Universal Imaging Corporation, Downingtown, PA) and the total fiber counts were calculated. Fifty fibers were randomly selected in each image (total of 250 fibers per nerve sample). The fiber and axon diameters were measured. The myelin thickness was derived from the difference between the fiber and axon diameter. Additionally, the g-value was calculated as a ratio of the axon diameter to the fiber diameter.

Group 6: Autologous Vein Wrap with Sutures (n ⴝ 8) The autologous vein segment was prepared as outlined above and wrapped around the repair site. It was secured to the epineurium using a single 10-O nylon suture proximally and distally. Following the above procedures, the wounds were closed using 4-O absorbable Vicryl sutures (Ethicon). Postoperatively, animals were housed in the Massachusetts General Hospital animal facility and had unlimited access to water and rat chow. Animals were permitted to mobilize freely.

Preparation of Human Amniotic Membrane Human placenta was obtained with the approval of the institutional ethics committee. The placenta was washed with Earle’s balanced salt solution to remove residual blood clots. The amniotic membrane was peeled away from the chorion, placed on nitrocellulose paper, and cut into segments. Segments were stored at ⫺80°C in medium consisting of a 1:1 solution of 100% glycerol and Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with 1% penicillin-streptomycin (Gibco). Immediately prior to use, the membranes were defrosted and rinsed in phosphate-buffered saline for 2 h to remove all glycerol.

Walking Track Analysis Functional recovery was assessed at biweekly intervals postoperatively using walking track analysis in all animals. The hind feet were dipped in dilute India ink and the rats were allowed to walk down a 10 ⫻ 40 cm corridor into a darkened box. The floor of the corridor was covered with a removable paper used to record the animals’ paw prints. Three clear paw prints were selected from each walking track and the parameters of print-length, toe-spread (distance between first and fifth toes), and intermediary toe-spread (distance between second and fourth toes) were measured. The contralateral paw print was used to determine the normal values. The formula of Bain and coworkers was used to calculate the sciatic function index (SFI) with a value of 0 representing normal function and a value of ⫺100 indicating complete loss of function [26]. The SFI for animals from each repair group were then averaged for comparison.

Histology and Histomorphometry At 12 wk post repair the animals were sacrificed with an intraperitoneal injection of pentobarbital (200 mg/kg). The right sciatic nerve was harvested and fixed in 4% paraformaldehyde at 4°C for 48 h. Specimens were post fixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in araldite. Cross-sections 1 ␮m in thick-

FIG. 1. Time course for functional recovery assessed by walking track analysis. (A) Group 1 is compared to groups 2, 3, and 4. (B) Group 1 is compared to groups 5 and 6. *P ⬍ 0.05 compared to group 1.

36

JOURNAL OF SURGICAL RESEARCH: VOL. 151, NO. 1, JANUARY 2009

RESULTS

repair site (Fig. 2). In contrast, nerves treated with amnion wraps sealed with PTB (group 3) did not demonstrate any gross evidence of scarring and there was a marked absence of adhesions between the repair site and the surrounding tissues (Fig. 3). Gross findings in the other experimental groups were variable and did not differ greatly from the standard neurorrhaphy control group.

Functional Recovery

Histology and Histomorphometry

Animals treated with PTB alone or amnion wrap sealed with PTB (experimental groups 2 and 3, respectively) showed better functional recovery compared to the other groups (Table 1, Fig. 1). The group with PTB alone (group 2) showed greater recovery compared to other groups at wk 8 to 12, and the group with amnion wrap and PTB (group 3) showed greater improvement at wk 6 to 12. At the time of sacrifice (12 wk postoperatively) groups 2 and 3 showed significant functional improvement compared to standard neurorrhaphy alone (⫺55.7 ⫾ 3.2 and ⫺60.8 ⫾ 6.8 versus ⫺70.8 ⫾ 4.2, P ⬍ 0.05 in both cases; Table 1). There was no statistical difference between the sciatic function indices of the other experimental groups where functional recovery followed a similar pattern to that seen in animals treated with standard neurorrhaphy alone (Fig. 1). Our previous study showed that the SFI was much lower (⫺84.7 ⫾ 9.4) when the nerve was prevented from rejoining and identical SFI for nerves treated with standard neurorrhaphy (⫺70.6 ⫾ 17.8) [8].

Toluidine blue stained sections showed the presence of regenerated myelinated axons both proximal and distal to the repair site in all of the experimental groups (Fig. 4). Table 2 details the histomorphometric analysis for the mean fiber and axonal diameters and myelin thickness for each experimental group. The regenerated nerve fibers were found to be significantly larger in nerves sealed with PTB and amnion wraps (group 3) compared to standard neurorrhaphy alone (8.42 ⫾ 2.09 versus 5.98 ⫾ 3.30 ␮m, P ⬍ 0.05). Myelin thickness was also significantly increased in group 3 (3.53 ⫾ 0.41 versus 2.58 ⫾ 0.66 ␮m, P ⬍ 0.05). These parameters were also increased in the nerves treated with PTB without a tissue wrap (group 2) but did not reach significance.

Statistics Analysis of the data was performed using Sigmastat for Windows v2.3 (Systat Software, Inc., San Jose, CA). Statistical significance was set at P ⬍ 0.05. Analysis of variance and Tukey’s pair-wise comparison tests were used to evaluate the differences between the study groups.

Gross Findings

Prior to harvest, treated nerves were exposed through the original surgical incision and inspected. All nerves were found to be in continuity at the time of sacrifice. In animals treated with standard neurorrhaphy alone (group 1), there was gross evidence of scarring with some constriction of the nerve at the

A

DISCUSSION

Our results indicate that sealing the peripheral nerve repair site using a combination of photochemical technology and human amniotic membrane can result in both functional and histological improvements compared to standard neurorrhaphy alone. Amniotic membrane has many attributes that make it appropriate for sealing neurorrhaphy sites in conjunction with PTB. Because amnion is a thin (20-␮m) membrane, it can be readily wrapped around a small diameter nerve. It is also translucent, comprising a basement membrane

B

FIG. 2. Gross appearance of repaired nerve at harvest (12 wk postoperatively). (A) Standard neurorrhaphy alone (group 1) showing evidence of scar formation and constriction of the nerve at the repair site. (B) Amnion wrap sealed with PTB (group 3) showing no obvious constriction of the nerve. (Color version of figure is available online.)

O’NEILL ET AL.: PHOTOCHEMICAL SEALING IMPROVES OUTCOME

37

B

A

FIG. 3. Adhesion formation after neurorraphy (12 wk postoperatively). (A) Adhesions were observed after standard neurorraphy alone (group 1). (B) Neurorrhaphy sites treated with PTB-sealed amnion wrap did not show adhesion formation. (Color version of figure is available online.)

and an avascular stroma, allowing the green activation light to penetrate to the epineural–amnion interface. Amnion is also nonimmunogenic and human amniotic cells do not express HLA-A, -B, -C, or -DR antigens of ␤ 2-microglobulin [25, 27]. The presence of both antiinflammatory and anti-angiogenic proteins has been confirmed in human amniotic membrane [28]. Amniotic membrane has been shown to reduce acute inflam-

1

2

3

4

5

6

FIG. 4. Cross-sections 5 mm distal to the repair site showing regeneration of myelinated axons in all repair groups (toluidine blue ⫻400). The number in each panel indicates the experimental group.

mation with increased apoptosis of polymorphonuclear neutrophils and CD20⫹ cells detected in treated wounds [29, 30]. Infiltration of neutrophils, lymphocytes, and macrophages is reduced in corneal perforations treated with amniotic membrane [31]. In addition, human amnion has been shown to contain a wide range of growth factors even after preservation [32]. These include basic fibroblast growth factor and transforming growth factors (TGF-␣, TGF-␤1, TGF-␤2, TGF-␤3), which have been implicated in the nerve repair process. The amnion has also been shown to be a suitable substratum for growing axons in the central nervous system [33]. These features have lead to the use of amnion in a wide range of tissue repair models [34 –39]. In our study, the beneficial effects of the amnion wrap on functional outcome and nerve regeneration were dependent on the photochemical sealing process. Neurorrhaphy sites to which amnion was secured only with sutures (group 4) did not show enhanced outcomes (Tables 1 and 2; Fig. 1). PTB has been shown to create an immediate water-tight seal between high collagen content tissues such as cornea [2, 4]. Our photochemical studies with Rose Bengal suggest that covalent cross-links are formed between collagen molecules on tissue surfaces [40]. The stromal layer of amnion and epineurium both largely comprise collagen, suggesting that a similar process occurs in the present study to seal off the endoneurium from the outside environment. We postulate that sealing of the repair site contains the milieu of growth factors within the neural microenvironment, maximizing their neurotrophic effect. Interestingly, treatment of the repair site with Rose Bengal followed by direct laser illumination (group 2) also led to improved function compared to the other treatment groups, suggesting that some sealing of the repair site occurred. In this approach we expected that

38

JOURNAL OF SURGICAL RESEARCH: VOL. 151, NO. 1, JANUARY 2009

TABLE 2 Histomorphometric Parameters 5 mm Distal to Repair Site in Each Group Group

Total fiber count (⫻0.01)

Fiber density (mm 2) (⫻0.1)

Axon diameter (␮m)

Fiber diameter (␮m)

Myelin thickness (␮m)

G-value

Standard neurorrhaphy (SN) SN ⫹ PTB SN ⫹ amnion ⫹ PTB SN ⫹ amnion ⫹ suture SN ⫹ vein ⫹ PTB SN ⫹ vein ⫹ suture

25.5 ⫾ 6.0 26.9 ⫾ 5.0 24.0 ⫾ 5.6 24.7 ⫾ 5.8 27.6 ⫾ 5.9 25.6 ⫾ 6.1

93.7 ⫾ 17.0 103.7 ⫾ 20.4 96.6 ⫾ 16.0 108.4 ⫾ 28.7 103.5 ⫾ 30.5 97.0 ⫾ 20.1

3.40 ⫾ 2.28 4.16 ⫾ 1.24 4.89 ⫾ 1.48* 3.51 ⫾ 2.16 3.39 ⫾ 1.85 3.68 ⫾ 2.61

5.98 ⫾ 3.30 6.99 ⫾ 1.75 8.42 ⫾ 2.09* 5.75 ⫾ 2.69 6.01 ⫾ 2.28 5.86 ⫾ 3.15

2.58 ⫾ 0.66 2.83 ⫾ 0.39 3.53 ⫾ 0.41* 2.24 ⫾ 0.63 2.62 ⫾ 0.58 2.18 ⫾ 0.66

0.56 ⫾ 0.02 0.59 ⫾ 0.06 0.58 ⫾ 0.03 0.61 ⫾ 0.03 0.56 ⫾ 0.02 0.62 ⫾ 0.05

* Denotes statistical significance P ⬍ 0.05 compared to standard neurorrhaphy.

uniform approximation of the epineurium would be difficult and unlikely to lead to a reliable, tight circumferential seal, indicating that sealing the amnion wrap with PTB over the repair site (group 3) would be the preferred approach. Autologous vein wraps, with or without PTB (groups 5 and 6), did not produce functional or histological benefit. The adventitial surface has a high collagen content, similar to that of amnion. However, amnion is thinner than the vein graft, allowing it to adhere more closely to the curved epineural surface. In addition, amnion is significantly more translucent than autologous vein, allowing greater light penetration, which initiates bonding between the wrap and the epineurium. These features ensure that amnion forms a reliable and reproducible bond. Variations in the bond quality achieved with autologous vein may explain its lack of therapeutic effect. At the time of harvest we noted a marked reduction in perineural adhesion formation in nerves treated with amnion wraps sealed with PTB. This is consistent with the findings of other studies reporting the clinical applications of amnion where it is reported to reduce adhesion formations [39]. Interestingly amnion wraps that were only sutured in place did not appear to result in reduced adhesions. This may be explained by the fact that the amnion was very loosely applied in these cases and may have permitted the invasion of perineural scar at the repair site. Similarly the nerves treated with amnion wraps sealed with PTB showed very little gross evidence of scar formation or constriction at the repair site. These findings require further more objective evaluation. Our results showed that the amnion wrap sealed with PTB had better functional and histological outcomes. These results may be due to early restoration of the blood–nerve barrier integrity. The blood–nerve barrier plays an essential role in maintaining a suitable physicochemical environment for the axons [41]. The blood–nerve barrier consists of tight junctions between both the perineurial cells and the endothelial cells in the endoneurium and serves as a diffusion barrier that isolates the endoneurium from the general extracellular space of the body. Following nerve tran-

section, there is an increase in blood–nerve barrier permeability permitting the influx of proteins into the endoneurium. This influx may be necessary for some elements of the regenerative sequence such as the entry of macrophages for myelin phagocytosis or increase of important trophic factors [42]. It has been suggested that the relationship between the blood–nerve barrier and axonal regeneration is somewhat symbiotic with the increased permeability aiding neural repair and the new advancing axons, in turn, restoring blood– nerve barrier integrity [43, 44]. While the exact relationship is undetermined, it is clear that restoration of endoneural homeostasis is necessary to permit effective function of the regenerated nerve [11]. Additional studies will be required to determine if there is a relationship between sealing of the nerve repair site and reconstitution of the blood–nerve barrier that may provide a favorable neural microenvironment. In conclusion, we have demonstrated that sealing of the peripheral nerve repair site using a photochemical technique is an effective adjunct to conventional suture neurorrhaphy, resulting in improved functional recovery and histomorphometric parameters. These findings may be attributable to early restoration of neural homeostasis and the optimization of the neurotrophic and neurotropic response to nerve injury. These encouraging results warrant further investigation and characterization in a larger animal model. ACKNOWLEDGMENTS The authors gratefully acknowledge support from the DOD Medical Free Electron Laser Program F4 9620-01-1-0014 and from CIMIT (Center for Integration of Medicine and Innovative Technology).

REFERENCES 1.

Bass L, Treat M. Laser tissue welding: A comprehensive review of current and future clinical applications. Lasers Surg Med 1995;17:315.

2.

Mulroy L, Kim J, Wu I, comprise. Photochemical keratodesmos for repair of lamellar corneal incisions. Invest Ophthalmol Vis Sci 2000;41:3335.

O’NEILL ET AL.: PHOTOCHEMICAL SEALING IMPROVES OUTCOME 3.

4.

5.

6.

7.

8.

9.

10.

11.

12. 13.

14. 15.

16.

17.

18.

19.

20.

21.

22.

23.

Proano C, Mulroy L, Jones E, et al. Photochemical keratodesmos for bonding corneal incisions. Invest Ophthalmol Vis Sci 2004;45:2177. Proano C, Azar D, Mocan M, et al. Photochemical keratodesmos (PKD) as an adjunct to sutures for bonding penetrating keratoplasty corneal incisions. J Cataract Refract Surg 2004;30: 2420. Chan B, Amann C, Yaroslavsky A, et al. Photochemical repair of Achilles tendon rupture in a rat model. J Surg Res 2005;124: 274. Kamegaya Y, Farinelli W, Echague AV, et al. Evaluation of photochemical tissue bonding for closure of skin incisions and excisions. Lasers Surg Med 2005;37:264. Chan B, Kochevar I, Redmond R. Enhancement of porcine skin graft adherence using a light activated process. J Surg Res 2002;108:77. Johnson TS, O’Neill AC, Motarjem PM, et al. Photochemical tissue bonding: A promising technique for peripheral nerve repair. J Surg Res 2007;143:224. Lundborg G. A 25-year prospective of peripheral nerve surgery: Evolving neuroscientific concepts and clinical significance. J Hand Surg 2000;25A:391. Raivich G, Makwana M. The making of successful axonal regeneration: genes, molecules and signal transduction pathways. Brain Res Rev 2007;53:287. Olsson Y. Microenvironment of the peripheral nervous system under normal and pathological conditions. Crit Rev Neurobiol 1990;5:265. Ide C. Peripheral nerve regeneration. Neurosci Res 1996;25:101. Grothe C, Haastert K, Jungnickel J. Physiological function and putative therapeutic impact of the FGF-2 system in peripheral nerve regeneration—lessons from in vivo studies in mice and rats. Brain Res Rev 2006;51:293. Sahenk Z, Seharaseyon J, Mendell J. CNTF potentiates peripheral nerve regeneration. Brain Res 1994;655:246. Dahlin L, Anagnostaki L, Lundborg G. Tissue response to silicone tubes used to repair human median and ulnar nerves. Scand J Plast Reconstr Surg Hand Surg 2001;35:29. Heijke G, Klopper P, Doorn IV, et al. Processed porcine collagen tubulization versus conventional suturing in peripheral nerve reconstruction: An experimental study in rabbits. Microsurgery 2001;21:84. Heijke G, Klopper P, Doorn IV, et al. Silicone rubber tubulization in peripheral sensory nerve reconstruction: An experimental study in rabbits. Microsurgery 2001;21:306. Lundborg G, Rosen B, Dahlin L, et al. Tubular versus conventional repair of median and ulnar nerves in the human forearm: Early results from a prospective, randomized, clinical study. J Hand Surg [Am] 1997;22:99. Lundborg G, Rosen B, Dahlin L, et al. Tubular repair of the median or ulnar nerve in the human forearm: A 5-year followup. J Hand Surg [Br] 2004;29:100. Varitimidis S, Vardakas D, Goebel F, et al. Treatment of recurrent compressive neuropathy of peripheral nerves in the upper extremity with an autologous vein insulator. J Hand Surg 2001; 26A:296. Xu J, Varitimidis S, Fischer K, et al. The effect of wrapping scarred nerves with autogenous vein graft to treat recurrent chronic nerve compression. J Hand Surg 2000;25A:93. Ruch D, Spinner R, Koman L, et al. The histologic effect of barrier vein wrapping of peripheral nerves. J Reconstruct Microsurg 1996;12:291. Xu J, Sotereanos D, Moller A, et al. Nerve wrapping with vein

24.

25.

26.

27. 28.

29.

30.

31.

32.

33. 34.

35. 36.

37.

38. 39.

40.

41.

42.

43.

44.

39

grafts in a rat model: A safe technique for the treatment of recurrent chronic compressive neuropathy. J Reconstruct Microsurg 1998;14:323. Ozgenel G, Filiz G. Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve. J Reconstruct Microsurg 2004;20:153. Akle C, Adinolfi M, Welsh K, et al. Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet 1981;2:1003. Bain J, Mackinnon S, Hunter D. Functional evaluation of complete sciatic, peroneal and posterior tibial nerve lesions in the rat. Plast Reconstruct Surg 1989;83:129. Adinolfi M. HLA typing of amniotic fluid cells. Prenat Diagn 1982;2:147. Hao Y, Hui-Kang D, Hwang D, et al. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membranes. Cornea 2000;19:348. Park W, Tseng S. Modulation of acute inflammation and keratocyte death by suturing, blood and amniotic membrane in PRK. Invest Ophthalmol Vis Sci 2000;41:2906. Shimmura S, Shimazaki J, Ohashi Y, et al. Antiinflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea 2001;20:408. Nobe J, Moura B, Robin J, et al. Results of penetrating keratoplasty for the treatment of corneal perforations. Arch Ophthalmol 1990;108:939. Koizumi N, Inatomi T, Sotozono C, et al. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 2000;20:173. Davis G. Human amnion membrane serves as a substratum for growing axons in vitro and in vivo. Science 1987;236:1106. Badawy S, Baggish M, ElBakry M, et al. Evaluation of tissue healing and adhesion formation after intraabdominal amniotic membrane graft in the rat. J Reproduct Med 1989;34:198. Brandt F, Albuquerque C, Lorenzato F. Female urethral reconstruction with amnion grafts. Intl J Surg Invest 2000;1:409. Erdener A, Ulman I, Ilhan H, et al. Amniotic membrane wrapping: An alternative method to the splenorrhaphy in the injured spleen. Eur J Pediatr Surg 1992;2:26. Ghanbari Z, Dahaghin M, Borna S. Long-term outcome of vaginal reconstruction with and without amnion grafts. Intl J Gynaecol Obstetr 2006;92:163. Gruss J, Jirsch D. Human amniotic membrane: A versatile wound dressing. Can Med Assoc J 1978;118:1237. Young R, Mason B, Cota J, et al. The use of amniotic membrane graft to prevent postoperative adhesions. Fertil Steril 1991;55: 624. Shen H, Spikes J, Kopeckova P, et al. Photodynamic crosslinking of proteins ii. Photocrosslinking a model protein-ribonuclease A. J Photochem Photobiol 1996;35:213. Rechtand E, Rapoport S. Regulation of the microenvironment of the peripheral nerve: Role of the blood-nerve barrier. Progr Neurobiol 1987;28:303. Stoll G, Trapp B, Griffin J. Macrophage function during Wallerian degeneration of rat optic nerve: Clearance of degenerating myelin and Ia expression. J Neurosci 1989;9:2327. Seitz R, Reiners K, Himmelmann F, et al. The blood nerve barrier in Wallerian degeneration: A sequential long term study. Muscle Nerve 1989;12:627. Sparrow J, Kiernan J. Endoneurial vascular permeability in degenerating and regenerating peripheral nerves. Acta Neuropathol 1981;53:181.