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Polymer electret guidance channels enhance peripheral nerve regeneration in mice
300
Brain Research, 480 (1989) 300--304
Elsevier BRE 23353
Polymer electret guidance channels enhance peripheral nerve regeneration in mice R.F. Valentini I, A.M. Sabatini 2, P. Dario 2 and P. Aebischer 1 IArtificial Organ Laboratory, Brown University, Providence, R! 0,"912 ( U.S.A. ) and 2Scuola Superiore S. Anna and Cemro 'E. Piaggio', University of Pisa, Pisa (Italy)
(Accepted 18 October 1988) Key wor&: Axotomy;Axonal regeneration; Guidance channel; Electret
Polytetrafluoroethylene(PTFE) tube~were prepared as electrets displayinga quasi-permanent surface charge due to the presence of 'rapped monopolar charge carriers. PTFE tubes containingeither positive or negativecharges and electricallyneutral PTFE tubes were used as nerve guidance channels for t~c repair of a ~ mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positivelyand negativelycharged PTFE electrets contained regenerated nerves with significantlymore myelinated axons than nerves regenerated in uncharged PTFE tuhes. Thi~ observation suggeststhat peripheral nerve regeneration can be enhanced by electrically charged nerve guidancechannels.
It has long been appreciated that the peripheral nervous system (PNS) is capable of regeneration following transection injury, although the precise mechanisms controlling regeneration are not known. A wide range of physical a , d chemical factors have been shown to influence the regeneration process. Recently, exogenously applied electrical fields have also been implicated in affecting neural regeneration. Several groups have reported that applied electric fields influence the extent and direction of neurite outgrowth from neurons cultured in vitro~'~°, although others have observed little or no effecte. In vivo regeneration following transection injury appears to be influenced by externally applied DC stimulation ~ and pulsed electromagnetic fields~.~.. Galvanotropic currents produced by silicone channels fitted with electrode cuffs have also been shown to enhance PNS regeneration in vivo~. Polymer electrets are a class of materials which may provide information regarding the effects of electrical charges on PNS regeneration. These materials can be fabricated to generate either transient or static electrical charges due to their physico-chemical Cor~,pondence: P. Aebischer, ArtificialOrgan ~ l o r y ,
properties 12. They are attractive f¢;r in vivo applications since they can be fabricated from biocompatible polymers and can generate electrical char?es without an external power so-J~e. Piezoelectric materials, such as polyvinylidene fluoride (PVDFj, are a class of electrets which depend primarily o~ dynamic mechanical deformation in order to generate transient charges on their surface 3. Chargo generation in piezoe!~'ctric polymers is due to the presence of stable, oriented molecular dipoles throughout the bulk of the material. In contrast, other polymers, such as polytetrafluoroethylen,; (PTFE), exhibit a different charge storage mechanism, relating primarily to monopolar charges entrapped in the material. PTFE electrets display a ~tatic surface charge whose distribution and stabihty are related to the method of fabrication. The term 'electret' usually defines a material displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers 4. In a previous study, we reported that piezoelectric PVDF guidance channels used to repair severed sciatic nerves in mice enhanced neural regeneration as
Box G, BrownUnivemly, Providence,RI 02912, U.S.A.
0006-8993/89/~y3.50C) 1989Elsevier SciencePublidm~ B.V. ( ~ !
Dividoa)
comps:red to non-piezoelectric PVDF tubes ~. Transient charges generated by random animal movements deforming the channel were thought to be thc factor underlying enhanced regeneration, but the e',tent of mechano-electrical transduction is difficult to quantify under ia vivo conditions. In order to investigate other mechanisms involved in the local electrical stimulation of nerve regeneration, and in particular a possible difference between predominantly dynamic (piezoelectric PVDF) and predominantly static (PTFE electret) charge generation, we compared the effect of either positively or negatively charged PTFE electret tubes on PNS regeneration in the mouse model with that of unpoled PTFE tubes of the same physico-chemical composition. Solid-walled PTFE tubes (Gore, Flagstaff, AZ) with an internal diameter of 0.9 mm were submitted to a corona poling procedure in order to inject electriced carriers into them. A brass wire fitted into the lumen of the tube was used as a reference electrode and a circumferential array of regularly spaced steel needles, at a distance of 2 mm from the outer wall of the tube, served to generate the high intensity electric field required for electret preparation. For positive corona discharge, the outer electrode array was connected to the positive output of a high voltage D.C. power supply with the inner electrode grounded; polarity was reversed for negative corona discharge. The corona poling process was performed at 150 °C to obtain electrets with high charge storage capabilities since, at high temperatures, the charge carriers pene,'rate more deeply into the polymer bulk, although rarely exceeding a few microns 12. The applied voltage was gradually increased to 14 kV and maintained at that level for 20 rain. The net surface charge density on the outer surface of each eiectret tube was measured using an induction-based method n;. A capacitative probe was placed 2 mm from the outer surface of the electret tube and connected to an electrometer (ketthley 610 C, Cleveland, OH). When exposed to the electric field produced by the electret, the probe acquires a charge, derived from the capacitance at the input of the electrometer. The meter voltage is thus directly related to the quantity of charge trapped in the electret. The average charge density meamred for positively poled tubes was 21 nC/cm2 and for negatively poled tubes was 9 nC/cm 2. This difference is attributed to the more limited pen
etration of negati ~e cha,g,~s into FT.:E t,ms t2. Identical PTFE tubes not submitted to electrical poling displayed no surface charge density and served as controls. All tubes were cleaned and sterilized similarly prior to implantation. The lef~ sciatic nerve of methoxyflurane-,nesthetized female CD-I mice (Charles River, Wilmington, MA) was exposed through an incision along the anterior-medial aspect of the upper thigh. A 3-4 m m segment of nerve proximal to the tibio-peroneal bifurcation was resected and discarded. A 4 mm nerve gap was created by anchoring the proximal and distal nerve stumps 4 mm apart within 6 mm long tubes using single 1043 nylon sutures. The tubes were prefilled with physiologic saline in order to prevent trapping of air bubbles within their lumens. Cohorts of 5 animals were implanted with positive and negatively charged PTFE electret tubes and control PTFE tubes. Animals were housed in climate-controlled rooms and received food and water ad libitum. Four weeks after implantation, the mice were deeply anesthetized with Nembutal and perfused transcardially with 5 ml of heparinized phosphatebuffered saline followed by 10 ml of a fixative containing 3.0% paraformaldehyde aad 2.5% glutaraldehyde at pH 7.4. The operative site wa ~ -eopened and the guidance channel and native scJa,c nerve removed. The specimens were then postfixed in a 10% osmium tetroxide solution, dehydrated, and embedded in Spurr resin. Transverse sections taken at the midpoint of the guidance channel were cut on a Sorvall MT-'~000 m;.crotome. Semi-thin and ultrathin sections were stained and prepared for light and electron microscopy. The cross-sectional area of the regenerated cable, the total blood vessel area, and the number of myelinated axons and blood vessels were measured with a Zeiss IM 35 microscope interfaced with a computerized morphometric system (CUE-2, Olympus, Lake Success, NY). The MannWhitney rank sum test was used to assess statistical difference between the various populations. All data are presented as mean _+S.E.M. Upon gross observation, all PTFE tubes were noted to elicit a minimal tissue reaction. This was later confirmed with histological analys/s which revealed several layers of fibroblasts and connective tissue surrounding the polymer. Regenerated tissue cables bridging the nerve stumps were visthalized
302 verse sections taken at the midpoint of ~he regenerated cable revealed numerous myelinated axons and blood vessels surrounded by a relatively thin epineurial sheath (Fig. 1A, C, E). Macrophages were often
through the translucent PTFE wall in all 15 implants. The regenerated cables were circular in shape and surrounded by a viscous gel, and never grew in contact with the inner wall of the PTFE tubes. Trans-
A
.%
C
,I
i-
I
z: Fi8. 1. Toluidine blue-stained transverse sectiom of nerves regenerated at the midlPointof unpoled (A), t g p ~ ~
(C), ~ ~ itively poled (E) polytetrafluoroethylene t u ~ (PTY'E)after 4 weeks of implantation (original magnification 118×). Note the numerous my©iinat~ axons surrounded by a fine epineur/al sheath and the mm'ked ~ differs-ricebetween the various rewesentative nerves. Higher power photomicrographs (original magnification 460x) of the same regenerated nerves reveal the more regular shape of myelinated axonsregenerated in unooled PTFE tubes (B) as compared to negatively (D) and positively (F) poled tubes.
303 noted lining the regenerated cables and the inner wall of the guidance channel. More detailed electron microscopic examination revealed presumptive Schwann cells and numerous microfascicles surrounded by perineurial-like tissue (Fig. 2). Numerous unmyelinated axons and myelinated axons at various stages of myelination were observed. Mast cells were occasionally seen within the regenerated cable. Quantitative analysis revealed significant differences among the 3 study groups. The cross-sectional area of nerves regenerated in positive PTFE electrets was significantly greater than for nerves regenerated in unpoled ~ tubes (Fig. 1A, E; Table I). Cables regenerated in negatively poled tubes showed intermediate values (Fig. 1C; Table I). The relative blood vessel area as compared to total cable area was greater in both poled tubes than in unpoled tubes (Table I). The number of myelinated axons regenerated in positive and negative electrets was significantly greater than observed in unpoled tubes, but there was no significant difference between positively and negatively charged electrets (Table lj. Qualitatively, the shape of individual myelinated axons regenerated in both types of poled tubes appeared to be quite irregular while those in unpoled tubes were rounder and more uniform in shape (Figs. 1B, D, F and 2). This study provides further evidence that electrically charged guidance channels enhance PNS regeneration. The fact that nerves regenerated in both positively and negatively poled electret tubes contained greater numbers of myelinated axons than unpoled tubes suggests that the polarity of electrical charges may not be critical in modifying the early events of the regeneration process. U:lder the conditions of the study, the influence of a net electrical field on the regenerating tissue may have enhanced regeneration. The number of myelinated axons seen in both TABLE
I
Quantitativeanalysisof regeneratednerve cables
Positive Cable area
(xIO4~)
16.7 +_1.5"
Negative 12.5 ± 1.4
Unpoled 10.2 _+1.2
Blood vesselarea 8.8+ 1.6" 7.2+2.2* 3.1 + 1.0 (% of total area) Myelinatedaxons 2,301:1:206* 2,118 + 181" 1,544+ 160 * Statistical si~niflcance(P < 0.05) as compared to unpoled PTFE tubes.
Fig. 2. Transmissionelectron micrographof a nerve fascicleregenerated in a positive PTFE electret 4 weeks postimplantatior. Note the presence of irregularlyshaped axons at different stages of myelinationin close contact ,vithpresumed Schwann cells. Numerous unmyelinated axons are also seen (original magnification2887<).
types of PTFE electret tubes at 4 weeks is similar to the number seen in piezoelectric PVDF tubes at 4 weeks as reported in an earlier study ~. It is interesting to note that numerous myelinated axons regenerated in PTFE robes displayed an irregular morphology similar to that seen in normal peripheral nerves. Rounder, more regularly shaped myelinated axons were observed in unpoled PTFE tubes as well as in unpoled and piezoeleo.,ic PVDF tubes t. This may be due to the different potterns of charge generation occurring in PTI.E electrets and piezoelectric PVDF tubes. Comparison of the results obtained with the PTFE versus the PVDF tubes should be made with caution since it is difficult to fabricate pure elcctret (i.e. trapped monopolar charges with no oriented dipoles) or pure piezoelectric (i.e. oriented dipoles with no trapped monopolar charges) materials. However, the fabrication techniques used in our studies insure a predominantly electret effect with poled PTFE tubes and a predominantly piezoelectric effect with PVDF tubes ~. The difference in the shape and number of myelinated axons in nerves regenerated in electrically poled versus non poled PTFE tubes, which arc otherwise identical, suggests that some type of electrical phenomenon influenced the regeneration process. It
304 has proven difficult, however, to characterize experimentally the precise charge storage and decay mechanisms associated with electret and piezoelectric materials ~2. With PTFE electrets, the primary source of electrical stimulation in vivo derives from the electrical field present in the lumen of the tube. The net field is generated by charges located throughout the wall of the PTFE tubes, but the charges may be due to several charge formation mechanisms. The configuration of our poling apparatus is such that charges are injected primarily on the outer surface of the tubes. Since charge carrier penetration is on the order of a few microns and the wall thickness of the PTFE polymer is about 175/tm, it is highly improbable that free charges migrate to the surface of the inn,:i .all. Rather, as in most PTFE electrets studied, a compensation charge equal in magnitude but opposite in sign to that of the monopolar charges injected on the outside appears on the opposite side 12. An ideal cylindrical elect, ret would exhibit no net luminal electrical field because of vectorial charge cancellation. Inhomogeneities in the charging process (i.e. unevenly distributed charge injection) and in the PTFE polymer (i.e. impurities and polymer chain irregularities) can lead to an uneven distribution of trapped charges and compensation charges within the bulk of the tube wall, resulting in a net electric field within the lumen of th ~ charged PTFE tubes. Slight mechanical deformation of the PTFE electrcts
1 Aebischer, P., Valentini, RF., Dario, "., Domenici, C. and Galletti, P.M., Piezoelectric guidance channels enhance regeneration in the mouse sciatic nerve after axotomy, Brain Research, 436 (1987) 165-168. 2 De Boni, U. ano Anderchek, K.E., Quantitative analysisof filopodial activity of mammalian neuronal growth cones in exogenous, electrical fields. In R. Nuccitelli (Ed.), Ionic Currents in Development, Liss, New York, 1986, pp. 285-293. 3 De Rossi, D., Galletti, P.M., Dario, P. and Richardson, P.D., The electromechanicai connection: piezoelectric polymersin artificialorgans, ASAIO J., 6 (1983) 1-11. 4 Hilczer, B. and Malecki, J., Electrets, Elsevier-PWN-Polish Scientific Publishers-Warszawa, (2nd edn.), 1986, 406 pP. 5 lto, H. and Bassett, C.A.L., Effect of weak, pulsing electromagnetic fields on neural regeneration in the rat, Clin. Orthop. Relat. Res., 181 (1983)283-290. 6 Jaffe, L.F. and Poo, M.M., Neurites grow faster toward the
causes charge displacement ~h.;ch may also contribute to the net electrical field in the lumen. Dtrect charge injection may also occur on the luminal surface of the tubes as a result of microdischarges from the snugly fitting inner reference electrode. The use of charged polymers may provide important information about the factors controlling regeneration. In vitro studies using non-mammalian neurons suggest that neurite extension may be affected by direct effects on the growth cone filiopodia or membrane receptors 6"1°. Further efforts should be directed toward determining how the magnitude, pattern, and polarity of static or transient electrical fields affect the regeneration process in vivo. Pat allel in vitro studies analyzing neurite outgrowth from aeurons cultured directly on pi,-zoelectric and electi~,t films should provide insight regarding the influence of electrical activity on cells and neurites in intimate contact with charged polymers. Such studies may lead to a better understanding of the mechanisms underlying neural regeneration and may also lead to the development of clinical devices to enhance recovery following nerve injury. This work was supported in part by a Whitaker Foundation grant and NIH Grant NS26159-01 to P.A. We would like to acknowledge the technical support of Riccardo Di Leonardo, Shelley Winn and Sarah Brace.
cathode than the anode in a steady field, J. Exp. Zoo,, 209 (1979) 115-128. 7 Kerns, J.M. and Freeman, J.A., D.C. electrical fields promote regeneration in the rat sciatic nerve efter axotomy, Soc. Neurosci. Abstr., 12 (1986) 13. 8 Millesi, H.. Reappraisal of nerw. repair, Surg. Clin. North Am., 61 (1981)321-340. 9 Nix, W.A. and Hopf, H.C., Electrical stimulation of regenerating nerve and its effect on motor recovery, Brain Research, 272 (1983)21-25. 10 Patel, N.B. and Poo, M.M., Orientation of neurite outgrowth by extracellular electric fields, J. Neurosei., 2 (1982) 483-496. 11 Raji, A.R.M. and Bowden, R.E.M., Effects of high peak l~u;sed electromagnetic field on the degeneration and regeneration of the common peroneal nerve in rats, J. Bone JointSurg. Br., Vol. 65 (1983)478-492. 12 Sessler, G.M., Electrets, 2nd edn., Springer, Berfin, 1987, 453 pp.
Brain Research, 480 (1989) 300--304
Elsevier BRE 23353
Polymer electret guidance channels enhance peripheral nerve regeneration in mice R.F. Valentini I, A.M. Sabatini 2, P. Dario 2 and P. Aebischer 1 IArtificial Organ Laboratory, Brown University, Providence, R! 0,"912 ( U.S.A. ) and 2Scuola Superiore S. Anna and Cemro 'E. Piaggio', University of Pisa, Pisa (Italy)
(Accepted 18 October 1988) Key wor&: Axotomy;Axonal regeneration; Guidance channel; Electret
Polytetrafluoroethylene(PTFE) tube~were prepared as electrets displayinga quasi-permanent surface charge due to the presence of 'rapped monopolar charge carriers. PTFE tubes containingeither positive or negativecharges and electricallyneutral PTFE tubes were used as nerve guidance channels for t~c repair of a ~ mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positivelyand negativelycharged PTFE electrets contained regenerated nerves with significantlymore myelinated axons than nerves regenerated in uncharged PTFE tuhes. Thi~ observation suggeststhat peripheral nerve regeneration can be enhanced by electrically charged nerve guidancechannels.
It has long been appreciated that the peripheral nervous system (PNS) is capable of regeneration following transection injury, although the precise mechanisms controlling regeneration are not known. A wide range of physical a , d chemical factors have been shown to influence the regeneration process. Recently, exogenously applied electrical fields have also been implicated in affecting neural regeneration. Several groups have reported that applied electric fields influence the extent and direction of neurite outgrowth from neurons cultured in vitro~'~°, although others have observed little or no effecte. In vivo regeneration following transection injury appears to be influenced by externally applied DC stimulation ~ and pulsed electromagnetic fields~.~.. Galvanotropic currents produced by silicone channels fitted with electrode cuffs have also been shown to enhance PNS regeneration in vivo~. Polymer electrets are a class of materials which may provide information regarding the effects of electrical charges on PNS regeneration. These materials can be fabricated to generate either transient or static electrical charges due to their physico-chemical Cor~,pondence: P. Aebischer, ArtificialOrgan ~ l o r y ,
properties 12. They are attractive f¢;r in vivo applications since they can be fabricated from biocompatible polymers and can generate electrical char?es without an external power so-J~e. Piezoelectric materials, such as polyvinylidene fluoride (PVDFj, are a class of electrets which depend primarily o~ dynamic mechanical deformation in order to generate transient charges on their surface 3. Chargo generation in piezoe!~'ctric polymers is due to the presence of stable, oriented molecular dipoles throughout the bulk of the material. In contrast, other polymers, such as polytetrafluoroethylen,; (PTFE), exhibit a different charge storage mechanism, relating primarily to monopolar charges entrapped in the material. PTFE electrets display a ~tatic surface charge whose distribution and stabihty are related to the method of fabrication. The term 'electret' usually defines a material displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers 4. In a previous study, we reported that piezoelectric PVDF guidance channels used to repair severed sciatic nerves in mice enhanced neural regeneration as
Box G, BrownUnivemly, Providence,RI 02912, U.S.A.
0006-8993/89/~y3.50C) 1989Elsevier SciencePublidm~ B.V. ( ~ !
Dividoa)
comps:red to non-piezoelectric PVDF tubes ~. Transient charges generated by random animal movements deforming the channel were thought to be thc factor underlying enhanced regeneration, but the e',tent of mechano-electrical transduction is difficult to quantify under ia vivo conditions. In order to investigate other mechanisms involved in the local electrical stimulation of nerve regeneration, and in particular a possible difference between predominantly dynamic (piezoelectric PVDF) and predominantly static (PTFE electret) charge generation, we compared the effect of either positively or negatively charged PTFE electret tubes on PNS regeneration in the mouse model with that of unpoled PTFE tubes of the same physico-chemical composition. Solid-walled PTFE tubes (Gore, Flagstaff, AZ) with an internal diameter of 0.9 mm were submitted to a corona poling procedure in order to inject electriced carriers into them. A brass wire fitted into the lumen of the tube was used as a reference electrode and a circumferential array of regularly spaced steel needles, at a distance of 2 mm from the outer wall of the tube, served to generate the high intensity electric field required for electret preparation. For positive corona discharge, the outer electrode array was connected to the positive output of a high voltage D.C. power supply with the inner electrode grounded; polarity was reversed for negative corona discharge. The corona poling process was performed at 150 °C to obtain electrets with high charge storage capabilities since, at high temperatures, the charge carriers pene,'rate more deeply into the polymer bulk, although rarely exceeding a few microns 12. The applied voltage was gradually increased to 14 kV and maintained at that level for 20 rain. The net surface charge density on the outer surface of each eiectret tube was measured using an induction-based method n;. A capacitative probe was placed 2 mm from the outer surface of the electret tube and connected to an electrometer (ketthley 610 C, Cleveland, OH). When exposed to the electric field produced by the electret, the probe acquires a charge, derived from the capacitance at the input of the electrometer. The meter voltage is thus directly related to the quantity of charge trapped in the electret. The average charge density meamred for positively poled tubes was 21 nC/cm2 and for negatively poled tubes was 9 nC/cm 2. This difference is attributed to the more limited pen
etration of negati ~e cha,g,~s into FT.:E t,ms t2. Identical PTFE tubes not submitted to electrical poling displayed no surface charge density and served as controls. All tubes were cleaned and sterilized similarly prior to implantation. The lef~ sciatic nerve of methoxyflurane-,nesthetized female CD-I mice (Charles River, Wilmington, MA) was exposed through an incision along the anterior-medial aspect of the upper thigh. A 3-4 m m segment of nerve proximal to the tibio-peroneal bifurcation was resected and discarded. A 4 mm nerve gap was created by anchoring the proximal and distal nerve stumps 4 mm apart within 6 mm long tubes using single 1043 nylon sutures. The tubes were prefilled with physiologic saline in order to prevent trapping of air bubbles within their lumens. Cohorts of 5 animals were implanted with positive and negatively charged PTFE electret tubes and control PTFE tubes. Animals were housed in climate-controlled rooms and received food and water ad libitum. Four weeks after implantation, the mice were deeply anesthetized with Nembutal and perfused transcardially with 5 ml of heparinized phosphatebuffered saline followed by 10 ml of a fixative containing 3.0% paraformaldehyde aad 2.5% glutaraldehyde at pH 7.4. The operative site wa ~ -eopened and the guidance channel and native scJa,c nerve removed. The specimens were then postfixed in a 10% osmium tetroxide solution, dehydrated, and embedded in Spurr resin. Transverse sections taken at the midpoint of the guidance channel were cut on a Sorvall MT-'~000 m;.crotome. Semi-thin and ultrathin sections were stained and prepared for light and electron microscopy. The cross-sectional area of the regenerated cable, the total blood vessel area, and the number of myelinated axons and blood vessels were measured with a Zeiss IM 35 microscope interfaced with a computerized morphometric system (CUE-2, Olympus, Lake Success, NY). The MannWhitney rank sum test was used to assess statistical difference between the various populations. All data are presented as mean _+S.E.M. Upon gross observation, all PTFE tubes were noted to elicit a minimal tissue reaction. This was later confirmed with histological analys/s which revealed several layers of fibroblasts and connective tissue surrounding the polymer. Regenerated tissue cables bridging the nerve stumps were visthalized
302 verse sections taken at the midpoint of ~he regenerated cable revealed numerous myelinated axons and blood vessels surrounded by a relatively thin epineurial sheath (Fig. 1A, C, E). Macrophages were often
through the translucent PTFE wall in all 15 implants. The regenerated cables were circular in shape and surrounded by a viscous gel, and never grew in contact with the inner wall of the PTFE tubes. Trans-
A
.%
C
,I
i-
I
z: Fi8. 1. Toluidine blue-stained transverse sectiom of nerves regenerated at the midlPointof unpoled (A), t g p ~ ~
(C), ~ ~ itively poled (E) polytetrafluoroethylene t u ~ (PTY'E)after 4 weeks of implantation (original magnification 118×). Note the numerous my©iinat~ axons surrounded by a fine epineur/al sheath and the mm'ked ~ differs-ricebetween the various rewesentative nerves. Higher power photomicrographs (original magnification 460x) of the same regenerated nerves reveal the more regular shape of myelinated axonsregenerated in unooled PTFE tubes (B) as compared to negatively (D) and positively (F) poled tubes.
303 noted lining the regenerated cables and the inner wall of the guidance channel. More detailed electron microscopic examination revealed presumptive Schwann cells and numerous microfascicles surrounded by perineurial-like tissue (Fig. 2). Numerous unmyelinated axons and myelinated axons at various stages of myelination were observed. Mast cells were occasionally seen within the regenerated cable. Quantitative analysis revealed significant differences among the 3 study groups. The cross-sectional area of nerves regenerated in positive PTFE electrets was significantly greater than for nerves regenerated in unpoled ~ tubes (Fig. 1A, E; Table I). Cables regenerated in negatively poled tubes showed intermediate values (Fig. 1C; Table I). The relative blood vessel area as compared to total cable area was greater in both poled tubes than in unpoled tubes (Table I). The number of myelinated axons regenerated in positive and negative electrets was significantly greater than observed in unpoled tubes, but there was no significant difference between positively and negatively charged electrets (Table lj. Qualitatively, the shape of individual myelinated axons regenerated in both types of poled tubes appeared to be quite irregular while those in unpoled tubes were rounder and more uniform in shape (Figs. 1B, D, F and 2). This study provides further evidence that electrically charged guidance channels enhance PNS regeneration. The fact that nerves regenerated in both positively and negatively poled electret tubes contained greater numbers of myelinated axons than unpoled tubes suggests that the polarity of electrical charges may not be critical in modifying the early events of the regeneration process. U:lder the conditions of the study, the influence of a net electrical field on the regenerating tissue may have enhanced regeneration. The number of myelinated axons seen in both TABLE
I
Quantitativeanalysisof regeneratednerve cables
Positive Cable area
(xIO4~)
16.7 +_1.5"
Negative 12.5 ± 1.4
Unpoled 10.2 _+1.2
Blood vesselarea 8.8+ 1.6" 7.2+2.2* 3.1 + 1.0 (% of total area) Myelinatedaxons 2,301:1:206* 2,118 + 181" 1,544+ 160 * Statistical si~niflcance(P < 0.05) as compared to unpoled PTFE tubes.
Fig. 2. Transmissionelectron micrographof a nerve fascicleregenerated in a positive PTFE electret 4 weeks postimplantatior. Note the presence of irregularlyshaped axons at different stages of myelinationin close contact ,vithpresumed Schwann cells. Numerous unmyelinated axons are also seen (original magnification2887<).
types of PTFE electret tubes at 4 weeks is similar to the number seen in piezoelectric PVDF tubes at 4 weeks as reported in an earlier study ~. It is interesting to note that numerous myelinated axons regenerated in PTFE robes displayed an irregular morphology similar to that seen in normal peripheral nerves. Rounder, more regularly shaped myelinated axons were observed in unpoled PTFE tubes as well as in unpoled and piezoeleo.,ic PVDF tubes t. This may be due to the different potterns of charge generation occurring in PTI.E electrets and piezoelectric PVDF tubes. Comparison of the results obtained with the PTFE versus the PVDF tubes should be made with caution since it is difficult to fabricate pure elcctret (i.e. trapped monopolar charges with no oriented dipoles) or pure piezoelectric (i.e. oriented dipoles with no trapped monopolar charges) materials. However, the fabrication techniques used in our studies insure a predominantly electret effect with poled PTFE tubes and a predominantly piezoelectric effect with PVDF tubes ~. The difference in the shape and number of myelinated axons in nerves regenerated in electrically poled versus non poled PTFE tubes, which arc otherwise identical, suggests that some type of electrical phenomenon influenced the regeneration process. It
304 has proven difficult, however, to characterize experimentally the precise charge storage and decay mechanisms associated with electret and piezoelectric materials ~2. With PTFE electrets, the primary source of electrical stimulation in vivo derives from the electrical field present in the lumen of the tube. The net field is generated by charges located throughout the wall of the PTFE tubes, but the charges may be due to several charge formation mechanisms. The configuration of our poling apparatus is such that charges are injected primarily on the outer surface of the tubes. Since charge carrier penetration is on the order of a few microns and the wall thickness of the PTFE polymer is about 175/tm, it is highly improbable that free charges migrate to the surface of the inn,:i .all. Rather, as in most PTFE electrets studied, a compensation charge equal in magnitude but opposite in sign to that of the monopolar charges injected on the outside appears on the opposite side 12. An ideal cylindrical elect, ret would exhibit no net luminal electrical field because of vectorial charge cancellation. Inhomogeneities in the charging process (i.e. unevenly distributed charge injection) and in the PTFE polymer (i.e. impurities and polymer chain irregularities) can lead to an uneven distribution of trapped charges and compensation charges within the bulk of the tube wall, resulting in a net electric field within the lumen of th ~ charged PTFE tubes. Slight mechanical deformation of the PTFE electrcts
1 Aebischer, P., Valentini, RF., Dario, "., Domenici, C. and Galletti, P.M., Piezoelectric guidance channels enhance regeneration in the mouse sciatic nerve after axotomy, Brain Research, 436 (1987) 165-168. 2 De Boni, U. ano Anderchek, K.E., Quantitative analysisof filopodial activity of mammalian neuronal growth cones in exogenous, electrical fields. In R. Nuccitelli (Ed.), Ionic Currents in Development, Liss, New York, 1986, pp. 285-293. 3 De Rossi, D., Galletti, P.M., Dario, P. and Richardson, P.D., The electromechanicai connection: piezoelectric polymersin artificialorgans, ASAIO J., 6 (1983) 1-11. 4 Hilczer, B. and Malecki, J., Electrets, Elsevier-PWN-Polish Scientific Publishers-Warszawa, (2nd edn.), 1986, 406 pP. 5 lto, H. and Bassett, C.A.L., Effect of weak, pulsing electromagnetic fields on neural regeneration in the rat, Clin. Orthop. Relat. Res., 181 (1983)283-290. 6 Jaffe, L.F. and Poo, M.M., Neurites grow faster toward the
causes charge displacement ~h.;ch may also contribute to the net electrical field in the lumen. Dtrect charge injection may also occur on the luminal surface of the tubes as a result of microdischarges from the snugly fitting inner reference electrode. The use of charged polymers may provide important information about the factors controlling regeneration. In vitro studies using non-mammalian neurons suggest that neurite extension may be affected by direct effects on the growth cone filiopodia or membrane receptors 6"1°. Further efforts should be directed toward determining how the magnitude, pattern, and polarity of static or transient electrical fields affect the regeneration process in vivo. Pat allel in vitro studies analyzing neurite outgrowth from aeurons cultured directly on pi,-zoelectric and electi~,t films should provide insight regarding the influence of electrical activity on cells and neurites in intimate contact with charged polymers. Such studies may lead to a better understanding of the mechanisms underlying neural regeneration and may also lead to the development of clinical devices to enhance recovery following nerve injury. This work was supported in part by a Whitaker Foundation grant and NIH Grant NS26159-01 to P.A. We would like to acknowledge the technical support of Riccardo Di Leonardo, Shelley Winn and Sarah Brace.
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