In vivo evaluation of poly(l -lactic acid) porous conduits for peripheral nerve regeneration

Biomaterials 20 (1999) 1109}1115

In vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration G.R.D. Evans *, K. Brandt
, M.S. Widmer, L. Lu, R.K. Meszlenyi, P.K. Gupta, A.G. Mikos, J. Hodges , J. Williams , A. GuK rlek , A. Nabawi , R. Lohman , C.W. Patrick Jr. Department of Plastic Surgery, The University of Texas, M.D. Anderson Cancer Center, Box 062, 1515 Holcombe Blvd., Houston, TX 77030, USA
The Division of Plastic Surgery, The University of Texas, Health Science Center at Houston, 6411 Fannin, Houston, TX 77030, USA Institute of Biosciences and Bioengineering, Rice University, 6100 Main, Houston, TX 77005, USA Received 24 June 1998; accepted 6 January 1999

Abstract The present study provides in vivo trials of poly(L-lactic acid) (PLLA) as a porous biodegradable nerve conduit using a 10 mm sciatic nerve defect model in rats. The PLLA conduits, fabricated by an extrusion technique, had an inner diameter of 1.6 mm, an outer diameter of 3.2 mm, and a length of 12 mm. They were highly porous with an interconnected pore structure (of 83.5% porosity and 12.1 lm mean pore size). The conduits were interposed into the right sciatic nerve defect of Sprague Dawley rats using microsurgical techniques; nerve isografts served as controls. Walking track analysis was performed after conduit placement monthly through 16 weeks. At the conclusion of 6 and 16 weeks, sections from the isograft/conduit and distal nerve were harvested for histomorphometric analysis. The right gastrocnemius muscle was also harvested and its weight was determined. All conduits remained intact without breakage. Moreover, no conduit elongated during the 16 weeks of placement. Walking track analysis and gastrocnemius muscle weight demonstrated increasing regeneration over the 16 weeks in both the conduit and isograft control groups, with control values signi"cantly greater. The nerve "ber density in the distal sciatic nerve for the PLLA conduits (0.16$0.07) was similar to that for the control isografts (0.19$0.05) at 16 weeks. The number of axons/mm in the distal sciatic nerve for the PLLA conduits was lower than that for the isografts (13 800$2500 vs. 10 700$4700) at 16 weeks. The results for PLLA were signi"cantly improved over those for 75 : 25 poly(DL-lactic-co-glycolic acid) of a previous study and suggest that PLLA porous conduits may serve as a sca!old for peripheral nerve regeneration.  1999 Elsevier Science Ltd. All rights reserved Keywords: Poly(L-lactic acid) (PLLA); Conduit; Nerve regeneration, Tissue engineering

1. Introduction Tumor extirpation, traumatic injuries and congenital anomalies may result in injury to or sacri"ce of critical nerves. Failure to restore injured nerves can result in the loss of muscle function, impaired sensation and/or painful neuropathies. Functional nerve defects have traditionally been reconstructed by the surgical transfer and sacri"ce of nerve from an uninjured location to the injured site. Allografts have been used in reconstruction but require systemic immunosuppression. Despite advances in nerve reconstruction, clinicians are still limited by the time necessary for regeneration and the morbidity associated with the harvest of autogenous nerve grafts. * Corresponding author. Tel.: 001 713 794 1247; fax: 001 713 794 5492; e-mail: GEvans@notes. mdacc.tmc.edu

Biodegradable nerve guidance conduits may eliminate the morbidity associated with autogenous nerve grafting and provide a way to modulate the in#uence of biological factors on peripheral nerve regeneration. The idea of developing a nerve conduit however is not new. GluK ck initially utilized decalci"ed bone as a conduit for nerve regeneration [1]. A variety of other substances have been employed including fascia, vein grafts and fallopian tubes. Wang et al. noted the jugular vein was a better conduit for nerve regeneration than femoral veins due its increased diameter [2, 3]. Silicone tubes have bridged 10 mm nerve gaps, with compound action potentials detectable by 6 weeks [4]. Pseudosynovial sheaths formed around a silicone rod have also been used to "ll small nerve gaps [5]. Unfortunately, these conduits were limited to reconstruction of small nerve defects ((10 mm) and functional nerve regeneration following

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their use was not assessed. Clinically, nerve defects up to and greater than 200 mm may require repair and restoration of function. Attempts to bridge larger nerve de"cits have used a variety of substances to "ll these conduits. Wang et al. used inverted jugular veins to enhance peripheral nerve regeneration by exposing a collagen rich adventitial surface to the regenerating axon [3]. Williams et al. noted that silicone tubes "lled with dialyzed plasma resulted in a three to "vefold increase in functional restitution detected at 8 weeks compared with chambers pre"lled with phosphated bu!ered saline (PBS) [6]. For a 20}25 mm sciatic nerve gap in rats, Madison et al. "lled silicone tubes with collagen or laminin and compared them with empty control tubes [7]. All of the tubes with additives demonstrated nerve regeneration extending up to 4}6 mm. Archibald et al. compared autografting and entubulation repair with collagen-based nerve guide conduits across 4 mm gaps in both rats and monkeys [8]. They noted that recovery of physiological response from target muscle and sensory nerve entubulation repair with a collagen-based nerve guide conduit was as e!ective as a standard nerve autograft over short nerve defects. Seckel et al. used hyaluronic acid, a compound associated with decreased scarring and improved "brin matrix formation, in an injectable polyethylene nerve guide in the rat sciatic nerve [9]. Better conduction velocity, higher axon counts, and a trend toward earlier myelination was demonstrated with hyaluronic acid compared with saline [9]. Contrary to these study results, Valentini et al. demonstrated that semipermeable guidance channels "lled with collagen or laminin displayed fewer myelinated axons than saline [10]. In an attempt to promote biodegradable materials as nerve conduits, a variety of substances have been employed [5, 11}14]. Previous nerve guides prepared from polyesters, speci"cally poly(DL-lactic acid) have demonstrated regeneration. Innervation of the distal nerve stump occurred in conduits providing the nerve gap was 10 mm or less [15]. Further studies utilized poly(L-lactic acid-co-e-caprolactone) copolymeric nerve guidance channels in the rat sciatic nerve. Nerve conduits were present 2 years after implantation. The mean "ber diameter was smaller in the conduit group compared with controls [16]. Kiyotani et al. studied sciatic nerve defects in seven cats (25 mm) and used a poly(glycolic acid)/collagen (type I) composite tube to bridge the gap. Four months after surgery, a population of myelinated "bers was observed within the conduit, however, the lumen was small and no comparison with autografts was performed [17]. Natural collagen matrices have also been employed to bridge sciatic nerve gaps [18]. Recent studies utilizing oxidized polypyrrole, an electrically conductive polymer, have demonstrated improved neurite outgrowth in PC12 cells [19]. Extracellular matrix analogues have also been developed by grafting glycosaminoglycan chains

onto type I collagen and by controlling the physicochemical properties of the resulting graft copolymer [20, 21]. This extracellular matrix analogue has been employed in biodegradable implants for sciatic nerve defects greater than 10 mm [21]. Alterations in the geometric con"guration of the conduit further appear to in#uence peripheral nerve regeneration [22, 23]. Synthetic guidance channels using silicone elastomer tubes have been divided into two compartments by a polymer strip 10 mm long with both a rough and a smooth surface. In the rough chambers with a 10 mm nerve gap, tissue adhered to both sides of the polymer strip. In the smooth chambers nerves demonstrated discrete single peripheral axons [22]. Similar studies have been reported by Aebischer et al. [23]. Previous studies in our laboratory utilized poly(DLlactic-co-glycolic acid) (PLGA) sca!olds (of 75 : 25 copolymer ratio of lactic acid to glycolic acid) in an attempt to guide peripheral nerve regeneration [24]. Results demonstrated adequate distal nerve innervation with an improved Sciatic Functional Index over 16 weeks. Axonal growth was not inhibited by polymer degradation [25]. Elongation and partial collapse, however, led to a search for a di!erent polymer as an alternative conduit for guided nerve regeneration. Poly(L-lactic acid) (PLLA) tissue guidance conduits have proven 10 times stronger and sti!er than 75 : 25 PLGA conduits of similar pore morphology over an 8-week in vitro degradation period [25]. The present study provides in vivo evaluation of PLLA as a porous biodegradable nerve conduit for peripheral nerve regeneration using the sciatic nerve model in rats. With the structural ability of PLLA to surpass PLGA, elongation and collapse of the conduit, we believe, could be avoided. The reported experiments with PLLA as a biodegradable conduit o!er several advantages to previous studies outlined above. These include: (1) a fully degradable conduit that will be replaced by myelinated axons at variable times, (2) a porous sca!old to allow vascularization, (3) a conduit that can be varied in length and luminal diameter while maintaining structural integrity and #exibility, and (4) a unique fabrication process that reproduces consistent geometric conformity and added stability, and (5) a functional model to test the conduit in an attempt to restore normalcy.

2. Materials and methods 2.1. Conduit fabrication Poly(L-lactic acid) (PLLA) (Birmingham Polymers, Birmingham, AL) was manufactured into porous biodegradable conduits using a combined solvent casting, extrusion, and particulate leaching technique [25]. The number average molecular weight (Mn) of the raw material was 46 500$2100 (n"5) [25] as measured by gel

G.R.D. Evans et al. / Biomaterials 20 (1999) 1109}1115

permeation chromatography. The PLLA conduits were fabricated with a salt weight fraction of 90%, a salt crystal size between 150 and 300 lm, and an extrusion temperature of 2753C. The resulted conduits had an inner diameter of 1.6 mm, an outer diameter of 3.2 mm, and a length of 12 mm. The PLLA conduits had an interconnected pore structure, and the porosity and mean pore size were measured by mercury porosimetry [25, 26] as 83.5$4.1% and 12.1$2.8 lm, respectively (n"3). The Mn of the processed PLLA decreased to 35 500$2700 (n"5) [25]. Tensile testing indicated a strength of 81.7$35.1 MPa, a modulus of 1.0$0.4 MPa, and a strain at break of 0.02$0.01 mm/mm (tested wet, n"10) [25]. The crystallinity of the PLLA conduits was 5.2$0.4% (n"6) [5]. The conduits degraded mainly by bulk degradation in vitro. After 8 weeks of degradation in phosphate bu!ered saline (PBS) solution (pH"7.4), the Mn decreased to 43% of the day 0 value [25]. The tensile properties remained relatively stable during degradation. The crystallinity of the conduits increased to 11.5$0.7% (n"6) after 8 weeks [25]. All the conduits maintained their weight, size, and shape throughout the time course. 2.2. Surgical technique The right sciatic nerves of 21 male Sprague Dawley (250 g) rats were implanted with the 12 mm PLLA conduits as previously described [24]. An additional 10 animals received nerve isografts to their right sciatic nerve from "ve donor animals and served as controls. Nerve isografts are nerves taken from similar immunologic animals (Sprague Dawley) and is a common control equating to nerve autografts; autografts are the current clinical gold standard. Brie#y, the animals were anesthetized and maintained by a 0.4 cm intramuscular injection of a premixed solution containing 95 mg/kg ketamine HCL (Keta-Sthetic2+, Boehringer Ingelheim, St. Joseph, MO), 6 mg/kg xylazine (Rompun2+, Miles, Shawnee Mission, KS), and 0.05 mg/kg atropine sulfate (Elkins-Sinn, Cherry Hill, NJ). The skin from the clipped lateral thigh was scrubbed in a routine fashion with antiseptic solution. The sciatic nerves were exposed by a muscle splitting incision and the sciatic nerve was divided near its origin to create an adequate distal segment. The 12 mm conduits were interposed between the ends of the sciatic nerve using 10-0 nylon sutures (Sharpoint Surgical Specialties Corp., Reading, PA) under microsurgical technique. The nerves were inserted into the conduits such that 1 mm of the nerve end remained within the tubular biodegradable sca!old. In a similar microsurgical technique, 12 mm nerve isografts harvested from donor animals were utilized to repair defects in the rat sciatic nerve. Nerve isografts were placed in a reverse fashion to prevent inadvertent axonal branching during proliferation through side branches from the donor nerve. Muscle and skin were closed using 4-0

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Dexon sutures (Davis#Geck, Wayne, NJ). Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health regulations and standards. 2.3. Functional assessment Walking track analysis was performed on all animals after conduit placement monthly through 16 weeks as previously described [24, 27]. This time schedule was chosen to allow adequate time for nerve regeneration, considering the length of the isograft/conduit and the distal nerve segment. Preoperatively, the animals were trained to walk down a 30;4 inch track into a darkened enclosure. Postoperatively, the rats' hind paws were painted with water soluble ink and any changes in their paw prints caused by nerve injury and denervation were recorded. Three di!erent parameters of the rodent's paw print were measured to determine the sciatic functional index [27]: Sciatic functional index"!38.3 (PLF)#109.5 (TSF) #13.3 (ITF)!8.8, PLF"Print length function"(Experimental PL !Normal PL)/Normal PL, TSF"Toe spread function"(Experimental TS !Normal TS)/Normal TS (1st}5th Toe), ITF"Intermedian toe spread function "(Experimental IT!Normal IT)/Normal IT (2nd}4th Toe). Changes in the sciatic functional index (SFI) correlate with changes in the rat paw print and are indicative of nerve injury and regeneration. Those SFI values approaching zero indicate better recovery. Although the SFI is an indirect measurement, it has been well established as an indicator of functional nerve restoration [27]. At 6 weeks, nine rats implanted with a PLLA conduit and "ve rats implanted with an isograft were euthanized. The remaining 12 rats implanted with a PLLA conduit and "ve rats implanted with an isograft were euthanized after 16 weeks. The medial and lateral gastrocnemius muscles were harvested and weighed to assess nerve reinnervation. The gastrocnemius muscle is supplied by the posterior tibial branch of the sciatic nerve. Once the nerve is severed, the muscle will begin to atrophy. As the nerve regenerates into the muscle, it will regain its mass proportional to the amount of reinnervation. This provides indirect evidence of nerve regeneration. Weight was

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determined by placing the muscle in pre-weighed sterile saline containers to prevent dehydration. The di!erence in weight of the containers before and after muscle placement determined the muscle weight. 2.4. Histological assessment At the conclusion of 6 and 16 weeks, sections of the midconduit/isograft and distal sciatic nerve from the same rats used for functional evaluation were harvested, "xed with 3% glutaraldehyde solution, embedded in epoxy resin, and stained with toluidine blue. The toluidine blue stained nerve sections were placed on the stage of an inverted microscope and viewed with phase optics. Images of the histological sections were digitized using a CCD camera and subsequently analyzed using standard image processing and analysis techniques [24]. Images were thresholded and segmented into individual axons. The number of axons per three randomly selected "elds measuring 80;60 lm were counted to give the number of axons/mm. The area of each axon was determined, summed together, and normalized with respect to the image area to give the nerve "ber density. This is a summation of myelination as the staining process identi"es myelin which is subsequently measured via image analysis.

was realized with previous PLGA conduits [24]. Proximal and distal nerve ends maintained their adherence to the conduit through the use of the microsuture. No signi"cant in#ammatory reactions could be identi"ed and no neuromas were apparent. Evaluation of the SFI for both control and conduit animals demonstrated improved functional recovery through 16 weeks indicating muscle reinnervation. Control animals had a lower SFI than the PLLA experimental animals by 16 weeks (Fig. 1). The weight of the gastrocnemius muscle was greater in the control isografts than the experimental PLLA conduits (Fig. 2).

2.5. Statistical analysis Statistical analysis was performed by two-sample t-test for independent samples with adjustment of variance and Wilcoxon rank sum tests, where appropriate. Statistical analysis for functional parameters was based on a mixed-e!ects model using the maximum likelihood method [28, 29]. The model includes a random subject e!ect to re#ect that rats are samples of a population and to induce correlation between measurements from the same rat. Since possible treatment di!erences are observed after the operation, the interaction e!ects in the model indicate the treatment e!ects assuming a common operation e!ect across all rats. To assess if the means of the SFI vary among groups of rats with di!erent treatments, the model allows each rat to have a random initial index as well as a random average decline in an index over several months. To determine the mean number of axons and the mean nerve "ber density, the model includes treatment and location e!ects, their interaction e!ects, as well as a random subject e!ect. The data were expressed as means$standard deviation. Statistical signi"cance was determined by P(0.05.

3. Results and discussion All conduits remained intact without breakage. No conduit elongated during the 16 weeks of placement as

Fig. 1. Sciatic functional index (SFI) for the control (isograft) and experimental (PLLA) animals performed monthly through 16 weeks. Control animals had a lower SFI (indicating improved reinnervation) compared to the PLLA animals by 16 weeks (P"0.000).

Fig. 2. Gastrocnemius muscle weight for animals implanted with control isografts and PLLA conduits and harvested at 6 and 16 weeks. The di!erence in gastrocnemius muscle weight was signi"cant at 6 weeks (P"0.005), but not 16 weeks (P"0.29).

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Fig. 3. Nerve "ber density calculated from sections at the midconduit/isograft and distal nerve for control isograft and PLLA conduit implants harvested at 6 and 16 weeks. The di!erence in nerve "ber density at the distal sciatic nerve was signi"cant at 6 weeks (P"0.010), but not 16 weeks (P"0.10). Also, the di!erence in nerve "ber density at the midconduit was signi"cant at 6 weeks (P"0.014), but not 16 weeks (P"0.06).

Fig. 4. Number of axons per unit area calculated from sections at the midconduit/isograft and distal nerve for control isograft and PLLA conduit implants harvested at 6 and 16 weeks. The di!erence in number of axons per unit area at the distal sciatic nerve was signi"cant at both 6 weeks (P"0.002) and 16 weeks (P"0.004). Also, the di!erence in number of axons per unit area at the implant midpoint was signi"cant at both 6 weeks (P"0.000) and 16 weeks (P"0.003).

Histomorphometry demonstrated axonal growth and nerve tissue advancement through the entire conduit and into the distal nerve stump at 6 and 16 weeks (Figs. 3 and 4). Compared to the control isografts, the number of axons per unit area and nerve "ber density in the distal nerve of the PLLA conduits were signi"cantly less except for the nerve "ber density at 16 weeks (Fig. 5a}c). It should also be noted that the temporal di!erences between 6 and 16 weeks in the distal nerve of the PLLA conduits were signi"cant for both the number of axons per unit area and the nerve "ber density. When evaluating the number of axons per unit area and nerve "ber density in the midconduit and control isograft, the values of the control isografts were greater than the PLLA conduits except at 16 weeks. The increased axon count and nerve "ber density in the midconduit compared to control isograft at 16 weeks may indicate pruning of regenerated "bers in the isografts. This occurs when elongating axons that do not reach their distal targets degenerate after a period of time. This pruning may occur sooner within the nerve isograft as axons may reach their target organs due to necessary factors present for axonal proliferation (support cells, induction factors). This may not be apparent at this time point within the PLLA conduits as axons may still be hunting for target innervations. This could be explained by the lack of aligned and support structures within the empty tube. Temporal changes between 6 and 16 weeks in the midconduit/isograft were also signi"cant (Figs. 3 and 4).

The present study provides in vivo trials of previously characterized PLLA conduits [25]. The "rst requirement for nerve regeneration is a conduit for guided axonal growth. PLLA polymers are attractive candidates for fabricating conduits because they are biocompatible, able to hold suture, can provide a porous sca!old for vascularization, and biodegrade into naturally metabolized products [30]. Degradation of the polymer does not appear to inhibit axonal growth. We believe that our experimental animal results are less than those for control animals because of the lack of induction factors and support cells, which are in place in a normal nerve graft. Harvest of the PLLA conduit required dissection of "rmly adherent muscle indicating tissue incorporation, vascularization, and polymer degradation. Histologic evaluation of the conduits noted traversing blood vessels (Fig. 6) which would allow nutritive support to advancing axons and provide delivery of induction factors. The signi"cant increase in axon number/mm in the distal sciatic nerve at 16 weeks over previously described results with 75 : 25 PLGA conduits [24] may be due to the absence of elongation and conduit collapse (10 700 axons/mm vs. 5800 axons/mm). Elongation was demonstrated with the use of PLGA conduits and is believed to be due to the degradation of the polymer and continued growth and movement of the animal. The mechanical stress placed on the conduit causes the conduit to elongate and subsequently collapse. This would inhibit the ability of axons to proliferate and is

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Fig. 5. Light micrographs demonstrating (a) the native sciatic nerve in the Sprague Dawley rats, (b) distal sciatic nerve at 16 weeks in control isografts, and (c) distal sciatic nerve at 16 weeks in the PLLA group. Magni"cation at 500;.

Fig. 6. Photomicrograph demonstrating vascular ingrowth through the side wall of a PLLA conduit at 16 weeks. Magni"cation at 500;. The Section includes an arrow indicating blood vessels within the conduit wall. No nerve tissue is identi"ed.

degradation has not been conducted and it is conceivable that degradation products (lactic acid) may adversely a!ect axonal growth and nerve function. This question is being addressed in future long-term studies. Further the critical nerve defect may not have been reached with our conduits. Most studies however suggest that this critical nerve defect is 10 mm [9, 15]. It was not the purpose of this study to evaluate nerve regeneration using a critical size defect but to assess the conducive ability of PLLA to serve as a potential conduit for peripheral nerve regeneration. Although results are less than those noted with control isografts, we believe PLLA can serve as a viable sca!old for axonal guidance. Modi"cations of the biologic environment with support cells and nerve induction factors may enhance nerve regeneration.

Acknowledgements a documented problem with nerve guidance channels [24]. PLLA conduits provided a more stable and optimal polymer for this purpose. Although the results are encouraging, certain limitations were recognized within the study. Full polymer

This work was supported in part by grants from the National Institutes of Health (R29-AR42639) (AGM), The University Cancer Foundation (GRDE) and the Thomas Cronin Fund (KB, GRDE).

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