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Peripheral nerve regeneration in the presence of collagen fibers: Effect of removal of the distal nerve stump
CIinicrri Mareriak 16 (1994) 73-80 1.0 1994 Elsevier Science Limited Printed in Great Britain. ELSEVlEK
Ail rights reserved 0267-6605!94/$7.00
Nerve Regeneration in the Pr ers: Effect of Removal of th urn Azam Department
izvi, George D. Pins & Frederick of Pathology,
(Received 12 March
UMDNJ-Robert
Wood
1993; sent for revision 22 October
Johnson
H. Silver* Medical
1993; accepted
School,
11 November
Piscataway,
NJ 08854, USA
1993)
Abstract: A previous report from our laboratory indicated that nerve regeneration through silicone tubes filled with self-assembled collagen fibers was affected by the biodegradation rate of the collagen. The present study was undertaken to evaluate the effect of removal of the distal nerve stump from the silicone tube filled with collagen fibers, Results obtained indicate that in the absence of the distal nerve stump, an implant containing collagen fibers facilitates Schwann cell migration and axonal elongation only when the implant does not biodegrade prior to nerve regeneration (by 8 weeks post-implantation). These results and results of previous studies suggest that the presence or absence of the distal nerve stump affects the extent of regeneration through a silicone tube filled with collagen fibers. Nerve regeneration occurs less frequently in the absence of the distal stump. This model may be a more sensitive test of regeneration in the presence of entubulation devices.
stump, preventing axonal misdirection and fibrosis at the suture site. Silicone tubes provide a shortterm, biocompatible microenvironment by which gaps as large as 10 mm can be repaired in rat sciatic nerve,4 but full functional recovery does not occur when silicone rubber tubes are used alone.5 The sequence of events that occur when a transected rat sciatic nerve regenerates across a silicone entubulated 10 mm interstump gap include: (1) secretion of a fibrin matrix, (2) migration of Schwann cells and fibroblasts, (3) axonal elongation and (4) nerve structure formation.6-9 In the silicone tube model, omission of the nerve stump from the distal end of th.e tube results in the absence of any nerve regeneration,” regardless of whether or not the tube is sealed. Under both conditions, the fluid filling the tubes contains high levels of the same neurotrophic factors normally observed.” Williams et a1.lo found that when the distal nerve insert was replaced by a 2 mm piece of skin, a matrix did not form and subsequent cell migration and axonal regeneration did not occur.
TRODUCTION Severe peripheral nerve injuries such as transection produce d&continuities of both the axons and the basement membrane tubes. Following transection, the axons located in the distal stump undergo Wallerian degeneration and are phagocytosed by both Schwann cells and macrophages. Schwann cells proliferate and the basement membrane tubes remain intact in the distal stump.“2 Axonal sprouting occurs from the proximal nerve stump, across the site of injury and into the distal stump. IMisdirection of the sprouting axons results in failure to reestablish contact with the original target organs and thus impairs functional recovery of the peripheral nerve.3 A variety of polymeric tubes consisting of silicone egradable polyesters have been used to bridge excised areas of peripheral nerve. These tubes align axonal growth toward the distal *To whom correspondence
should be addressed.
73
74
A.H. Rizvi, G.D. Pins, F.H. Silver
When a 2 mm piece of tendon was inserted, a matrix did form at one week, although a nerve structure across the gap was not seen at later time periods. The matrix was believed to have either dissolved before cells could enter the tube or it did not promote cellular migration and subsequent axonal regeneration. Weis and Schroder” found fibrin matrix formation and subsequent axonal regeneration when fat tissue was used as a distal insert. Previously, we have reported that reconstituted type I collagen fibers (isolated from bovine corium) facilitate early Schwann cell and fibroblast migration and subsequent axonal regeneration of transected entubulated rat sciatic nerve (10 mm interstump gap) when the fibers biodegraded by 6 weeks post-implantation.‘2 When the fibers remained intact, axonal regeneration was inhibited. The purpose of this study is to evaluate axonal regeneration in a silicone tube containing reconstituted type I collagen fibers in the absence of the distal nerve stump.
MATERIALS
AND METHODS
Insoluble collagen preparation
Insoluble type I collagen from fresh uncured bovine corium was obtained from Devro, Inc. (Somerville, NJ, USA) after fragmentation into pieces. The corium was swollen in acid, precipitated, washed in distilled water and isopropanol, lyophilized, and stored at -30°C as described previously. l3 The composition of the collagen o chains was characterized by SDSpolyacrylamide gel electrophoresis as a-l(I) and a-2(1), beta, gamma and higher molecular weight components of type I collagen after reduction and heat denaturation. The results of amino acid analysis were consistent with the composition of bovine type I collagen, as described previously.i3 Prosthesis preparation
In order to produce collagen fibers, a 1% (w/v) dispersion of type I collagen in dilute hydrochloric acid (pH 2.0) was extruded through polyethylene tubing (inner diameter 0.28 mm) into a bath of aqueous fiber formation buffer containing 135 mM NaCl, 30 mM TES and 30 mM sodium phosphate dibasic (pH 7.4) at 37°C as described by Kato et aLi After immersion in the buffer for 45 min, the fibers were dehydrated in isopropanol for 4 h, rinsed
CONTROL Proximal
Distal Stump 1
15mmSilicone Tube
COLLAGEN
\ Silicone Plug
FIBER TREATED
120 Collagen Fibers \
-15
e-10
mm+ mm -
Fig. 1. Schematic diagram illustrating a silicone tube 15 mm long with a diameter of l-5 mm. The proximal nerve stump is sutured into one end of the tube while the other end is sealed with medical-grade silicone adhesive.
in distilled water for 15 min, and allowed to air dry overnight. The collagen fibers were crosslinked by exposure to a condition of severe dehydration at 110°C and a vacuum of 0.1 mtorr for 72 h. Approximately 120 of these fibers (diameter 100 pm, length 10 mm) were inserted into a silicone tube (inner diameter 1.5 mm, length 15 mm) in order to form a longitudinally aligned fibrous collagen plug. The silicone tube was sealed at its distal end using medical-grade silicone adhesive which was formed into a plug (Fig. 1). Two groups of implants were prepared. Group 1 was crosslinked using a combination of severe dehydration at 110°C for 3 days and treatment with 1% (w/v) cyanamide (carbodiimide), pH 5.5, in water. The other group was treated with glutaraldehyde vapor at room temperature as described previously. l2 Prostheses were then immersed in Alcide ExsporTM (Alcide Corporation, Norwalk, CT, USA) for sterilization prior to surgery as described previously.’ 5 The extent of crosslinking of the collagen fibers prepared with glutaraldehyde or carbodiimide was characterized by determining the rate of enzymatic degradation using collagenase. Collagenase degradation assay
In order to characterize the degradation rates of the collagen fibers produced from these two groups,
Peripheral nerve regeneration in the presence of collagen jbers
a time-controlled collagenase enzyme assay was conducted. Glass coverslips with collagen fibers from both batches were placed in a 12-well tissue culture plate as described by Wong et aZ.16 The fibers (n = 40) were enzymatically digested in a solution containing 300 units/ml collagenase (Sigma Chemical Co., C-0130 at 300 units/mg), 5 rnM CaC12 and 50 mM Tris-HCl at pH 7.6. The number of fibrils exposed per collagen fiber was recorded at 2 h time intervals under an Olympus CK2 phase contrast microscope. The fibril numbers were plotted versus time for a period of 14 h and compared in order to determine the difference between the degradation rates of both collagen batches.
Thirty-four adult female Sprague-Dawley rats, each weighing between 250 and 350 g, were anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg/kg). Under aseptic conditions, a lateral skin incision was made on the right thigh. In order to expose the sciatic nerve, blunt dissection was performed through the avascular fascial plane between the vastus lateralis and hamstring muscles. Following exposure, the nerve was transected with microsurgical scissors and then the proximal stump was placed into the open end of the silicone tube. The stump was secured in place by suturing the nerve’s outer epineural sheath to the silicone tube with 9-O nylon suture. The muscle layers and skin opening were closed with 9-O nylon and 4-O chromic gut sutures, respectively. An identical surgical procedure was performed on a control animal group. In the control group, silicone tubes without the collagen fibers were used to bridge the gap. After intervals of 4 and 8 weeks, the regenerated nerve tissue from each group was explanted and studied by electron microscopy.
After time intervals of 4 and 8 weeks, the animals were reanesthetized and the newly regenerated nerve tissue in the silicone tube was fixed in 1.5% (v/v> glutaraldehyde, 4% (w/v) paraformaldehyde in 0.1 M cacodylate and 4 mM CaClz. Immediately after removal, the silicone tube was slit longitudinally while immersed in fixative. The regenerated nerve tissue was photographed, then removed and transferred to fresh fixative
75
overnight. The tissue was postfixed in 1% (w/v> osmium tetroxide and block stained in 1% (w/v> aqueous uranyl acetate solution. It was then processed through an ascending series of alcohols and embedded in Polybed 812 resin. Ultrathin cross-sections of the nerve were cut one-quarter (proximally or 2.5 mm), midway or 5 -0 mm, and three-quarters (distally or 7.5 mm) along each processed explant. The sections were stained with 5% (w/v> aqueous many1 acetate and lead citrate,17 then subsequently analyzed and photographed with a Phillips Model 420 transmission electron microscope. Schwann cells were identified by their close association with myelinated and unmyelinated axons, membrane characteristics and dark-staining nuclei. Fibroblast identification was based on elongated cell and nuclear structures, prominent rough endoplasmic reticulum and characteristic nuclear staining properties. Perineurial cells were distinguished by their multjlayered, flattened appearance comprising the fascicles surrounding groups of axons.
RESULTS
A total of 11 animals were used in each of the collagen fiber treated groups (5 sacrificed 4 weeks post-implantation and 6 at 8 weeks post-implantation). Twelve animals were used for the control group (6 sacrificed at 4 and 8 weeks post~impla~tation). Tissue regeneration at 4 weeks
At 4 weeks post-implantation, no nerve tissue was visible between the roximal nerve stump and the distal silicone plug the control group. The control explants contained no ganized structure in the 10 mm gap region; only id was visible. Of the 6 animals implanted with the control prosthesis, none showed any evidence of nerve tissue regeneration. In the group containing ~arbodiimide-treated collagen fibers (CARB), 4 of the 5 explants showed evidence of nerve regeneration at 4 weeks postimplantation. The distance the nerves regenerated in each explant in descending order was 3 - 5, 3 *O, 2.5 and 0-O mm, respectively. Thus, the average length of nerve growth in the CAR collagen fiber group wa,s 2.4 & 1.3 mm (Fig. 2). Grossly, the implanted collagen fibers were observable only up to the point where new nervous
A.H. Rizvi, G.D. Pins, F.H. Silver
76
Implantation Duration
Control i / 0
? ?BWeeks. ??4Weeks I
z
4
6
8
1
Length (mm) of nerve regeneration
Fig. 2. Distance diimide
(CARB)
of nerve regeneration in sealed silicone tubes containing treated collagen fibers. Glutaraldehyde (GLUT), carbotreated collagen fiber implants and control silicone tubes (Control) were observed at 4 and 8 weeks postimplantation.
tissue appeared. Thus, the collagen fibers which were 10 mm in length at the time of implantation had biodegraded to 30% of their initial lengths by 4 weeks. Histologically, islands of unmyelinated axons and their associated Schwann cells were observed in the areas between the collagen
Fig. 3. Transmision
fibers (Fig. 3). Very few myelinated axons were visible and few blood vessels were observed. The formation of fascicles around the axons and Schwann cells by alternating layers of perineurial cells and connective tissue was not visible at this stage.
electron micrograph showing unmyelinated axons (U) and their associated Schwann cells (SC) observed presence of carbodiimide crosslinked collagen fibers (CF) 4 weeks post-implantation.
in the
Peripheral
nerve regeneration
In the glutaraldehyde-treated collagen fiber group (GLUT), all the explants showed evidence of nerve regeneration at 4 weeks post-implantation. The distance the nerve regenerated in each of the explants was 5.0, 4.5, 4.5, 4-O and 4-O mm, respectively. The average length of regeneration among the 5 specimens in this group was 4”4 f O”42 mm (Fig. 2). Grossly, the collagen fibers were visible from the proximal stump to the distal silicone plug. The fibers displayed no evidence of degradation or change in structure. Also, the regenerated tissue could be identified by the darker contrast material visible within the confines of the implanted collagen fibers. Histologically, the structure of the regenerated nerve was similar to the tissue observed in the implant containing carbodiimide-treated collagen fibers at 4 weeks. Group s of unmyelinated axons were visible with few myelinated axons and blood vessels. ost-implantation S, no regenerated nerve tissue was visible in the control group explants. Similar to the explants at 4 weeks post-implantation, no organized structure in she 10 mm gap region could be seen. Only fluid was observed. Of the 6 animals
in the presence
ofcollagen
Jibers
implanted with the control prost of tissue regeneration was obser any animal. In the carbodiimide-treated collagen fiber implant group, none of the 6 explants showed tissue regeneration at 8 weeks cost-i~~~lantatio~. The implanted collagen fi ers which were originally 10 mm in length we no longer visible by 8 weeks. In their place, fluid was observed. This observation suggests that the carbodiimidetreated collagen fiber implant had completely degraded by 8 weeks. In the glutaraldehyde-treated collagen fiber group, all 6 explants showed evidence of nerve regeneration at 8 weeks. The regeneration distance in each of the explants was 8-5, 8.0, 7.5, 7-5, 7.5 and 7-O mm, respectively. Thus, the average regeneration distance among the 6 explants was 7.7 f Oa52 mm (Fig. 2). As in the 4-week glutaraldehyde-treated collagen pi implanted collagen fibers spanned the 10 mm gap. No evidence of fiber degradation was observed. The newly regenerated tissue could by the darker area observed within the collagen fiber scaffold. Histologically, the newly regenerated nerve tissue at 8 weeks differed from the tissue observed at 4 weeks post-i~l~~la~tation. More
Fig. 4. Transmission Aectron micrograph showing myelinated (M) and unmyelinated axons (U) and S&warm ceils in the tissue regenerated in the presence of glutaraldehyde crosslinked collagen fibers (CF) 8 weeks post-implantation.
78
A.H. Rizvi, G.D. Pins, F.H. Silver
myelinated axons and fewer unmyelinated axons were observed. Also, fascicularization of the axons and their associated Schwann cells was visible indicating greater maturity in the &week explant when compared to the 4-week specimen (Fig. 4). In the 4-week carbodiimide- and glutaraldehydetreated collagen fiber implant groups, the longitudinally aligned collagen fibers appeared roughly parallel to the long axis of the regenerating tissue. At the EM level (Fig. 5) regenerating nerve cells extended their axons between collagen fibers in a longitudinal or linear direction. Collagenase degradation assay
The carbodiimide and glutaraldehyde crosslinked collagen fibers degraded at different rates. At 8 weeks post-implantation, the carbodiimide crosslinked collagen fibers had disappeared but the glutaraldehyde crosslinked collagen fibers remained intact and appeared to be minimally degraded. A time-controlled collagenase enzyme assay was conducted by measuring the number of fibrils exposed in each fiber per unit of time, in order to compare the degradation rates of the carbodiimide and glutaraldehyde crosslinked collagen fibers (Fig. 6). The rate of appearance of fibrils is initially propor-
tional to the rate of collagenase degradation of collagen fibers; in the long term the number of flbrils decreases as the collagen is totally removed. The results indicated that the difference in the collagen degradation rate in vivo correlated with the collagenase degradation rate of the fibers in vitro, i.e. glutaraldehyde crosslinked collagen fibers remained intact longer than carbodiimide-treated fibers.
DISCUSSION
In a previous rat study12 using slowly and rapidly degrading collagen fibers, nerve regeneration was facilitated or inhibited depending on whether the fibers biodegraded by 6 weeks post-implantation (facilitated) or remained visibly intact (inhibited). It was concluded that in the presence of the distal stump and rapidly degrading collagen fibers, fibroblast and Schwann cell migration and axon regeneration is enhanced and more efficient since 100% of the collagen-treated animals showed regeneration. In contrast, the fibrin matrix that formed between opposing stumps was able to support regeneration in only 40% of the control tubes. l2 The purpose of the present study was to investi-
Fig. 5. Transmission electron micrograph of a longitudinal section of a glutaraldehyde-treated collagen fiber implant at 4 weeks postimplantation. Myelinated axons (M) and Schwann cells (SC) can be observed along the path of the collagen fibers (CF).
Peripheral
nerve regeneration
in the presence
of collagen fibers
DHT3X-linkedCollagenFibers Glut X-linkedCollagenFibers
0
0
2
4
6
Time (hours) Fig. 6. Graph i&tstrating collagenase digestion time of crosslinked glutaraldehyde (Glut.) and carbodiimide (DHT3) collagen fibers. Degradation time was determined by counting the number of fibrils exposed at each time interval. The increased rate of fibrils exposed as a function of time denotes greater collagen degradation.
gate axonal regeneration through a silicone tube containing collagen fibers in the absence of the distal nerve stump. Histological and morphological evaluations indicated that no axonal ingrowth had occurred in any of the control tubes that bad been sealed at the distal end with silicone adhesive. Similar observations were noted after 8 weeks in the implants which had contained carbodiimide-treated collagen fibers. In contrast, regeneration was observed at 4 weeks in implants which employed carbodiimide-treated collagen fibers and at 4 and 8 weeks in stents containing glutaraldehyde-treated fibers. Additionally, the rate of biodegradation of carbodiimide-treated fibers was significantly greater than glutaraldehyde-treated fibers both in vivo and in vitro. The results of the current studies, in which the distal end of the tube was blocked, are inconsistent with the conclusions of our previous entubulation experiments Results of entubulation experiments employing both the proximal and distal stumps indicated that nerve regeneration through a silicone tube was possible regardless of the presence of collagen fibers, but rapidly degrading collagen fibers enhanced Schwann cell migration and axonal ingrowth.i2 This and other investigations also at a fibrin matrix may help facilitate regeneration silicone sciatic nerve through tubes.s;i2 In the present studies where the distal stump was replaced with a silicone plug, sciatic
nerve regeneration was observed only in the presence of glutaraldehyde-treated collagen fibers. Empty silicone tubes and tubes where the collagen fibers had completely degraded (carbodiimidetreated fibers implanted for 8 weeks) contained only fluid. Lundborg et aI4 observed similar results when attempting to transect 10 mm gaps with silicone tubes. These findings su.ggest that a fibrin matrix cannot serve as a template for axonal elongation and Schwann cell migration unless both the proximal and distal nerve stumps are in close proximity. These results also imply that the relationship between the collagen fiber biodegradation rate and the extent of regeneration is dependent upon the entubulation model. In the absence of the distal stump, nerve regeneration occurs less frequently. In conclusion, an implant containing collagen fibers without a distal stump facilitates Schwarm cell migration and axonal elongation only when the implant does not biodegrade prior to nerve regeneration. In contrast, when the implants utilize both the proximal and distal sturn eration is inhibited by collagen fibers which remain intact for periods greater than the time required for regeneration to be completed. These data suggest that nerve entubulation in the absence of the distal stump leads less frequently to nerve regeneration in controls and may therefore be a more sensitive model of regeneration.
80
A.H.
Rizvi,
G.D. Pins,
REFERENCES 1. Carbonetto, S., The extracellular matrix of the nervous system. Trends Neurosci., 7 (1982) 382-7. 2. Aguayo, A.J. & Bray, G.M., Experimental nerve grafts. In Nerve Repair and Regeneration: Its Clinical and Experimental Basis, ed. D.L. Jewett & H.R. McCaroll Jr. C.V. Mosby Co., St Louis, MO, 1980, pp. 68-76. 3. Lundborg, G., Nerve regeneration and repair. Acta Orthop. Stand., 58 (1987) 145-69. 4.
Lundborg, G., Dahlin, L.B., Danielson, N., Gelberman, R., Longo, F., Powell, H. & Varon, S., Nerve regeneration in silicone chambers: influence of gap length and of distal stump components, Exp. Neural., 76 (1982) 361-75. 5. Ashur, H., Vilner, Y., Finsterbush, A., Rousso, M., Weinberg, H. & Devor, M., Extent of fiber regeneration after peripheral nerve repair: silicone splint versus suture, gap repair versus graft. Exp. NeuroZ., 97 (1987) 365-74. 6. Williams, L.R., Longo, F.M. & Powell, H.C., Spatialtemporal progress of peripheral nerve regeneration within a silicone chamber: parameters for a bioassay. J. Comp. Neuvol., 228 (1983) 460-70. 7. Longo, F.M., Skaper, S.D., Manthorpe, M., Williams, L.R., Lundborg, G. & Varon, S., Temporal changes of neuronotrophic activities accumulating in vivo within nerve regeneration chambers. Exp. NeuroZ., 81 (1983) 756669. 8. Longo, F.M., Hayman, E.G., Davis, G.E., Ruoslahti, E., Engvall, E., Manthorpe, M. & Varon, S., Neuritepromoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers. Brain Res., 309 (1984) 105-17. 9.
Longo, F.M., Manthorpe,
M., Skaper, S.D., Lundborg, G.
F.H. Silver
& Varon, S., Neuronotrophic activities accumulate in vivo within silicone nerve regeneration chambers. Brain Res., 261(1983) 109-17. 10. Williams, L.R., Powell, H.C., Lundborg,
G. & Varon, S., Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber, Brain Res., 293 (1984) 201-11.
11. Weis, J. & Schroder, J.M., Differential effects of nerve, muscle, and fat tissue on regenerating nerve fibers in viva, Muscle and Nerve, 12 (1989) 123-34. 12. Rizvi, A.H., Wasserman, A.J., Zazanis, G. & Silver, F.H.,
Evaluation of peripheral nerve regeneration in the presence of longitudinally aligned collagen fibers. Cells Mater., 1 (1991) 279-89. 3. Weadock, K.S., Olson, R.M. & Silver, F.H., Evaluation of collagen crosslinking techniques Biomater. Med. Dev. Artif. Organs, 11 (1984)293-318. 4. Kato, Y.P., Christiansen, D.L., Hahn, R.A., Shieh, S.J.,
Goldstein, J.D. & Silver, F.H., Mechanical properties of collagen fibers: a comparison of reconstituted and rat tail tendon fibres. Biomaterials, 10 (1989) 38-42. 15. Glasgold, M.J., Kato, Y.P., Christiansen, D.C., Hauge, J.A., Glasgold, A.I. & Silver, F.H., Mechanical properties of septal cartilage homografts. Otolaryngol. Head Neck Surg., 99 (1988) 374-9.
16. Wong, E., Christiansen, D., Rizvi, A., Geller, H.M. & Silver, F.H., A method of preparation of etched collagen fibers that support neurite outgrowth. J. Appl. Biomater., 1 (1990) 225-32.
17. Reynolds, ES., The use of lead citrate at high pH as an electron-opaque stain in electromicroscopy. J. Cell Biol., 17 (1963) 208815.
Ail rights reserved 0267-6605!94/$7.00
Nerve Regeneration in the Pr ers: Effect of Removal of th urn Azam Department
izvi, George D. Pins & Frederick of Pathology,
(Received 12 March
UMDNJ-Robert
Wood
1993; sent for revision 22 October
Johnson
H. Silver* Medical
1993; accepted
School,
11 November
Piscataway,
NJ 08854, USA
1993)
Abstract: A previous report from our laboratory indicated that nerve regeneration through silicone tubes filled with self-assembled collagen fibers was affected by the biodegradation rate of the collagen. The present study was undertaken to evaluate the effect of removal of the distal nerve stump from the silicone tube filled with collagen fibers, Results obtained indicate that in the absence of the distal nerve stump, an implant containing collagen fibers facilitates Schwann cell migration and axonal elongation only when the implant does not biodegrade prior to nerve regeneration (by 8 weeks post-implantation). These results and results of previous studies suggest that the presence or absence of the distal nerve stump affects the extent of regeneration through a silicone tube filled with collagen fibers. Nerve regeneration occurs less frequently in the absence of the distal stump. This model may be a more sensitive test of regeneration in the presence of entubulation devices.
stump, preventing axonal misdirection and fibrosis at the suture site. Silicone tubes provide a shortterm, biocompatible microenvironment by which gaps as large as 10 mm can be repaired in rat sciatic nerve,4 but full functional recovery does not occur when silicone rubber tubes are used alone.5 The sequence of events that occur when a transected rat sciatic nerve regenerates across a silicone entubulated 10 mm interstump gap include: (1) secretion of a fibrin matrix, (2) migration of Schwann cells and fibroblasts, (3) axonal elongation and (4) nerve structure formation.6-9 In the silicone tube model, omission of the nerve stump from the distal end of th.e tube results in the absence of any nerve regeneration,” regardless of whether or not the tube is sealed. Under both conditions, the fluid filling the tubes contains high levels of the same neurotrophic factors normally observed.” Williams et a1.lo found that when the distal nerve insert was replaced by a 2 mm piece of skin, a matrix did not form and subsequent cell migration and axonal regeneration did not occur.
TRODUCTION Severe peripheral nerve injuries such as transection produce d&continuities of both the axons and the basement membrane tubes. Following transection, the axons located in the distal stump undergo Wallerian degeneration and are phagocytosed by both Schwann cells and macrophages. Schwann cells proliferate and the basement membrane tubes remain intact in the distal stump.“2 Axonal sprouting occurs from the proximal nerve stump, across the site of injury and into the distal stump. IMisdirection of the sprouting axons results in failure to reestablish contact with the original target organs and thus impairs functional recovery of the peripheral nerve.3 A variety of polymeric tubes consisting of silicone egradable polyesters have been used to bridge excised areas of peripheral nerve. These tubes align axonal growth toward the distal *To whom correspondence
should be addressed.
73
74
A.H. Rizvi, G.D. Pins, F.H. Silver
When a 2 mm piece of tendon was inserted, a matrix did form at one week, although a nerve structure across the gap was not seen at later time periods. The matrix was believed to have either dissolved before cells could enter the tube or it did not promote cellular migration and subsequent axonal regeneration. Weis and Schroder” found fibrin matrix formation and subsequent axonal regeneration when fat tissue was used as a distal insert. Previously, we have reported that reconstituted type I collagen fibers (isolated from bovine corium) facilitate early Schwann cell and fibroblast migration and subsequent axonal regeneration of transected entubulated rat sciatic nerve (10 mm interstump gap) when the fibers biodegraded by 6 weeks post-implantation.‘2 When the fibers remained intact, axonal regeneration was inhibited. The purpose of this study is to evaluate axonal regeneration in a silicone tube containing reconstituted type I collagen fibers in the absence of the distal nerve stump.
MATERIALS
AND METHODS
Insoluble collagen preparation
Insoluble type I collagen from fresh uncured bovine corium was obtained from Devro, Inc. (Somerville, NJ, USA) after fragmentation into pieces. The corium was swollen in acid, precipitated, washed in distilled water and isopropanol, lyophilized, and stored at -30°C as described previously. l3 The composition of the collagen o chains was characterized by SDSpolyacrylamide gel electrophoresis as a-l(I) and a-2(1), beta, gamma and higher molecular weight components of type I collagen after reduction and heat denaturation. The results of amino acid analysis were consistent with the composition of bovine type I collagen, as described previously.i3 Prosthesis preparation
In order to produce collagen fibers, a 1% (w/v) dispersion of type I collagen in dilute hydrochloric acid (pH 2.0) was extruded through polyethylene tubing (inner diameter 0.28 mm) into a bath of aqueous fiber formation buffer containing 135 mM NaCl, 30 mM TES and 30 mM sodium phosphate dibasic (pH 7.4) at 37°C as described by Kato et aLi After immersion in the buffer for 45 min, the fibers were dehydrated in isopropanol for 4 h, rinsed
CONTROL Proximal
Distal Stump 1
15mmSilicone Tube
COLLAGEN
\ Silicone Plug
FIBER TREATED
120 Collagen Fibers \
-15
e-10
mm+ mm -
Fig. 1. Schematic diagram illustrating a silicone tube 15 mm long with a diameter of l-5 mm. The proximal nerve stump is sutured into one end of the tube while the other end is sealed with medical-grade silicone adhesive.
in distilled water for 15 min, and allowed to air dry overnight. The collagen fibers were crosslinked by exposure to a condition of severe dehydration at 110°C and a vacuum of 0.1 mtorr for 72 h. Approximately 120 of these fibers (diameter 100 pm, length 10 mm) were inserted into a silicone tube (inner diameter 1.5 mm, length 15 mm) in order to form a longitudinally aligned fibrous collagen plug. The silicone tube was sealed at its distal end using medical-grade silicone adhesive which was formed into a plug (Fig. 1). Two groups of implants were prepared. Group 1 was crosslinked using a combination of severe dehydration at 110°C for 3 days and treatment with 1% (w/v) cyanamide (carbodiimide), pH 5.5, in water. The other group was treated with glutaraldehyde vapor at room temperature as described previously. l2 Prostheses were then immersed in Alcide ExsporTM (Alcide Corporation, Norwalk, CT, USA) for sterilization prior to surgery as described previously.’ 5 The extent of crosslinking of the collagen fibers prepared with glutaraldehyde or carbodiimide was characterized by determining the rate of enzymatic degradation using collagenase. Collagenase degradation assay
In order to characterize the degradation rates of the collagen fibers produced from these two groups,
Peripheral nerve regeneration in the presence of collagen jbers
a time-controlled collagenase enzyme assay was conducted. Glass coverslips with collagen fibers from both batches were placed in a 12-well tissue culture plate as described by Wong et aZ.16 The fibers (n = 40) were enzymatically digested in a solution containing 300 units/ml collagenase (Sigma Chemical Co., C-0130 at 300 units/mg), 5 rnM CaC12 and 50 mM Tris-HCl at pH 7.6. The number of fibrils exposed per collagen fiber was recorded at 2 h time intervals under an Olympus CK2 phase contrast microscope. The fibril numbers were plotted versus time for a period of 14 h and compared in order to determine the difference between the degradation rates of both collagen batches.
Thirty-four adult female Sprague-Dawley rats, each weighing between 250 and 350 g, were anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg/kg). Under aseptic conditions, a lateral skin incision was made on the right thigh. In order to expose the sciatic nerve, blunt dissection was performed through the avascular fascial plane between the vastus lateralis and hamstring muscles. Following exposure, the nerve was transected with microsurgical scissors and then the proximal stump was placed into the open end of the silicone tube. The stump was secured in place by suturing the nerve’s outer epineural sheath to the silicone tube with 9-O nylon suture. The muscle layers and skin opening were closed with 9-O nylon and 4-O chromic gut sutures, respectively. An identical surgical procedure was performed on a control animal group. In the control group, silicone tubes without the collagen fibers were used to bridge the gap. After intervals of 4 and 8 weeks, the regenerated nerve tissue from each group was explanted and studied by electron microscopy.
After time intervals of 4 and 8 weeks, the animals were reanesthetized and the newly regenerated nerve tissue in the silicone tube was fixed in 1.5% (v/v> glutaraldehyde, 4% (w/v) paraformaldehyde in 0.1 M cacodylate and 4 mM CaClz. Immediately after removal, the silicone tube was slit longitudinally while immersed in fixative. The regenerated nerve tissue was photographed, then removed and transferred to fresh fixative
75
overnight. The tissue was postfixed in 1% (w/v> osmium tetroxide and block stained in 1% (w/v> aqueous uranyl acetate solution. It was then processed through an ascending series of alcohols and embedded in Polybed 812 resin. Ultrathin cross-sections of the nerve were cut one-quarter (proximally or 2.5 mm), midway or 5 -0 mm, and three-quarters (distally or 7.5 mm) along each processed explant. The sections were stained with 5% (w/v> aqueous many1 acetate and lead citrate,17 then subsequently analyzed and photographed with a Phillips Model 420 transmission electron microscope. Schwann cells were identified by their close association with myelinated and unmyelinated axons, membrane characteristics and dark-staining nuclei. Fibroblast identification was based on elongated cell and nuclear structures, prominent rough endoplasmic reticulum and characteristic nuclear staining properties. Perineurial cells were distinguished by their multjlayered, flattened appearance comprising the fascicles surrounding groups of axons.
RESULTS
A total of 11 animals were used in each of the collagen fiber treated groups (5 sacrificed 4 weeks post-implantation and 6 at 8 weeks post-implantation). Twelve animals were used for the control group (6 sacrificed at 4 and 8 weeks post~impla~tation). Tissue regeneration at 4 weeks
At 4 weeks post-implantation, no nerve tissue was visible between the roximal nerve stump and the distal silicone plug the control group. The control explants contained no ganized structure in the 10 mm gap region; only id was visible. Of the 6 animals implanted with the control prosthesis, none showed any evidence of nerve tissue regeneration. In the group containing ~arbodiimide-treated collagen fibers (CARB), 4 of the 5 explants showed evidence of nerve regeneration at 4 weeks postimplantation. The distance the nerves regenerated in each explant in descending order was 3 - 5, 3 *O, 2.5 and 0-O mm, respectively. Thus, the average length of nerve growth in the CAR collagen fiber group wa,s 2.4 & 1.3 mm (Fig. 2). Grossly, the implanted collagen fibers were observable only up to the point where new nervous
A.H. Rizvi, G.D. Pins, F.H. Silver
76
Implantation Duration
Control i / 0
? ?BWeeks. ??4Weeks I
z
4
6
8
1
Length (mm) of nerve regeneration
Fig. 2. Distance diimide
(CARB)
of nerve regeneration in sealed silicone tubes containing treated collagen fibers. Glutaraldehyde (GLUT), carbotreated collagen fiber implants and control silicone tubes (Control) were observed at 4 and 8 weeks postimplantation.
tissue appeared. Thus, the collagen fibers which were 10 mm in length at the time of implantation had biodegraded to 30% of their initial lengths by 4 weeks. Histologically, islands of unmyelinated axons and their associated Schwann cells were observed in the areas between the collagen
Fig. 3. Transmision
fibers (Fig. 3). Very few myelinated axons were visible and few blood vessels were observed. The formation of fascicles around the axons and Schwann cells by alternating layers of perineurial cells and connective tissue was not visible at this stage.
electron micrograph showing unmyelinated axons (U) and their associated Schwann cells (SC) observed presence of carbodiimide crosslinked collagen fibers (CF) 4 weeks post-implantation.
in the
Peripheral
nerve regeneration
In the glutaraldehyde-treated collagen fiber group (GLUT), all the explants showed evidence of nerve regeneration at 4 weeks post-implantation. The distance the nerve regenerated in each of the explants was 5.0, 4.5, 4.5, 4-O and 4-O mm, respectively. The average length of regeneration among the 5 specimens in this group was 4”4 f O”42 mm (Fig. 2). Grossly, the collagen fibers were visible from the proximal stump to the distal silicone plug. The fibers displayed no evidence of degradation or change in structure. Also, the regenerated tissue could be identified by the darker contrast material visible within the confines of the implanted collagen fibers. Histologically, the structure of the regenerated nerve was similar to the tissue observed in the implant containing carbodiimide-treated collagen fibers at 4 weeks. Group s of unmyelinated axons were visible with few myelinated axons and blood vessels. ost-implantation S, no regenerated nerve tissue was visible in the control group explants. Similar to the explants at 4 weeks post-implantation, no organized structure in she 10 mm gap region could be seen. Only fluid was observed. Of the 6 animals
in the presence
ofcollagen
Jibers
implanted with the control prost of tissue regeneration was obser any animal. In the carbodiimide-treated collagen fiber implant group, none of the 6 explants showed tissue regeneration at 8 weeks cost-i~~~lantatio~. The implanted collagen fi ers which were originally 10 mm in length we no longer visible by 8 weeks. In their place, fluid was observed. This observation suggests that the carbodiimidetreated collagen fiber implant had completely degraded by 8 weeks. In the glutaraldehyde-treated collagen fiber group, all 6 explants showed evidence of nerve regeneration at 8 weeks. The regeneration distance in each of the explants was 8-5, 8.0, 7.5, 7-5, 7.5 and 7-O mm, respectively. Thus, the average regeneration distance among the 6 explants was 7.7 f Oa52 mm (Fig. 2). As in the 4-week glutaraldehyde-treated collagen pi implanted collagen fibers spanned the 10 mm gap. No evidence of fiber degradation was observed. The newly regenerated tissue could by the darker area observed within the collagen fiber scaffold. Histologically, the newly regenerated nerve tissue at 8 weeks differed from the tissue observed at 4 weeks post-i~l~~la~tation. More
Fig. 4. Transmission Aectron micrograph showing myelinated (M) and unmyelinated axons (U) and S&warm ceils in the tissue regenerated in the presence of glutaraldehyde crosslinked collagen fibers (CF) 8 weeks post-implantation.
78
A.H. Rizvi, G.D. Pins, F.H. Silver
myelinated axons and fewer unmyelinated axons were observed. Also, fascicularization of the axons and their associated Schwann cells was visible indicating greater maturity in the &week explant when compared to the 4-week specimen (Fig. 4). In the 4-week carbodiimide- and glutaraldehydetreated collagen fiber implant groups, the longitudinally aligned collagen fibers appeared roughly parallel to the long axis of the regenerating tissue. At the EM level (Fig. 5) regenerating nerve cells extended their axons between collagen fibers in a longitudinal or linear direction. Collagenase degradation assay
The carbodiimide and glutaraldehyde crosslinked collagen fibers degraded at different rates. At 8 weeks post-implantation, the carbodiimide crosslinked collagen fibers had disappeared but the glutaraldehyde crosslinked collagen fibers remained intact and appeared to be minimally degraded. A time-controlled collagenase enzyme assay was conducted by measuring the number of fibrils exposed in each fiber per unit of time, in order to compare the degradation rates of the carbodiimide and glutaraldehyde crosslinked collagen fibers (Fig. 6). The rate of appearance of fibrils is initially propor-
tional to the rate of collagenase degradation of collagen fibers; in the long term the number of flbrils decreases as the collagen is totally removed. The results indicated that the difference in the collagen degradation rate in vivo correlated with the collagenase degradation rate of the fibers in vitro, i.e. glutaraldehyde crosslinked collagen fibers remained intact longer than carbodiimide-treated fibers.
DISCUSSION
In a previous rat study12 using slowly and rapidly degrading collagen fibers, nerve regeneration was facilitated or inhibited depending on whether the fibers biodegraded by 6 weeks post-implantation (facilitated) or remained visibly intact (inhibited). It was concluded that in the presence of the distal stump and rapidly degrading collagen fibers, fibroblast and Schwann cell migration and axon regeneration is enhanced and more efficient since 100% of the collagen-treated animals showed regeneration. In contrast, the fibrin matrix that formed between opposing stumps was able to support regeneration in only 40% of the control tubes. l2 The purpose of the present study was to investi-
Fig. 5. Transmission electron micrograph of a longitudinal section of a glutaraldehyde-treated collagen fiber implant at 4 weeks postimplantation. Myelinated axons (M) and Schwann cells (SC) can be observed along the path of the collagen fibers (CF).
Peripheral
nerve regeneration
in the presence
of collagen fibers
DHT3X-linkedCollagenFibers Glut X-linkedCollagenFibers
0
0
2
4
6
Time (hours) Fig. 6. Graph i&tstrating collagenase digestion time of crosslinked glutaraldehyde (Glut.) and carbodiimide (DHT3) collagen fibers. Degradation time was determined by counting the number of fibrils exposed at each time interval. The increased rate of fibrils exposed as a function of time denotes greater collagen degradation.
gate axonal regeneration through a silicone tube containing collagen fibers in the absence of the distal nerve stump. Histological and morphological evaluations indicated that no axonal ingrowth had occurred in any of the control tubes that bad been sealed at the distal end with silicone adhesive. Similar observations were noted after 8 weeks in the implants which had contained carbodiimide-treated collagen fibers. In contrast, regeneration was observed at 4 weeks in implants which employed carbodiimide-treated collagen fibers and at 4 and 8 weeks in stents containing glutaraldehyde-treated fibers. Additionally, the rate of biodegradation of carbodiimide-treated fibers was significantly greater than glutaraldehyde-treated fibers both in vivo and in vitro. The results of the current studies, in which the distal end of the tube was blocked, are inconsistent with the conclusions of our previous entubulation experiments Results of entubulation experiments employing both the proximal and distal stumps indicated that nerve regeneration through a silicone tube was possible regardless of the presence of collagen fibers, but rapidly degrading collagen fibers enhanced Schwann cell migration and axonal ingrowth.i2 This and other investigations also at a fibrin matrix may help facilitate regeneration silicone sciatic nerve through tubes.s;i2 In the present studies where the distal stump was replaced with a silicone plug, sciatic
nerve regeneration was observed only in the presence of glutaraldehyde-treated collagen fibers. Empty silicone tubes and tubes where the collagen fibers had completely degraded (carbodiimidetreated fibers implanted for 8 weeks) contained only fluid. Lundborg et aI4 observed similar results when attempting to transect 10 mm gaps with silicone tubes. These findings su.ggest that a fibrin matrix cannot serve as a template for axonal elongation and Schwann cell migration unless both the proximal and distal nerve stumps are in close proximity. These results also imply that the relationship between the collagen fiber biodegradation rate and the extent of regeneration is dependent upon the entubulation model. In the absence of the distal stump, nerve regeneration occurs less frequently. In conclusion, an implant containing collagen fibers without a distal stump facilitates Schwarm cell migration and axonal elongation only when the implant does not biodegrade prior to nerve regeneration. In contrast, when the implants utilize both the proximal and distal sturn eration is inhibited by collagen fibers which remain intact for periods greater than the time required for regeneration to be completed. These data suggest that nerve entubulation in the absence of the distal stump leads less frequently to nerve regeneration in controls and may therefore be a more sensitive model of regeneration.
80
A.H.
Rizvi,
G.D. Pins,
REFERENCES 1. Carbonetto, S., The extracellular matrix of the nervous system. Trends Neurosci., 7 (1982) 382-7. 2. Aguayo, A.J. & Bray, G.M., Experimental nerve grafts. In Nerve Repair and Regeneration: Its Clinical and Experimental Basis, ed. D.L. Jewett & H.R. McCaroll Jr. C.V. Mosby Co., St Louis, MO, 1980, pp. 68-76. 3. Lundborg, G., Nerve regeneration and repair. Acta Orthop. Stand., 58 (1987) 145-69. 4.
Lundborg, G., Dahlin, L.B., Danielson, N., Gelberman, R., Longo, F., Powell, H. & Varon, S., Nerve regeneration in silicone chambers: influence of gap length and of distal stump components, Exp. Neural., 76 (1982) 361-75. 5. Ashur, H., Vilner, Y., Finsterbush, A., Rousso, M., Weinberg, H. & Devor, M., Extent of fiber regeneration after peripheral nerve repair: silicone splint versus suture, gap repair versus graft. Exp. NeuroZ., 97 (1987) 365-74. 6. Williams, L.R., Longo, F.M. & Powell, H.C., Spatialtemporal progress of peripheral nerve regeneration within a silicone chamber: parameters for a bioassay. J. Comp. Neuvol., 228 (1983) 460-70. 7. Longo, F.M., Skaper, S.D., Manthorpe, M., Williams, L.R., Lundborg, G. & Varon, S., Temporal changes of neuronotrophic activities accumulating in vivo within nerve regeneration chambers. Exp. NeuroZ., 81 (1983) 756669. 8. Longo, F.M., Hayman, E.G., Davis, G.E., Ruoslahti, E., Engvall, E., Manthorpe, M. & Varon, S., Neuritepromoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers. Brain Res., 309 (1984) 105-17. 9.
Longo, F.M., Manthorpe,
M., Skaper, S.D., Lundborg, G.
F.H. Silver
& Varon, S., Neuronotrophic activities accumulate in vivo within silicone nerve regeneration chambers. Brain Res., 261(1983) 109-17. 10. Williams, L.R., Powell, H.C., Lundborg,
G. & Varon, S., Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber, Brain Res., 293 (1984) 201-11.
11. Weis, J. & Schroder, J.M., Differential effects of nerve, muscle, and fat tissue on regenerating nerve fibers in viva, Muscle and Nerve, 12 (1989) 123-34. 12. Rizvi, A.H., Wasserman, A.J., Zazanis, G. & Silver, F.H.,
Evaluation of peripheral nerve regeneration in the presence of longitudinally aligned collagen fibers. Cells Mater., 1 (1991) 279-89. 3. Weadock, K.S., Olson, R.M. & Silver, F.H., Evaluation of collagen crosslinking techniques Biomater. Med. Dev. Artif. Organs, 11 (1984)293-318. 4. Kato, Y.P., Christiansen, D.L., Hahn, R.A., Shieh, S.J.,
Goldstein, J.D. & Silver, F.H., Mechanical properties of collagen fibers: a comparison of reconstituted and rat tail tendon fibres. Biomaterials, 10 (1989) 38-42. 15. Glasgold, M.J., Kato, Y.P., Christiansen, D.C., Hauge, J.A., Glasgold, A.I. & Silver, F.H., Mechanical properties of septal cartilage homografts. Otolaryngol. Head Neck Surg., 99 (1988) 374-9.
16. Wong, E., Christiansen, D., Rizvi, A., Geller, H.M. & Silver, F.H., A method of preparation of etched collagen fibers that support neurite outgrowth. J. Appl. Biomater., 1 (1990) 225-32.
17. Reynolds, ES., The use of lead citrate at high pH as an electron-opaque stain in electromicroscopy. J. Cell Biol., 17 (1963) 208815.