Journal of Investigative Surgery, Early Online, 1–10, 2014 C 2014 Informa Healthcare USA, Inc. Copyright ISSN: 0894-1939 print / 1521-0553 online DOI: 10.3109/08941939.2014.906688
ORIGINAL RESEARCH
Novel Technique for Repair of Severed Peripheral Nerves in Rats Using Polyurea Crosslinked Silica Aerogel Scaffold Firouzeh Sabri,1 David Gerth,2 George-Rudolph M. Tamula,1 Thien-Chuong N. Phung,1 Kyle J. Lynch,1 John D. Boughter Jr3
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1
Department of Physics, University of Memphis, Memphis, Tennessee, USA, 2 Department of Otolaryngology Head and Neck Surgery, UTHSC, Memphis, Tennessee, USA, 3 Department of Anatomy and Neurobiology, UTHSC, Memphis, Tennessee, USA
ABSTRACT Purpose/Aim: To design, synthesize, and test in vivo an aerogel-based top-open peripheral nerve scaffold to simultaneously support and guide multiple completely severed peripheral nerves in a rat model. Also, to explore options for immobilizing severed nerves on the aerogel material without the use of sutures resulting in reduced surgical time. Materials and Method: A novel material and approach was developed for the reattachment of severed peripheral nerves. Nerve confinement and alignment in this case relies on the surface properties of a lightweight, highly porous, polyurea crosslinked silica aerogel scaffold. The distal and proximal ends of completely transected nerve terminals were positioned inside prefabricated “top-open” corrugated channels that cradled approximately two thirds of the circumference of the nerve trunk and connectivity of the severed nerves was evaluated using sciatic function index (SFI) technique for five months post-surgery on 10 female Sprague–Dawley rats then compared with the gold standard for peripheral nerve repair. The interaction of nerves with the surface of the scaffold was investigated also. Results and Conclusion: Multichannel aerogel-based nerve support scaffold showed similar SFI recovery trend as the case suture repair technique. Usage of an adhesion-promoting coating reduced the friction between the nerve and the scaffold leading to slippage and lack of attachment between nerve and surface. The aerogel scaffold used in this study did not collapse under pressure during the incubation period and allowed for a rapid and non-invasive peripheral nerve repair approach without the demands of microsurgery on both time and surgical expertise. This technique may allow for simultaneous repair and reconnection of multiple severed nerves particularly relevant to nerve branching sites. Keywords: aerogel; scaffold; nerve; peripheral; repair; conduit; porous; crosslinked
INTRODUCTION
and training [5, 10–12]. Therefore, sutureless nerve repair techniques [13–18] have also been investigated in recent years with mixed degrees of effectiveness. Polyurea crosslinked silica aerogels are highly porous, mechanically strong, translucent materials that have demonstrated a promising future for in vivo and in vitro biomedical applications [19–21]. Their highly textured nanoscopic surfaces are expected to contribute to enhanced cell attachment and proliferation [22–25]. Additionally, the distinct ultrasonic behavior of aerogels allows for easy in vivo tracking of implants as shown in a recent study [26]. These advantages point to the potential use of this material in sutureless nerve repair. In this study, a peripheral nerve repair scaffold
Trauma patients suffer the majority of peripheral nerve injuries and the cost for U.S. peripheral nerve repair has exceeded $1 billion in recent years [1, 2]. Today, the gold standard in nerve repair consists of surgeons employing sutures to repair the severed nerves when the gap between distal and proximal terminals is short [3–5]. Complications associated with autografts and allografts have limited the performance of these techniques [6, 7] and thus paved the way for tubular conduits [7–9]. Complications associated with use of sutures include hemorrhaging, extended surgery time, and the need for a high level of surgeon skill Received 7 December 2013; accepted 17 March 2014.
Address correspondence to Firouzeh Sabri, Department of Physics, University of Memphis, Memphis, TN 38152, USA. E-mail: fsabri@ memphis.edu
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2 F. Sabri et al. was designed from polyurea crosslinked silica (X-Si) aerogels and tested on a widely used and accepted nerve model, the sciatic nerve of Sprague–Dawley rats [27–30]. In additional experiments, we investigated the effect of synthetic and biologic adhesives on the interaction and amount of available mobility of the peripheral nerve with the scaffold surface.
MATERIALS AND METHODS
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Preparation of Aerogel Implants Pigmented and clear polyurea crosslinked silica aerogels were synthesized according to our previously described formulation [20]. The aerogel implants were cut and roughly shaped from the bulk material using a diamond tipped abrasive disk mounted in a rotary tool. For the deep muscle study (sciatic nerve location and facial nerve location), samples were polished by hand to the final desired size while the nerve confinement channels were created by a fine-tipped rotary tool. All implants used for in vivo procedures were sterilized for 24 hr by a standard ethylene oxide sterilization process in an Amprolene system (Anderson Products) prior to surgery. Implants used for in vitro and cadaver testing were kept in an airtight container and rinsed with isopropyl alcohol immediately prior to usage. Figure 1 shows examples of aerogel scaffolds with various possible channel configurations and implant geometries. All samples were tested for mechanical integrity after the machining and shaping process was completed. In Figure 2, cross-sectional view of aerogel scaffolds specifically used in this study is shown both in the dou-
ble channel (Figure 2a) and single channel (Figure 2b) mode, while embedded in tissue or paraffin.
In Vivo Aerogel Scaffold Experiments All surgical procedures described in this section were performed on 10 female Sprague–Dawley rats weighing 200–300 g. Rats were anesthetized 15 min prior to surgery via an initial intramuscular injection of Telazol (0.03–0.05 ml at 0.3–0.5 mg/kg), followed by isoflurane inhalation at 1%–2% as described previously [19]. During the post-surgery recovery period none of the animals were restrained and they were all allowed to continue with their normal grooming routine. They were fed a routine diet and kept under close observation for signs of infection or abnormal behavior. Rats were euthanized post implant recovery by an overdose of carbon dioxide. This study was approved by the Animal Care and Use Committee at the University of Memphis. Sciatic Nerve Procedure Figure 3 shows a schematic diagram of the aerogel scaffold nerve support concept used in this study drawn for the sciatic nerve location. The top-open channel design demonstrates preexisting channels to support bifurcation of the sciatic nerve post-surgical severance. Each rat was weighed before surgery and administered carprofen at the dose of 5 mg/kg subcutaneously before the surgery. All surgeries were performed aseptically. Specifically, the surgical area (left hind limb) was shaved, cleaned with a betadine soap, and wiped with 70% isopropyl alcohol after undergoing
FIGURE 1 Aerogel scaffold configurations: Optical images of different pigmented and clear X-Si aerogel scaffold configurations. (a) Tubular conduit with Au thread passing through, (b) “top-open” 1-D channel with Cu thread passing through, (c) “closed” multichannel, and (d) closed configuration with a “snake” channel configuration. All implants were tested for mechanical integrity after channel formation and no defects were found. Scale bars = 5 mm. Journal of Investigative Surgery
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Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study
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FIGURE 2 Embedded aerogel scaffolds: Cross-sectional images of (a) dual channel and (b) single channel X-Si scaffold embedded in (a) paraffin and (b) tissue, prior to sectioning. The channels are formed along the longitudinal axis of the implant (not shown). Scale bars = 5 mm.
anesthesia in the surgical prep area. After the animal was moved into the surgery room, the surgical area was draped with a sterile drape. The surgeon wore a mask, head cover, and clean scrubs. He used sterile gloves as well as sterilized instruments for each surgery. Under a dissecting microscope, a transverse incision was made through the skin of the limb halfway between the iliac crest and the femur’s articulation with the tibia. A self-retracting retainer was replaced, and dissection was carried down between the biceps femoris and gluteus muscle until the sciatic nerve was identified. The
sciatic nerve of the Sprague–Dawley rats were exposed and severed sharply at the branching site. At this stage five different procedures were performed that will be described below: (I) Physical immobilization with X-Si aerogel scaffold: Once the sciatic nerve was severed, the doublechannel (Figure 2a) aerogel implant was positioned between the muscle and the sciatic nerve branches such that the nerve terminals were in direct contact with the surface of the implant and
FIGURE 3 Aerogel scaffold for nerve support: Illustration showing the concept of aerogel scaffold with preexisting channels specifically formed for the sciatic nerve bifurcation region and the positioning of the nerves in the channels, at the time of surgery. C
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cedures. The rats were observed at least twice a day for the next three days to observe if the rats were eating and drinking normally as well as assessing use of the rear leg. No antibiotics were given. There were no complications following any of the surgeries.
FIGURE 4 Aerogel insert at sciatic nerve surgical site: In vivo optical image of the multi-channel X-Si aerogel scaffold prior to insertion at the sciatic nerve surgical site. Nerves were severed prior to positioning of the implants.
(II)
(III)
(IV)
(V)
were positioned inside the tracks previously described. No adhesive or sutures were used for immobilizing the aerogel implant in this case and nerve positioning took approximately 5 min. The skin was then closed with staples and the animal was allowed to emerge from anesthesia. Physical immobilization with laminin-coated X-Si scaffold: The sciatic nerve was exposed and severed as previously described. The free nerve terminals were then positioned inside the tracks of a double-channel implant that was coated with a 50 μl drop of laminin (Sigma-Aldrich, St. Louis, MO) applied with a syringe tip (Figure 4). Nerves and implant were held together for approximately 30 s until the laminin drop began to gel. The skin was closed and animal cared for as described in the previous section. Suture repair: The sciatic nerve branches were exposed once again and transected sharply as described above. The nerve segments were then coapted using standard epineural suture technique with interrupted 9–0 nylon sutures. This process took approximately 18 min. No repair: The sciatic nerve branches were exposed and severed, however, nerve endings were then abandoned and no coaptation was attempted. Healthy rat-control: A healthy female Sprague– Dawley rat of the same age and weight with no procedures performed was used as a positive control.
After each surgery every rat was given 2 ml of Lactated Ringer’s subcutaneously. Carprofen was injected subcutaneously at the dose of 5 mg/kg/day for the next two days. Carprofen is an NSAID that is commonly given to rodents for pain following surgical pro-
Post-surgical Testing A custom-built wooden walking track with adequate confinement and lined with chart paper was used for animal gait evaluation. Non-toxic water-soluble paint was first applied to the rats’ hind paws and animals were (individually) encouraged to move toward a darkened cubicle, at the end of the track. Upon confinement of the rat, the prints were promptly removed from the chamber and allowed to dry before analysis. Rats from all groups were tested periodically, at least once a week for a total of five month post-surgery. Paw print data were analyzed using the sciatic function index (SFI) using the Bain—Mackinnon–Hunter (BMH) formula [31, 32]. At the conclusion of the study, rats were euthanized via CO2 asphyxiation.
Mechanical Testing Procedures In Vitro The strength of attachment of cadaveric rat sciatic nerves to aerogel implants were tested in vitro by means of the Lap shear test [33] for several biological and non-biological adhesives. Adhesives tested include: commercially available dental adhesives AllBond SE and One-Step (Bisco Inc., Schaumburg, IL), Super glue, Cell-Tak (BD Biosciences, CA), and basement membrane extract (BME) (R&D Systems, Minneapolis, MN). Earlier, adhesives were prepared and mixed according to the individual manufacturers’ recommendations. Sciatic nerves were removed from the left and right hind legs of cadaveric Sprague–Dawley rats immediately prior to testing. Tests were performed by applying two coats of the adhesive under observation to one side of a disk-shaped aerogel implant prepared in Section 2.1. Before curing/gelling of each type of adhesive, one end of the extracted sciatic nerve was positioned in the center of the coated area of the aerogel disk and allowed to attach. In each case the area of the nerve overlapping the adhesive area was kept constant. The top clamp of a Mark-10 bench top tensile tester was attached to the free end of the aerogel disk while the bottom clamp held onto the free end of the sciatic nerve in each case. The tensile tester loaded with a 20lb force gauge was programmed to “pull” at a rate of 30 mm/min for all measurements. The tensile behavior of the cadaver sciatic nerves alone was also tested. All cadaver nerves used for this part of the study were extracted from cadavers of the same age, weight, and species for consistency. Experiments were repeated for Journal of Investigative Surgery
Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study
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each type of adhesive at least twice and in some cases three times. In Vivo The sciatic nerve of a rat cadaver was exposed and the length of the intact nerve was measured using a digital caliper while the limb was in full extension. The nerve was then severed at one end and it instantly retracted to a final length that was measured again as previously mentioned. The free end of the sciatic nerve was then attached using appropriate clips to a handheld portable force gauge. The nerve was stretched back to its original length prior to severance and the force gauge reading was recorded. The spring constant of the sciatic nerve was then calculated. The full length of the nerve was removed from the rat and stored in a 4◦ C refrigerator for one week. The experiment was repeated on the “aged” sciatic nerve and the spring constant calculated again. Friction Studies In order to simulate the effect of a high water content interfacial layer (e.g., laminin) on the adhesion and frictional behavior of two surfaces, the following experiment was performed: a mesh number 220 strip of sandpaper (aerogel stimulant) was attached to a glass microscope slide and then positioned in the bottom clamp of a Mark 10 bench-top tensile tester. Sciatic nerves were removed as described previously from freshly euthanized rats and immediately transferred to the tensile tester. Approximately 3 mm of the length of the sciatic nerve was held by the top clamp. The free length of the sciatic nerve was brought into contact with the surface of the sandpaper and the nerve was dragged along the sandpaper at a rate of 30 mm/min until the two surfaces had completely lost contact. The experiment was repeated and all parameters were kept the same as before with the exception of a 50 ml drop of water, coating the longitudinal axis of the sandpaper, immediately before the nerve segment making contact with the surface of the sandpaper. The experiment was repeated twice and a fresh nerve was used each time. The force magnitude required to pull the nerve along the length of the sandpaper surface with and without the water drop was compared and registered.
RESULTS
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averages of SFI values collected weekly for each rat. The error bars reflect the standard error of the mean for each month. The BMH index is expected to theoretically vary between 0 (a completely functional nerve) and −100 (a completely dysfunctional nerve) [31, 32]. The gait of the healthy rat with no surgical intervention (V) is shown in Figure 5a with SFI values between −10 and −25 serving as a positive control. In this case, SFI values are closest to the zero mark among the various groups and indicate the baseline errors in the measurement technique. The rat with severed nerve and no surgical intervention (IV) served as the negative control for the study and the SFI collected from this subject is shown in Figure 5b. The SFI for this group, theoretically, was expected to be around −100. Although there were few outliers, the data in Figure 5b show a concentration of large and negative SFI values, which agrees with previously established literature. The animal’s gait is clearly impaired initially and continues to demonstrate a wide “swing.” The current gold standard for nerve repair is suture repair (III) and SFI values from this group are shown in Figure 5c for the same recovery period. The initial values for the SFI are significantly lower than previously seen, indicating an advantage over groups V and IV. As the months progressed, collected SFI values showed a concentration between −70 and −40. The initial SFI values for this case (Figure 5d) show a behavior slightly more impacted by nerve damage than the suture-repaired case (Figure 5c, group III). Overtime however, the SFI values for the two groups reach a similar range and they plateau at a value of −39. This fact may suggest that the sutureless polyurea crosslinked aerogel technique may have assisted in the healing process of the sciatic nerve and could be a possible alternative to nerve suturing. Upon extraction of the implant(s), the scaffold was located at the original surgery site and despite the lack of sutures, implant had not traveled. Physical immobilization of severed nerves was also tested with a laminin-coated X-Si scaffold (II) where a thin layer of laminin was sandwiched between the nerves and the channels. The gait recovery was expected to out-perform the behavior of group I but demonstrated a poorer recovery as a function of time as can be seen in Figure 5e. Over time, the animals’ gait became significantly wider and was no longer registered on the graph paper. Upon implant extraction it was noticed that the implant had shifted from the initial position and caused an obstruction.
Walking Track Analysis In all surgical cases, the operated legs were paralyzed and contractions of the hind paws of the operated leg were not present immediately after surgery. SFI values generated for each test group (I, II, III, IV, and V) are shown in Figure 5 for five month post-surgery until the rats were euthanized. Each plot represents monthly C
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Tensile Behavior of Cadaveric Nerves The tensile behavior of sciatic nerves extracted from three different cadaveric rats of similar weight and age is shown in Figure 6. Figure 6a shows the elastic region of all tested nerves as well as their rupture
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FIGURE 5 Sciatic function index (SFI) recovery: Comparison of gait recovery over five month post-surgery for groups (a) no surgery-positive control (V), (b) severed with no surgery-negative control (IV), (c) suture repair-gold standard (III), (d) X-Si scaffold (I), and (e) X-Si scaffold + laminin (II). SFI values represent monthly average values with standard error of the mean reflecting the error bars.
Journal of Investigative Surgery
Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study
culated using classical Hookean relationship and the spring constant value for a 2 hr old nerve was calculated to be 2.29 N/cm. This value appears to be consistent with values reported in literature, for example, a value of 1.96 N/cm for a tibial nerve [34]. After one week of “aging,” the spring constants were measured again and registered an average value of 0.228 N/cm clearly demonstrating the impact of aging on the tensile properties of the nerves.
5.0
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Load (N)
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2.5 2.0 1.5
Analysis of Nerve–Substrate Attachment Behavior
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(b) FIGURE 6 Tensile behavior of sciatic nerves: Room temperature tensile behavior of cadaver rats sciatic nerves. (a) The sciatic nerves show an elastic behavior up to ∼ 4 N above which nerve rupture commences and deviations in individual nerves become apparent. (b) Zoom on lower points showing similar elongation behavior for all three nerves tested.
behavior at load values between 3.5 and 5N. Some variation in the load tolerance for each nerve is expected since the tightness of the grips, the fibrous nature of the tissue, and the age of the nerve do impact the rupture load behavior. Figure 6b shows the linear region of the graph where the three different nerves show consistent load-elongation behavior. The graph suggests that the mechanism used for nerve attachment should not exceed force values of 3N otherwise damage to the nerve trunk could occur and nerve regrowth and extension may be suppressed. An average value for spring constant of sciatic nerve for a Sprague–Dawley rat was cal C
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These results are summarized in Table 1 and suggest that the implant for group II did not show an SFI profile similar to group I (or better) due to the presence of the laminin leading to “slippage” of the nerves from the channels surface. These results suggest that with adequate traction and friction between the two surfaces in contact it is possible to retain connection if the implant surface is engineered to posses sufficient texture without the use of adhesives. The attachment strength of sciatic nerves to aerogel implants by means of non-biological adhesives such as superglue, All-Bond, and One-Step exceeded the natural load tolerance of the nerve fibers and the nerves ruptured prior to failure of the attachment. For these experiments, the nerve fibers tore at load values close to the values determined for the nerve while nerve fibers embedded in the adhesive region remained “trapped.” In the case of biological adhesives (BME and Cell-Tak) however, all attachments failed at very small loads and the sciatic nerves slipped off the implant surfaces with little effort, again, suggesting that moderate amounts of friction and “traction” between nerve body and the surface of interest will play an important role in the attachment of the two entities The non-slippage behavior of severed nerve trunks when placed in direct contact with aerogel scaffold was confirmed again using a branch of the facial nerve. The severed ends of the facial nerve of a freshly euthanized Sprague–Dawley rat were positioned inside the single channel opening of an X-Si implant and the two terminals remained in place, without retracting and slipping from the channel. The gap present between the two ends shown in Figure 7 is due to thickness of the implant.
TABLE 1 Effect of a water-based coating on attachment: Summary of two trails comparing the force required to sustain motion on a “dry” surface versus a “wet” surface
Trial 1 Trial 2
F = F2 −F1 (N)
Pull rate (mm/min)
0.15 0.2
30 30
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TABLE 2 Summary of adhesion tests: Table summarizing the adhesives tested and the outcome of the tests. Biological adhesives with a high water content acted as a friction-reducing agent leading to slippage of nerves while non-biological adhesives completely immobilized the nerves with no opportunity for mobility and migration
FIGURE 7 Aerogel insert at facial nerve surgical site: Optical image of the single channel X-Si aerogel scaffold prior to insertion at the facial nerve surgical site in a freshly euthanized cadaver rat. The facial nerve was severed immediately prior to positioning of the implants. Channel does not contain any coatings or adhesives and nerve segments remain attached to the channel due to frictional forces.
Sample tested
N
X-Si Aerogel + All Bond + Nerve X-Si Aerogel +One-Step + Nerve X-Si Aerogel+ superglue + Nerve X-Si Aerogel + Cell-Tak + Nerve X-Si Aerogel + BME + Nerve
2 2 3 3 2
Comments Nerve ruptured before attachment failure Nerve ruptured before attachment failure Nerve ruptured before attachment failure Attachment failed before nerve ruptured Attachment failed before nerve ruptured
DISCUSSION AND CONCLUSION In Figure 8, typical force-travel behavior for a “wet” surface is compared with a “dry” surface demonstrating the lack of “traction” when the surface is coated with a high water content coating, while on a “dry” surface significant force is required to travel along the length of the surface. The measurement was stopped once the full length of the nerve had reached the other end of the travel path. Table 2 shows the results of two trials demonstrating at least 0.15N of force required to sustain motion in the “dry” case while in the “wet” case hardly any force was needed leading to slippage and lack of traction between the two surfaces that are in contact.
FIGURE 8 Effect of friction on nerve mobility: Example behavior of sustaining nerve motion and mobility on a “wet” surface compared to a “dry” surface.
We designed a novel scaffold for peripheral nerve repair, constructed from X-Si aerogel with a high degree of surface friction and physical confinement capabilities. Silica aerogels are a lightweight, mesoporous, strong, and biocompatible material with great promise for medical applications. Given that aerogels can be easily shaped to contain multiple channels with the desired channel configurations, it can be developed for any of the peripheral nerves. Here, we constructed and evaluated a multi-channel X-Si aerogel scaffold for sciatic nerve connection and repair in rats. Walking track analyses carried out during recovery confirm that surgical intervention and proper reapproximation is clearly necessary for the correct reconnection of severed nerve terminals. Assessment of recovery of function was made using SFI computations; collection of prints using this method suffers from inconsistencies that are often hard to control. The SFI “trend” however for each experimental group is clear over the five month period with suture repair and scaffold with no laminin showing similar recovery trends. The surgical repair method presented here offers insight into the development of a rapid and non-invasive peripheral nerve repair approach that may offer an alternative to allograft/autograft without the demands of microsurgery on both time and surgical expertise. As shown in this study, aerogel scaffold surface properties play an important role in nerve attachment and nerve mobility where a moderate amount of friction can aid in preventing nerve slippage and the absolute need for sutures. Physical immobilization of severed nerves with X-Si aerogel scaffold (I) containing preexisting channels was significantly easier and faster than the traditional suture repair procedure. Processing and surgical time was reduced significantly by using the X-Si aerogel scaffold (less than 5 min) in comparison with the suturing technique (approximately 18 min). The Journal of Investigative Surgery
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Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study presence of the laminin layer did not enhance nerve recovery. Instead, it acted as a lubricant and reduced the friction between the two surfaces (nerve + channel) hence leading to an implant repositioning occurring. This hypothesis was confirmed when tensile tests performed on a wet versus dry surface showed a noticeable difference in the force required to pull a sciatic nerve on the surface, simulating the migration of nerves on a scaffold with/without a coating layer (discussed in Section 3.3). Tests performed on the facial nerve (Figure 7) also showed good adhesion between the nerve and the substrate. Therefore, while waterbased biological “adhesion-promoting” layers such as BME, laminin, and Cell-Tak are necessary for the attachment and proliferation of individual cells, they significantly reduce the surface friction between the nerves’ exterior and the surface of the scaffold. It is therefore likely that the mesoporous nature of the aerogel scaffold directly impacts the nerve–substrate interaction. While the relationship between scaffold pore size and cell activity (attachment) is currently poorly understood, it is clear that if pores are too large (μm size) there is a decrease in the specific surface area available which in turn limits cell attachment [35]. As a result, mesoporous substrates offer a unique advantage over other materials providing extra “anchoring” opportunities. In a recent study, cells demonstrated improved distribution and adhesion to mesoporous substrates compared with featureless smooth surfaces [36], which supports the current hypothesis. While it is expected that the optimal pore size will vary with different cell types [37], this study suggests that further investigation into aerogel-based scaffolds will facilitate the design of new nerve repair conduits and scaffolds.
ACKNOWLEDGMENTS The first author would like to acknowledge John Daffron, Department of Physics, for all support with instrumentation design and fabrication. The authors are also grateful to Dr. Karyl Buddington for all surgical procedures that were conducted at the University of Memphis Animal Care Facility. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
FUNDING This study was funded by the FedEx Innovation Grant, FedEx Institute of Technology, University of Memphis. C
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[22] Fana YW, Cuia FZ, Chenb LN, et al. Adhesion of neural cells on silicon wafer with nano-topographic surface. Appl Surface Sci. 2002;187:313–318. [23] Gentile F, Tirinatob L, Battistac E, et al. Cells preferentially grow on rough substrates. Biomaterials. 2010;31:7205–7212. [24] Long Y, Zhang N, Huang Y, et al. Formation of highly aligned grooves on inner surface of semi-permeable hollow fiber membrane for directional axonal outgrowth. ASME J Manuf Sci Eng. 2008;130:1–8. [25] Sorkin R, Greenbaum A, David-Pur M, et al. Process entanglement as a neuronal anchorage mechanism to rough surfaces. Nanotechnology. 2009;20:1–8. [26] Sabri F, Sebelik ME, Meacham R, et al. In vivo ultrasonic detection of polyurea crosslinked silica aerogel implants. PLoS ONE. 2013;8(6):e66348. doi:10.1371/journal.pone.0066348. [27] Dellon AL, Mackinnon SE. Selection of the appropriate parameter to measure neural regeneration. Ann Plast Surg. 1989; 23:197–202. [28] Mackinnon SE, Dellon AL. A study of nerve regeneration across synthetic (Maxon) and biologic (collagen) nerve conduits for nerve gaps up to 5 cm in the primate. J Reconstr Microsurg. 1990;6:117–121. [29] Shen N, Zhu J. Application of sciatic functional index in nerve functional assessment. Microsurgery. 1995; 16:552–555.
[30] Dijkstra JR, Meek MF, Robinson PH, et al. Methods to evaluate functional nerve recovery in adult rats: walking track analysis, video analysis and the withdrawal reflex. J Neurosci Methods. 2000;96:89–96. [31] Bain JR, Mackinnon SE, Hunter DA. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg. 1989;83:129– 136. [32] Varejao ASP, Meek MF, Ferreira AJA, et al. Functional evaluation of peripheral nerve regeneration in the rat: walking track analysis. J Neurosci Methods. 2001;108:1–9. [33] Pocius A. Adhesion and Adhesives Technology 2E: An Introduction. Cincinnati, Ohio: Hanser; 2002. [34] Haftek J. Stretch injury of peripheral nerve. acute effects of stretching on rabbit nerve. J Bone Joint Surg Br. 1970;52:354–365. [35] Murphy CM, O’Brien FJ. Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds. Cell Adhes Migration. 2010;4:377–381. [36] Gentile F, La Rocca R, Marinaro G, et al. Differential cell adhesion on mesoporous silicon substrates. Appl Mater Interfaces. 2012;4:2903–2911. [37] O’Brien FJ, Harley BA, Yannas IV, et al. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials. 2005;26:433–441.
Journal of Investigative Surgery
ORIGINAL RESEARCH
Novel Technique for Repair of Severed Peripheral Nerves in Rats Using Polyurea Crosslinked Silica Aerogel Scaffold Firouzeh Sabri,1 David Gerth,2 George-Rudolph M. Tamula,1 Thien-Chuong N. Phung,1 Kyle J. Lynch,1 John D. Boughter Jr3
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1
Department of Physics, University of Memphis, Memphis, Tennessee, USA, 2 Department of Otolaryngology Head and Neck Surgery, UTHSC, Memphis, Tennessee, USA, 3 Department of Anatomy and Neurobiology, UTHSC, Memphis, Tennessee, USA
ABSTRACT Purpose/Aim: To design, synthesize, and test in vivo an aerogel-based top-open peripheral nerve scaffold to simultaneously support and guide multiple completely severed peripheral nerves in a rat model. Also, to explore options for immobilizing severed nerves on the aerogel material without the use of sutures resulting in reduced surgical time. Materials and Method: A novel material and approach was developed for the reattachment of severed peripheral nerves. Nerve confinement and alignment in this case relies on the surface properties of a lightweight, highly porous, polyurea crosslinked silica aerogel scaffold. The distal and proximal ends of completely transected nerve terminals were positioned inside prefabricated “top-open” corrugated channels that cradled approximately two thirds of the circumference of the nerve trunk and connectivity of the severed nerves was evaluated using sciatic function index (SFI) technique for five months post-surgery on 10 female Sprague–Dawley rats then compared with the gold standard for peripheral nerve repair. The interaction of nerves with the surface of the scaffold was investigated also. Results and Conclusion: Multichannel aerogel-based nerve support scaffold showed similar SFI recovery trend as the case suture repair technique. Usage of an adhesion-promoting coating reduced the friction between the nerve and the scaffold leading to slippage and lack of attachment between nerve and surface. The aerogel scaffold used in this study did not collapse under pressure during the incubation period and allowed for a rapid and non-invasive peripheral nerve repair approach without the demands of microsurgery on both time and surgical expertise. This technique may allow for simultaneous repair and reconnection of multiple severed nerves particularly relevant to nerve branching sites. Keywords: aerogel; scaffold; nerve; peripheral; repair; conduit; porous; crosslinked
INTRODUCTION
and training [5, 10–12]. Therefore, sutureless nerve repair techniques [13–18] have also been investigated in recent years with mixed degrees of effectiveness. Polyurea crosslinked silica aerogels are highly porous, mechanically strong, translucent materials that have demonstrated a promising future for in vivo and in vitro biomedical applications [19–21]. Their highly textured nanoscopic surfaces are expected to contribute to enhanced cell attachment and proliferation [22–25]. Additionally, the distinct ultrasonic behavior of aerogels allows for easy in vivo tracking of implants as shown in a recent study [26]. These advantages point to the potential use of this material in sutureless nerve repair. In this study, a peripheral nerve repair scaffold
Trauma patients suffer the majority of peripheral nerve injuries and the cost for U.S. peripheral nerve repair has exceeded $1 billion in recent years [1, 2]. Today, the gold standard in nerve repair consists of surgeons employing sutures to repair the severed nerves when the gap between distal and proximal terminals is short [3–5]. Complications associated with autografts and allografts have limited the performance of these techniques [6, 7] and thus paved the way for tubular conduits [7–9]. Complications associated with use of sutures include hemorrhaging, extended surgery time, and the need for a high level of surgeon skill Received 7 December 2013; accepted 17 March 2014.
Address correspondence to Firouzeh Sabri, Department of Physics, University of Memphis, Memphis, TN 38152, USA. E-mail: fsabri@ memphis.edu
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2 F. Sabri et al. was designed from polyurea crosslinked silica (X-Si) aerogels and tested on a widely used and accepted nerve model, the sciatic nerve of Sprague–Dawley rats [27–30]. In additional experiments, we investigated the effect of synthetic and biologic adhesives on the interaction and amount of available mobility of the peripheral nerve with the scaffold surface.
MATERIALS AND METHODS
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Preparation of Aerogel Implants Pigmented and clear polyurea crosslinked silica aerogels were synthesized according to our previously described formulation [20]. The aerogel implants were cut and roughly shaped from the bulk material using a diamond tipped abrasive disk mounted in a rotary tool. For the deep muscle study (sciatic nerve location and facial nerve location), samples were polished by hand to the final desired size while the nerve confinement channels were created by a fine-tipped rotary tool. All implants used for in vivo procedures were sterilized for 24 hr by a standard ethylene oxide sterilization process in an Amprolene system (Anderson Products) prior to surgery. Implants used for in vitro and cadaver testing were kept in an airtight container and rinsed with isopropyl alcohol immediately prior to usage. Figure 1 shows examples of aerogel scaffolds with various possible channel configurations and implant geometries. All samples were tested for mechanical integrity after the machining and shaping process was completed. In Figure 2, cross-sectional view of aerogel scaffolds specifically used in this study is shown both in the dou-
ble channel (Figure 2a) and single channel (Figure 2b) mode, while embedded in tissue or paraffin.
In Vivo Aerogel Scaffold Experiments All surgical procedures described in this section were performed on 10 female Sprague–Dawley rats weighing 200–300 g. Rats were anesthetized 15 min prior to surgery via an initial intramuscular injection of Telazol (0.03–0.05 ml at 0.3–0.5 mg/kg), followed by isoflurane inhalation at 1%–2% as described previously [19]. During the post-surgery recovery period none of the animals were restrained and they were all allowed to continue with their normal grooming routine. They were fed a routine diet and kept under close observation for signs of infection or abnormal behavior. Rats were euthanized post implant recovery by an overdose of carbon dioxide. This study was approved by the Animal Care and Use Committee at the University of Memphis. Sciatic Nerve Procedure Figure 3 shows a schematic diagram of the aerogel scaffold nerve support concept used in this study drawn for the sciatic nerve location. The top-open channel design demonstrates preexisting channels to support bifurcation of the sciatic nerve post-surgical severance. Each rat was weighed before surgery and administered carprofen at the dose of 5 mg/kg subcutaneously before the surgery. All surgeries were performed aseptically. Specifically, the surgical area (left hind limb) was shaved, cleaned with a betadine soap, and wiped with 70% isopropyl alcohol after undergoing
FIGURE 1 Aerogel scaffold configurations: Optical images of different pigmented and clear X-Si aerogel scaffold configurations. (a) Tubular conduit with Au thread passing through, (b) “top-open” 1-D channel with Cu thread passing through, (c) “closed” multichannel, and (d) closed configuration with a “snake” channel configuration. All implants were tested for mechanical integrity after channel formation and no defects were found. Scale bars = 5 mm. Journal of Investigative Surgery
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Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study
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FIGURE 2 Embedded aerogel scaffolds: Cross-sectional images of (a) dual channel and (b) single channel X-Si scaffold embedded in (a) paraffin and (b) tissue, prior to sectioning. The channels are formed along the longitudinal axis of the implant (not shown). Scale bars = 5 mm.
anesthesia in the surgical prep area. After the animal was moved into the surgery room, the surgical area was draped with a sterile drape. The surgeon wore a mask, head cover, and clean scrubs. He used sterile gloves as well as sterilized instruments for each surgery. Under a dissecting microscope, a transverse incision was made through the skin of the limb halfway between the iliac crest and the femur’s articulation with the tibia. A self-retracting retainer was replaced, and dissection was carried down between the biceps femoris and gluteus muscle until the sciatic nerve was identified. The
sciatic nerve of the Sprague–Dawley rats were exposed and severed sharply at the branching site. At this stage five different procedures were performed that will be described below: (I) Physical immobilization with X-Si aerogel scaffold: Once the sciatic nerve was severed, the doublechannel (Figure 2a) aerogel implant was positioned between the muscle and the sciatic nerve branches such that the nerve terminals were in direct contact with the surface of the implant and
FIGURE 3 Aerogel scaffold for nerve support: Illustration showing the concept of aerogel scaffold with preexisting channels specifically formed for the sciatic nerve bifurcation region and the positioning of the nerves in the channels, at the time of surgery. C
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cedures. The rats were observed at least twice a day for the next three days to observe if the rats were eating and drinking normally as well as assessing use of the rear leg. No antibiotics were given. There were no complications following any of the surgeries.
FIGURE 4 Aerogel insert at sciatic nerve surgical site: In vivo optical image of the multi-channel X-Si aerogel scaffold prior to insertion at the sciatic nerve surgical site. Nerves were severed prior to positioning of the implants.
(II)
(III)
(IV)
(V)
were positioned inside the tracks previously described. No adhesive or sutures were used for immobilizing the aerogel implant in this case and nerve positioning took approximately 5 min. The skin was then closed with staples and the animal was allowed to emerge from anesthesia. Physical immobilization with laminin-coated X-Si scaffold: The sciatic nerve was exposed and severed as previously described. The free nerve terminals were then positioned inside the tracks of a double-channel implant that was coated with a 50 μl drop of laminin (Sigma-Aldrich, St. Louis, MO) applied with a syringe tip (Figure 4). Nerves and implant were held together for approximately 30 s until the laminin drop began to gel. The skin was closed and animal cared for as described in the previous section. Suture repair: The sciatic nerve branches were exposed once again and transected sharply as described above. The nerve segments were then coapted using standard epineural suture technique with interrupted 9–0 nylon sutures. This process took approximately 18 min. No repair: The sciatic nerve branches were exposed and severed, however, nerve endings were then abandoned and no coaptation was attempted. Healthy rat-control: A healthy female Sprague– Dawley rat of the same age and weight with no procedures performed was used as a positive control.
After each surgery every rat was given 2 ml of Lactated Ringer’s subcutaneously. Carprofen was injected subcutaneously at the dose of 5 mg/kg/day for the next two days. Carprofen is an NSAID that is commonly given to rodents for pain following surgical pro-
Post-surgical Testing A custom-built wooden walking track with adequate confinement and lined with chart paper was used for animal gait evaluation. Non-toxic water-soluble paint was first applied to the rats’ hind paws and animals were (individually) encouraged to move toward a darkened cubicle, at the end of the track. Upon confinement of the rat, the prints were promptly removed from the chamber and allowed to dry before analysis. Rats from all groups were tested periodically, at least once a week for a total of five month post-surgery. Paw print data were analyzed using the sciatic function index (SFI) using the Bain—Mackinnon–Hunter (BMH) formula [31, 32]. At the conclusion of the study, rats were euthanized via CO2 asphyxiation.
Mechanical Testing Procedures In Vitro The strength of attachment of cadaveric rat sciatic nerves to aerogel implants were tested in vitro by means of the Lap shear test [33] for several biological and non-biological adhesives. Adhesives tested include: commercially available dental adhesives AllBond SE and One-Step (Bisco Inc., Schaumburg, IL), Super glue, Cell-Tak (BD Biosciences, CA), and basement membrane extract (BME) (R&D Systems, Minneapolis, MN). Earlier, adhesives were prepared and mixed according to the individual manufacturers’ recommendations. Sciatic nerves were removed from the left and right hind legs of cadaveric Sprague–Dawley rats immediately prior to testing. Tests were performed by applying two coats of the adhesive under observation to one side of a disk-shaped aerogel implant prepared in Section 2.1. Before curing/gelling of each type of adhesive, one end of the extracted sciatic nerve was positioned in the center of the coated area of the aerogel disk and allowed to attach. In each case the area of the nerve overlapping the adhesive area was kept constant. The top clamp of a Mark-10 bench top tensile tester was attached to the free end of the aerogel disk while the bottom clamp held onto the free end of the sciatic nerve in each case. The tensile tester loaded with a 20lb force gauge was programmed to “pull” at a rate of 30 mm/min for all measurements. The tensile behavior of the cadaver sciatic nerves alone was also tested. All cadaver nerves used for this part of the study were extracted from cadavers of the same age, weight, and species for consistency. Experiments were repeated for Journal of Investigative Surgery
Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study
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each type of adhesive at least twice and in some cases three times. In Vivo The sciatic nerve of a rat cadaver was exposed and the length of the intact nerve was measured using a digital caliper while the limb was in full extension. The nerve was then severed at one end and it instantly retracted to a final length that was measured again as previously mentioned. The free end of the sciatic nerve was then attached using appropriate clips to a handheld portable force gauge. The nerve was stretched back to its original length prior to severance and the force gauge reading was recorded. The spring constant of the sciatic nerve was then calculated. The full length of the nerve was removed from the rat and stored in a 4◦ C refrigerator for one week. The experiment was repeated on the “aged” sciatic nerve and the spring constant calculated again. Friction Studies In order to simulate the effect of a high water content interfacial layer (e.g., laminin) on the adhesion and frictional behavior of two surfaces, the following experiment was performed: a mesh number 220 strip of sandpaper (aerogel stimulant) was attached to a glass microscope slide and then positioned in the bottom clamp of a Mark 10 bench-top tensile tester. Sciatic nerves were removed as described previously from freshly euthanized rats and immediately transferred to the tensile tester. Approximately 3 mm of the length of the sciatic nerve was held by the top clamp. The free length of the sciatic nerve was brought into contact with the surface of the sandpaper and the nerve was dragged along the sandpaper at a rate of 30 mm/min until the two surfaces had completely lost contact. The experiment was repeated and all parameters were kept the same as before with the exception of a 50 ml drop of water, coating the longitudinal axis of the sandpaper, immediately before the nerve segment making contact with the surface of the sandpaper. The experiment was repeated twice and a fresh nerve was used each time. The force magnitude required to pull the nerve along the length of the sandpaper surface with and without the water drop was compared and registered.
RESULTS
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averages of SFI values collected weekly for each rat. The error bars reflect the standard error of the mean for each month. The BMH index is expected to theoretically vary between 0 (a completely functional nerve) and −100 (a completely dysfunctional nerve) [31, 32]. The gait of the healthy rat with no surgical intervention (V) is shown in Figure 5a with SFI values between −10 and −25 serving as a positive control. In this case, SFI values are closest to the zero mark among the various groups and indicate the baseline errors in the measurement technique. The rat with severed nerve and no surgical intervention (IV) served as the negative control for the study and the SFI collected from this subject is shown in Figure 5b. The SFI for this group, theoretically, was expected to be around −100. Although there were few outliers, the data in Figure 5b show a concentration of large and negative SFI values, which agrees with previously established literature. The animal’s gait is clearly impaired initially and continues to demonstrate a wide “swing.” The current gold standard for nerve repair is suture repair (III) and SFI values from this group are shown in Figure 5c for the same recovery period. The initial values for the SFI are significantly lower than previously seen, indicating an advantage over groups V and IV. As the months progressed, collected SFI values showed a concentration between −70 and −40. The initial SFI values for this case (Figure 5d) show a behavior slightly more impacted by nerve damage than the suture-repaired case (Figure 5c, group III). Overtime however, the SFI values for the two groups reach a similar range and they plateau at a value of −39. This fact may suggest that the sutureless polyurea crosslinked aerogel technique may have assisted in the healing process of the sciatic nerve and could be a possible alternative to nerve suturing. Upon extraction of the implant(s), the scaffold was located at the original surgery site and despite the lack of sutures, implant had not traveled. Physical immobilization of severed nerves was also tested with a laminin-coated X-Si scaffold (II) where a thin layer of laminin was sandwiched between the nerves and the channels. The gait recovery was expected to out-perform the behavior of group I but demonstrated a poorer recovery as a function of time as can be seen in Figure 5e. Over time, the animals’ gait became significantly wider and was no longer registered on the graph paper. Upon implant extraction it was noticed that the implant had shifted from the initial position and caused an obstruction.
Walking Track Analysis In all surgical cases, the operated legs were paralyzed and contractions of the hind paws of the operated leg were not present immediately after surgery. SFI values generated for each test group (I, II, III, IV, and V) are shown in Figure 5 for five month post-surgery until the rats were euthanized. Each plot represents monthly C
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Tensile Behavior of Cadaveric Nerves The tensile behavior of sciatic nerves extracted from three different cadaveric rats of similar weight and age is shown in Figure 6. Figure 6a shows the elastic region of all tested nerves as well as their rupture
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FIGURE 5 Sciatic function index (SFI) recovery: Comparison of gait recovery over five month post-surgery for groups (a) no surgery-positive control (V), (b) severed with no surgery-negative control (IV), (c) suture repair-gold standard (III), (d) X-Si scaffold (I), and (e) X-Si scaffold + laminin (II). SFI values represent monthly average values with standard error of the mean reflecting the error bars.
Journal of Investigative Surgery
Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study
culated using classical Hookean relationship and the spring constant value for a 2 hr old nerve was calculated to be 2.29 N/cm. This value appears to be consistent with values reported in literature, for example, a value of 1.96 N/cm for a tibial nerve [34]. After one week of “aging,” the spring constants were measured again and registered an average value of 0.228 N/cm clearly demonstrating the impact of aging on the tensile properties of the nerves.
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Analysis of Nerve–Substrate Attachment Behavior
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(b) FIGURE 6 Tensile behavior of sciatic nerves: Room temperature tensile behavior of cadaver rats sciatic nerves. (a) The sciatic nerves show an elastic behavior up to ∼ 4 N above which nerve rupture commences and deviations in individual nerves become apparent. (b) Zoom on lower points showing similar elongation behavior for all three nerves tested.
behavior at load values between 3.5 and 5N. Some variation in the load tolerance for each nerve is expected since the tightness of the grips, the fibrous nature of the tissue, and the age of the nerve do impact the rupture load behavior. Figure 6b shows the linear region of the graph where the three different nerves show consistent load-elongation behavior. The graph suggests that the mechanism used for nerve attachment should not exceed force values of 3N otherwise damage to the nerve trunk could occur and nerve regrowth and extension may be suppressed. An average value for spring constant of sciatic nerve for a Sprague–Dawley rat was cal C
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These results are summarized in Table 1 and suggest that the implant for group II did not show an SFI profile similar to group I (or better) due to the presence of the laminin leading to “slippage” of the nerves from the channels surface. These results suggest that with adequate traction and friction between the two surfaces in contact it is possible to retain connection if the implant surface is engineered to posses sufficient texture without the use of adhesives. The attachment strength of sciatic nerves to aerogel implants by means of non-biological adhesives such as superglue, All-Bond, and One-Step exceeded the natural load tolerance of the nerve fibers and the nerves ruptured prior to failure of the attachment. For these experiments, the nerve fibers tore at load values close to the values determined for the nerve while nerve fibers embedded in the adhesive region remained “trapped.” In the case of biological adhesives (BME and Cell-Tak) however, all attachments failed at very small loads and the sciatic nerves slipped off the implant surfaces with little effort, again, suggesting that moderate amounts of friction and “traction” between nerve body and the surface of interest will play an important role in the attachment of the two entities The non-slippage behavior of severed nerve trunks when placed in direct contact with aerogel scaffold was confirmed again using a branch of the facial nerve. The severed ends of the facial nerve of a freshly euthanized Sprague–Dawley rat were positioned inside the single channel opening of an X-Si implant and the two terminals remained in place, without retracting and slipping from the channel. The gap present between the two ends shown in Figure 7 is due to thickness of the implant.
TABLE 1 Effect of a water-based coating on attachment: Summary of two trails comparing the force required to sustain motion on a “dry” surface versus a “wet” surface
Trial 1 Trial 2
F = F2 −F1 (N)
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TABLE 2 Summary of adhesion tests: Table summarizing the adhesives tested and the outcome of the tests. Biological adhesives with a high water content acted as a friction-reducing agent leading to slippage of nerves while non-biological adhesives completely immobilized the nerves with no opportunity for mobility and migration
FIGURE 7 Aerogel insert at facial nerve surgical site: Optical image of the single channel X-Si aerogel scaffold prior to insertion at the facial nerve surgical site in a freshly euthanized cadaver rat. The facial nerve was severed immediately prior to positioning of the implants. Channel does not contain any coatings or adhesives and nerve segments remain attached to the channel due to frictional forces.
Sample tested
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X-Si Aerogel + All Bond + Nerve X-Si Aerogel +One-Step + Nerve X-Si Aerogel+ superglue + Nerve X-Si Aerogel + Cell-Tak + Nerve X-Si Aerogel + BME + Nerve
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Comments Nerve ruptured before attachment failure Nerve ruptured before attachment failure Nerve ruptured before attachment failure Attachment failed before nerve ruptured Attachment failed before nerve ruptured
DISCUSSION AND CONCLUSION In Figure 8, typical force-travel behavior for a “wet” surface is compared with a “dry” surface demonstrating the lack of “traction” when the surface is coated with a high water content coating, while on a “dry” surface significant force is required to travel along the length of the surface. The measurement was stopped once the full length of the nerve had reached the other end of the travel path. Table 2 shows the results of two trials demonstrating at least 0.15N of force required to sustain motion in the “dry” case while in the “wet” case hardly any force was needed leading to slippage and lack of traction between the two surfaces that are in contact.
FIGURE 8 Effect of friction on nerve mobility: Example behavior of sustaining nerve motion and mobility on a “wet” surface compared to a “dry” surface.
We designed a novel scaffold for peripheral nerve repair, constructed from X-Si aerogel with a high degree of surface friction and physical confinement capabilities. Silica aerogels are a lightweight, mesoporous, strong, and biocompatible material with great promise for medical applications. Given that aerogels can be easily shaped to contain multiple channels with the desired channel configurations, it can be developed for any of the peripheral nerves. Here, we constructed and evaluated a multi-channel X-Si aerogel scaffold for sciatic nerve connection and repair in rats. Walking track analyses carried out during recovery confirm that surgical intervention and proper reapproximation is clearly necessary for the correct reconnection of severed nerve terminals. Assessment of recovery of function was made using SFI computations; collection of prints using this method suffers from inconsistencies that are often hard to control. The SFI “trend” however for each experimental group is clear over the five month period with suture repair and scaffold with no laminin showing similar recovery trends. The surgical repair method presented here offers insight into the development of a rapid and non-invasive peripheral nerve repair approach that may offer an alternative to allograft/autograft without the demands of microsurgery on both time and surgical expertise. As shown in this study, aerogel scaffold surface properties play an important role in nerve attachment and nerve mobility where a moderate amount of friction can aid in preventing nerve slippage and the absolute need for sutures. Physical immobilization of severed nerves with X-Si aerogel scaffold (I) containing preexisting channels was significantly easier and faster than the traditional suture repair procedure. Processing and surgical time was reduced significantly by using the X-Si aerogel scaffold (less than 5 min) in comparison with the suturing technique (approximately 18 min). The Journal of Investigative Surgery
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Polyurea Crosslinked Silica Aerogel Scaffold—A Pilot Study presence of the laminin layer did not enhance nerve recovery. Instead, it acted as a lubricant and reduced the friction between the two surfaces (nerve + channel) hence leading to an implant repositioning occurring. This hypothesis was confirmed when tensile tests performed on a wet versus dry surface showed a noticeable difference in the force required to pull a sciatic nerve on the surface, simulating the migration of nerves on a scaffold with/without a coating layer (discussed in Section 3.3). Tests performed on the facial nerve (Figure 7) also showed good adhesion between the nerve and the substrate. Therefore, while waterbased biological “adhesion-promoting” layers such as BME, laminin, and Cell-Tak are necessary for the attachment and proliferation of individual cells, they significantly reduce the surface friction between the nerves’ exterior and the surface of the scaffold. It is therefore likely that the mesoporous nature of the aerogel scaffold directly impacts the nerve–substrate interaction. While the relationship between scaffold pore size and cell activity (attachment) is currently poorly understood, it is clear that if pores are too large (μm size) there is a decrease in the specific surface area available which in turn limits cell attachment [35]. As a result, mesoporous substrates offer a unique advantage over other materials providing extra “anchoring” opportunities. In a recent study, cells demonstrated improved distribution and adhesion to mesoporous substrates compared with featureless smooth surfaces [36], which supports the current hypothesis. While it is expected that the optimal pore size will vary with different cell types [37], this study suggests that further investigation into aerogel-based scaffolds will facilitate the design of new nerve repair conduits and scaffolds.
ACKNOWLEDGMENTS The first author would like to acknowledge John Daffron, Department of Physics, for all support with instrumentation design and fabrication. The authors are also grateful to Dr. Karyl Buddington for all surgical procedures that were conducted at the University of Memphis Animal Care Facility. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
FUNDING This study was funded by the FedEx Innovation Grant, FedEx Institute of Technology, University of Memphis. C
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