ORIGINAL ARTICLE A Tissue-Engineered Conduit for Peripheral Nerve Repair

Tessa Hadlock, MD; Jennifer Elisseeff, BS; Robert Langer, ScD; Joseph Vacanti, MD; Mack Cheney, MD

Background: Peripheral nerve repair using autograft ma- formed using a dip-molding technique. They were cre- terial has several shortcomings, including donor site mor- ated containing 1, 2, 4, or 5 sublumina, or “fascicular ana- bidity, inadequate return of function, and aberrant re- logs.” Populations of Schwann cells were isolated, ex- generation. Recently, peripheral nerve research has panded in culture, and plated onto these polymer films, focused on the generation of synthetic nerve guidance where they demonstrated excellent adherence to the poly- conduits that might overcome these phenomena to im- mer surfaces. Regeneration was demonstrated through prove regeneration. In our laboratory, we use the unique several constructs. chemical and physical properties of synthetic polymers in conjunction with the biological properties of Schwann Conclusions: A tubular nerve guidance conduit pos- cells to create a superior prosthesis for the repair of mul- sessing the macroarchitecture of a polyfascicular periph- tiply branched peripheral nerves, such as the facial nerve. eral nerve was created. The establishment of resident Schwann cells onto poly-L- and polylactic–co- Objectives: To create a polymeric facial nerve analog surfaces was demonstrated, and the feasi- approximating the fascicular architecture of the extra- bility of in vivo regeneration through the conduit was temporal facial nerve, to introduce a population of shown. It is hypothesized that these tissue-engineered de- Schwann cells into the analog, and to implant the vices, composed of widely used biocompatible, biode- prosthesis into an animal model for assessment of gradable polymer materials and adherent Schwann cells, regeneration. will be useful in promoting both more robust and more precisely directed peripheral nerve regeneration. Results: Tubes of poly-L-lactic acid (molecular weight, 100 000) or polylactic–co-glycolic acid were Arch Otolaryngol Head Neck Surg. 1998;124:1081-1086

ERIPHERAL NERVE injuries sults are obtained.1,2 While this approach continue to be among the incurs some donor site morbidity and usu- most challenging problems ally involves a secondary surgical site, its faced by surgeons. Within main drawback is insufficient functional otolaryngology, it has been outcome. In the case of the facial nerve, particularlyP difficult to develop appropri- autografted repairs rarely, if ever, result in ate strategies for the management of fa- a recovery that is better than House- From the Department of cial paralysis, recurrent laryngeal nerve in- Brackmann grade III.3 Otolaryngology, Massachusetts Eye and Ear Infirmary jury, and the morbidity associated with Attention has long been directed at al- (Drs Hadlock and Cheney), insensate oropharyngeal and esophageal ternatives to autografting. Researchers in and the Laboratory of flap reconstructions after ablative sur- the past have attempted to bridge severed Transplantation and Tissue gery for malignancy. The development of nerve endings with a wide variety of au- Engineering, Children’s a synthetic nerve guidance conduit would tologous biological tubular structures, in- Hospital (Drs Hadlock and be of enormous utility in the manage- cluding artery, vein, inside-out vein con- Vacanti), Boston, Mass, and the ment of these and other nerve-related duits, and decalcified channels.4-8 Department of Chemical problems in otolaryngology. Some have used cadaveric nerve allografts Engineering and the The standard approach to repair a pe- or xenografts to bridge long nerve de- Harvard–Massachusetts ripheral nerve when a gap is present is to fects.9 Others have used skeletal muscle Institute of Technology Division 10-12 of Health Sciences and bridge the severed ends with a segment of grafts for this purpose. While investi- Technology, Massachusetts autologous donor nerve. When the cali- gators have achieved promising results us- Institute of Technology, ber of the donor autograft matches that of ing many of these techniques, to our knowl- Cambridge (Ms Elisseeff the recipient nerve, in both total diam- edge none has matched or surpassed those and Dr Langer). eter and average density, superior re- achieved by autograft repair.

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 MATERIALS AND METHODS collagenase and dispase, the crude cell suspension was plated in Dulbecco modified Eagle medium, enriched with 10% POLYMER PREPARATION heat-inactivated fetal bovine serum and antibiotics. The cells were treated for the ensuing 2 to 3 days with cytosine ara- Poly-L-lactic acid with a molecular weight (MW) of 100 000 binoside for the elimination of . On day 5, a thy1.1 and a copolymer of PLGA with a lactide-glycolide ratio of 85:15 antibody purification was used to remove residual fibro- were purchased commercially (Polysciences, Worthington, blasts, and the purified population was ex- Pa). Stock solutions of 2.5% to 5% (wt/vol) of the polymers panded on polylysine-coated dishes over a period of 6 weeks were generated by dissolution in either chloroform (CHCl3) using mitogenic media enriched with a cyclic AMP activa- or methylene chloride (CH2Cl2). Tubes were prepared by a tor (Forskolin, Sigma Chemical Co, St Louis, Mo) and glial modification of a standard dip-molding technique. Briefly, growth factor (Biomedical Technologies, Wilmington, for the single lumen conduits, a thin-walled polytef sheath Mass). Cultures were assessed for purity using an immu- (outer diameter, 1.8 mm) was passed over a glass capillary nocytochemical stain against S100 protein, a glial-specific tube. Inside the fume hood, the tube was secured to a dip- marker. ping apparatus and systematically dipped into the polymer For adult Schwann cell isolation a previously de- suspension every 15 seconds, for a total of 20 dips. The tubes scribed explant technique was used.32 Lewis rats (weigh- were placed into and removed from the solution at a con- ing 200-250 g) were killed, and the sciatic were nerves re- stant rate and were suspended for a total of 10 seconds be- moved. The epineurial tissue was microscopically peeled tween each subsequent dip for solvent evaporation. After the away, and the remaining tissue was cut into 1-mm seg- dipping procedure, the tubes remained in the fume hood over- ments. These segments were placed on tissue culture plas- night for evaporation of solvent at atmospheric pressure, fol- tic in Dulbecco modified Eagle medium and explanted on lowed by an additional 24-hour period under low vacuum a weekly basis. After 6 weeks, the explants were enzymati- for the removal of residual solvent, and then stored in a des- cally digested, purified, and expanded as described above. sicator for later characterization. Yields from these explants were much lower, as expected, A similar approach was taken for the creation of tubes and Schwann cells for the majority of the ensuing studies with multiple sublumina. Smaller polytef sheaths (OD, 600 were performed using the neonatal preparations. µm) were passed over stainless steel wires rather than glass For the adherence studies, polymer films prepared and capillary tubes. Each sublumen was made separately, as de- sterilized as described above were placed onto tissue cul- scribed above, and then bundled together for creation of ture plates. A Schwann cell suspension was then plated onto the final multiluminal channel. For example, to create a the films, and the cultures were examined using light mi- 5-lumen conduit, 5 tubes were prepared separately. The croscopy on a daily basis for observations of adherence, sur- group of tubes was then bound together with a suture at vival, and proliferation behavior. either end and dipped as a bundle for a total of 20 addi- tional dips to achieve a single prosthesis with 5 separate IN VIVO STUDIES compartments. The bundled conduits were redried and stored as described above. The polymer conduits, with or without inhabitant synge- Polymer films for the plating of Schwann cells were neic Schwann cell populations, were implanted into 20-mm prepared from the same polymer stock solutions as the con- gaps in the left sciatic nerve of 10 adult Lewis rats. After duits. They were formed by a standard solvent casting tech- induction of anesthesia with inhalational methoxyfluor- nique. Several milliliters of the polymer suspension was ane, the animal was shaved and prepped. An incision was placed into a 100-mm glass Petri dish in the fume hood, made over the left hind limb, and the gluteal muscles were and the solvent was allowed to slowly evaporate. The re- divided to expose the sciatic nerve. Under the operating sultant films were gas sterilized with ethylene oxide and microscope, a 20-mm segment of nerve was removed, and used for the plating of Schwann cells. the proximal and distal stumps were secured to either end of the polymer conduit using several 9-0 nylon microsu- POLYMER CHARACTERIZATION tures. The muscle and skin were closed in layers, and the animals recovered in a controlled environment. They were The tubes were examined grossly for mechanical strength given food and water ad libitum and examined for signs of and pliability. Scanning electron microscopy using an en- autotomy. Institutional guidelines regarding animal ex- vironmental scanning electron microscope (Jeol USA Inc, perimentation were followed. After 3 months, the animals Peabody, Mass) was used to examine luminal surfaces and were evaluated for functional sensory recovery using stan- porosity. permeation chromatography using a peristal- dard pinch testing and perfused with 3% glutaraldehyde, tic pump (Hewlett-Packard Co, Palo Alto, Calif) and com- and the implants were harvested. Observations were made mercially purchased columns (Polymers Inc, Burlington, regarding tubular continuity both along the length and at Vt) was carried out to determine the MW of the processed the anastomoses. The specimens were plastic embedded, polymer. cut into 1-µm-thick sections, and stained with toluidine blue for assessment of both the axonal contents of the conduit CELLULAR STUDIES and the degree of surrounding inflammation. For com- parative purposes, 2 control animals underwent 20-mm au- Schwann cells from both neonatal and adult Lewis rats were tografts rather than tubular implants. Surgical and harvest isolated and expanded in culture using previously de- techniques were identical to those described above, scribed methods.31 Briefly, for neonatal cell harvests, 2 except that when the 20-mm segment of sciatic nerve was litters of day 2 Lewis pups were killed, and their sciatic removed, it was rotated 180° and sewn in as a reversed nerves were removed. After enzymatic digestion with autograft.

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Entubulation repair, by which the proximal and dis- moting substances over time and to harbor Schwann cells tal ends of the severed nerve are inserted into either end would exceed that of simple hollow conduits. This was of a hollow conduit, has been attempted extensively.13-19 accomplished by creating several thin-walled compart- It is hypothesized that isolation of the local regenerative ments within the conduit. This increase in surface area milieu may encourage more focused regeneration to- will ultimately allow more extensive delivery of neuro- ward the distal stump. Mechanisms for this increased re- trophins throughout the conduit and may allow the se- generation include decreased access of inflammatory cells lective delivery of different into different to the stumps and decreased diffusion of distally elabo- regions of the conduit. The larger surface area provided rated neurotrophins into surrounding tissues, which re- by the introduction of subluminal architecture also sig- sults in increased local concentrations of these sub- nificantly increases the surface area available for Schwann stances at the proximal stump, where they exert their cell adherence. effects. This entubulation model has helped to delineate We also studied the adherence of both neonatal and the molecular and cellular events that occur in the en- adult Schwann cells to poly-L-lactic acid (PLLA) and poly- vironment of the injured and regenerating peripheral lactic–co-glycolic acid (PLGA) surfaces to discern whether nerve. the surface environment provided by conduits of these Studies of regenerating peripheral nerves have un- materials would be favorable to inhabitance by Schwann covered a variety of influences that affect the behavior cells. While neonatal cell populations expand more rap- of individual nerve growth cones at the proximal stump. idly and give higher yields, it is important for tissue- For example, it was discovered that the presence of elec- engineering applications to demonstrate the feasibility trical field stimulation promotes robust exten- of cell harvest from adult tissues, as implant devices would sion20 and that certain neurotropic and neurotrophic sub- have to be developed from biopsy specimens. We also stances are secreted by the cut nerve ends after injury.21,22 explored in vivo regeneration through the conduits over A very large number of seemingly unrelated molecules a long gap compared with autografts. and peptides also possess neurotrophic properties or promote axonal elongation after injury, including ex- RESULTS tracellular matrix components, testosterone, vasoactive intestinal peptide, and the immunosuppressive agent Both PLLA and PLGA conduits were created containing tacrolimus, among others.23-28 1 to 5 sublumina (Figure 1). All conduits displayed fa- Recently, focus has been directed toward the devel- vorable gross pliability characteristics. They could be de- opment of nerve guidance conduits made from syn- formed approximately 30° without kinking or fractur- thetic biomaterials. Investigators now hope to use the con- ing. We found that the materials were soft enough to pass trollable properties of synthetic materials to create 9-0 nylon suture through readily, but were thick enough regenerative environments that provide known neuro- that the sutures held without tearing. trophic influences, including electroconductivity and the Electron micrographs demonstrated smooth- ability to release nerve growth–promoting substances over walled, nonporous tubular structures, as expected from time. It is also feasible to manipulate conduit architec- solvent casting preparation techniques (Figure 2). Wall ture, surface properties, porosity, and biodegradability thickness ranged from 50 to 100 µm. Internal lumen di- to optimize the regenerative conditions. ameters correlated precisely with the outer diameter of Entubulation repair with polymeric conduits pos- the polytef sheaths used in the conduit preparation, sug- sessing electroconductive properties has demonstrated gesting that no shrinkage or deformation of the tubular increased regeneration over neutral controls. Polymeric conduits took place during drying. Molecular weight de- channels engineered to slowly release growth factors from terminations using gel permeation chromatography dem- their walls have also proven beneficial. The addition of onstrated MWs close to those of the starting materials. Schwann cells to the lumen of a hollow guidance chan- nel has provided a benefit,29,30 based on the fact that PLLA PLGA Schwann cells express both surface proteins and elabo- Starting MW 100 (Reported) 64.7 rate soluble factors conducive to the extension of the re- Processed MW 80.3 41 Polydispersity 1.44 1.48 generating axon tips. The beneficial presence of Schwann cells was found to be concentration dependent, with tubes This similarity suggests that no significant material loaded with higher numbers of cells leading to in- breakdown occurs during the polymer processing tech- creased axonal bridging through conduits. Certain lumen- nique. occupying substances, including com- Both neonatal and adult Lewis rat Schwann cell cul- ponents, soluble neurotrophins, or combinations of these, tures appeared to contain more than 90% Schwann cells have also proven useful to the bridging of neuronal gaps. after the purification steps. The cells adhered equally well However, despite findings that convincingly demon- to films of either pure PLLA or the PLGA copolymer. strate the benefit of these influences that are able to be When plated in mitogenic media, the cells divided to reach introduced through biomaterials, no hollow conduit confluence over the same time course as similar cells model been able to bridge a neural gap more effectively plated onto polylysine-coated tissue culture plastic. When than control autografts. plated in ordinary media, the cells extended processes In this study, we used a polymer dip-molding tech- and were morphologically indistinguishable from cells nique to create a biocompatible, biodegradable polymer plated onto control dishes of polylysine-coated tissue cul- conduit whose ability both to release growth-pro- ture plastic (Figure 3).

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Figure 2. Electron micrographs of polymer conduit (original magnification ϫ790). Top, Junction of 3 lumina. Bottom, View inside 1 lumen. Note smooth, nonporous surface.

Figure 1. Cross section of polymer conduits (original magnification ϫ30). Top, Single-lumen conduit (1-mm scale). Bottom, Five-lumen conduit. fect axonal elongation. It has been well demonstrated that a number of growth factors and other small molecules Implantation studies revealed that 1 of the tubular promote neurite extension, and polymer technology has conduits had become dislodged at the proximal anasto- been shown to be capable of delivering a number of these mosis, and 2 of the single-lumen channels had fractured substances effectively. at the junction of the proximal one third and the distal The current model for delivery of neurotrophins in- two thirds. All other conduits remained intact, as did the volves an entubulation of the nerve into a single-lumen control autografts. conduit. Growth-promoting substances are then admin- Histologic evaluation revealed minimal inflamma- istered in 1 of 3 ways. Either they are loaded into the walls tory reaction in all specimens. A thin fibrotic band was of the hollow conduit, so that they are slowly released seen to surround the outer surface of the tubes. Cross in a circumferential fashion, or they are loaded into an sections of the conduits, at a point 5 mm from the proxi- aqueous medium and delivered into the lumen of the con- mal anastomosis, revealed the presence of a regenerat- duit at the time of surgery. Alternatively, they have been ing axon cable in all the intact single-lumen channels, delivered systemically or regionally by injection. as well as the autografts (Figure 4). Figure 5 shows We have designed a fully biodegradable polymer con- the percentage of neural tissue in these regenerating cables. duit whose multiple-channel architecture is conducive The multiluminal conduits demonstrated sparse islands to a more effective local slow release of neurotrophins. of regenerating , although there were too few to There is enormous benefit to the use of of quantify. Sensory testing at 12 weeks revealed with- lactic and glycolic acid, because the rate of biodegrada- drawal to nociceptive stimulus at the fifth digit of all ex- tion can be tightly controlled by altering the ratio of the perimental animals, but withdrawal to nociceptive stimu- 2 monomers. Degradation, in turn, controls the rate of lus of the third digit occurred in only 1 animal in the release of growth-promoting substances, as they are re- autograft group. leased primarily in association with degradation of the polymer walls. COMMENT A second advantage to this polyluminal conduit is that it provides increased area for Schwann cell adher- Synthetic polymer conduits are a promising approach to ence. While several studies have introduced Schwann cells artificial nerve guide development. They possess many into the conduit prior to implantation, the surface area properties that can be finely manipulated in order to ef- for Schwann cell adherence through these hollow con-

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Figure 3. Light micrographs of Schwann cells cultured onto Figure 4. Toluidine blue–stained cross sections of regenerated nerve cable polylactic–co-glycolic acid polymer films. Top, In a mitogenic medium, the (original magnification ϫ660). Top, Single-lumen conduit. Bottom, cells are nearly confluent (original magnification ϫ50). Bottom, In an Autograft. ordinary medium, the cells are adherent, extending processes (original magnification ϫ100).

50 hind limb. The tubes experienced some fracturing and 45 anastomotic disruption, suggesting that the 100-µm- 40 thick walls may be unacceptably thin for these long 35 peripheral nerve gaps. The presence of a neural regen- 30 erate in many of the conduits is a starting point from 25 which we may make improvement with the addition 20 of neurotrophic substances. It is possible that the lack

% Neural Tissue 15 of contiguous neural cables through the multiluminal conduits stems from the lack of porosity or the higher 10 residual solvent presence in these conduits compared 5 with the single-lumen conduits. Both these issues are 0 12345 currently being addressed in our laboratory. We have Figure 5. Graph illustrating the percentage of the cross section represented begun to use highly porous tubular structures and by neural tissue (axons and sheaths) for the 3 single-lumen conduits have eliminated chlorinated hydrocarbons from our (bars 1-3) and the 2 autografts (bars 4 and 5). preparation technique. We have also developed an extensive battery of behavioral tests that will offer more information regarding the functional recovery duits has been a limiting factor. We have demonstrated we seek to achieve. We recognize the limitations of the in this study that Schwann cells adhere, survive, and can rat sciatic nerve model, in which neural regeneration be directed to divide on polymer surfaces of PLLA and occurs more robustly than in higher species, and expect PLGA. In our 5-lumen conduits, the surface area for to move to more analogous mammalian species once Schwann cell adherence rises from 1.1 to 1.9 cm2, a fac- the architecture and properties of the prosthesis have tor of nearly 2. By decreasing the internal diameter of each been optimized. of the sublumina and increasing their number, it is fea- We hypothesize that these polyluminal structures sible to increase surface area for cellular adherence by will have more thorough release profiles as much as an order of magnitude. and higher inhabitant Schwann cell numbers, and will The in vivo implantation study demonstrated that ultimately lead to the bridging of longer gaps than cur- the polymeric substances were well tolerated in the rat rently possible using existent artificial nerve conduits.

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Accepted for publication June 23, 1998. 15. Williams L, Varon S. Modification of fibrin matrix formation in situ enhances nerve The authors wish to thank Daniel A. Hunter, RT, for regeneration in silicone chambers. J Comp Neurol. 1985;231:209-220. 16. Brushart T, Mathur V, Sood R, et al. Dispersion of regenerating axons across preparation of the histologic specimens. enclosed neural gaps. J Hand Surg Am. 1995;20:557-564. Reprints: Tessa Hadlock, MD, Department of Otolar- 17. Madison R, Dasilva C, Dikkes P. Entubulation repair with protein additives in- yngology, Massachusetts Eye and Ear Infirmary, 243 Charles creases the maximum nerve gap distance successfully bridged with tubular pros- St, Boston, MA 02114. theses. Brain Res. 1988;447:325-334. 18. Archibald S, Krarup C, Shefner J, et al. 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Editorial Footnote

his technique appears very promising but cer- tainly is no better than cable nerve auto- T graphs. With further study in primates, it may well fit into our surgical armamentarium in the future.

Harold C. Pillsbury III, MD Chapel Hill, NC

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