The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.     

Fresh and predegenerate nerve allografts and isografts in trembler mice

In order to investigate whether Schwann cell or myelin was the principal antigen responsible for nerve graft reiection, fresh nerve grafts and those in which myelin had been previously allowed to degenerate (predegenerate grafts) from both isogeneic BALB/c and allogeneic C57/B1 mice were inserted into trembler BALB/c mice. Schwann cells within nerve allografts from C57/B1 mice were rejected, whether or not the grafts contained myelin. Nerve isografts from normal BALB/c animals produced normally myelinated trembler axons within the grafted segments, and across these segments conduction velocity was restored towards the normal value. It is concluded that Schwann cells, not myelin, constitute the principlal antigen within nerve allografts and it is Schwann-cell rejection that limits the sucessful use of nerve allografts.     

Muscle allograft survival after cyclosporin A immunosuppression

In normal rats, autografts of skeletal muscle survive, whereas allografts of muscle are immunologically rejected. In the present study we sought to determine whether or not the new immunosuppressive drug cyclosporin A (CyA) would prevent the rejection of muscle allografts. Autografts and allografts of rat extensor digitorum longus muscles were made into recipients that went untreated (i.e., no treatment or vehicle injected) or that received daily injections of CyA (5 mg/kg). Autografts of muscle survived throughout the 12-week period of study. On the other hand, untreated rats rejected muscle allografts within 2 weeks, whereas allografts in CyA-treated recipients survived, even at 12 weeks when the experiment was terminated. In another study, CyA therapy was discontinued after 4 weeks in rats that received bilateral muscle allografts, both of which had survived; one was removed at that time. The second muscle allograft from these rats was examined 2 months later, and all were found rejected. CyA was also effective in preventing the rejection of muscle allografts in rats that were previously sensitized to the same transplantation antigens present on the allogeneic muscle cells. These results demonstrated that CyA can prevent the rejection of muscle allografts but only if drug treatment is continued. We conclude that CyA is a potent immunosuppressive agent which could prove useful in clinical muscle allotransplantation or in experimental animal studies that require immunosuppression.     

Failure of host axons to regenerate through a once successful but later rejected long nerve allograft

Host axons will regenerate through a long nerve allograft in an immunologically tolerant rat. However, if tolerance is abolished, rejection occurs and allogeneic cells (e.g., Schwann, vascular, perineurial, etc.) as well as regenerated host axons disappear from the allograft. Because following tolerance abolition host axons begin to regenerate into the connective tissue remnants of the rejected nerve allografts, the extent of this renewed axonal growth was investigated. It was found that in a tolerance-abolished rat, host axons only regrew into the proximal 1 cm of a 4-cm allograft which in a fully tolerant recipient would have had numerous allogeneic Schwann cell-myelinated axons throughout its length. It is concluded that viable allogeneic cells (i.e., Schwann, fibroblast, and vascular) together with their connective tissue matrix provide the best way to aid host nerve fiber regeneration through a long nerve allograft.     

Failure of cyclosporin-A to induce immunological unresponsiveness to nerve allografts

Although some allografts bearing major and minor transplantation antigens can survive after the cessation of immunosuppression with cyclosporin-A (Cy-A), nerve allografts do not. In an attempt to induce immunological unresponsiveness to nerve allografts, we used grafts containing only minor transplantation antigens and varied the duration of Cy-A therapy from 2 to 12 weeks. Our results demonstrated that nerve allografts survived in rats during Cy-A therapy, but when the drug administration ceased, the allografts were rejected. Other factors besides the degree of histoincompatibility and duration of Cy-A treatment must be involved in determining whether or not unresponsiveness develops to allografts after Cy-A withdrawal. We conclude that nerve allograft immunosuppression generated by Cy-A requires regular administration of the drug.     

Basement membrane component changes in nerve allografts and isografts

This study describes immunocytochemical changes in laminin, which is an integral basement membrane (BM) component, during axonal regeneration through antigenic nerve allografts and nonantigenic nerve isografts. In normal rat nerve, laminin was localized in the BM of Schwann cells and the perineurium. During nerve allograft rejection, the perineurium and Schwann cells disappeared. However, the Schwann cell BMs persisted and became distorted and collapsed. In isografted nerves, the perineurium and Schwann cells were present, and only a few Schwann cell BMs appeared to be distorted; however, the staining for laminin was faint, indicating a possible BM breakdown. A new BM appeared as small rings around the Schwann cells after they had become associated with regenerated axons. Because only a limited axonal regeneration occurred in allografts as compared to isografts, it is concluded that the viable Schwann cells, and their BM architecture, are essential for regeneration through long nerve grafts.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

The fate of neurons and neurilemmal cells in allografts of ganglia in the spinal cord of normal and immunologically tolerant rats

Neurons and neurilemmal cells (i.e., satellite cells and Schwann cells) in allografts (i.e., grafts between genetically different members of the same species) of ganglia are rejected after transplantation into the anterior chamber of the eye or into muscle of normal rats, but not when transplanted into immunologically tolerant animals. These results demonstrate that nervous tissue allografts are immunogenic and that they are not afforded any protection against rejection even when transplanted into a putative immunologically privileged graft site (i.e., a graft site where an allograft may survive indefinitely without recourse to immunosuppression) such as the eye. Because the central nervous system is regarded as a privileged transplantation site, the fate of isografts (i.e., grafts between genetically identical members of the same species) and allografts transplanted to this location has been determined. The allografts were incompatible with respect to both major and minor, or only to minor, transplantation antigens. The grafts were transplanted into the cervical spinal cord of normal and immunologically tolerant rats and their survival after 65 days was appraised histologically. It was found that, whereas neurons and neurilemmal cells survived in all ganglia isografts and in all allografts to tolerant hosts, allograft survival in normal recipients depended on the magnitude of the histoincompatibility. When both major and minor incompatibilities prevailed, all neurons were rejected in allografts except for two which had a few neurons remaining. Neurilemmal cells in all these ganglia were markedly reduced. No obvious rejection of neurological cells occurred in allografts when only minor antigenic differences existed. It is concluded that the immunological rejection of neurological cells in allografts can occur in the spinal cord of normal rats when major antigenic differences exist but that this rejection is either delayed or absent in situations where the graft is incompatible only with regard to minor antigens. Moreover, all rejections can be prevented by rendering the hosts immunologically tolerant.     

Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)     

The fate and functional capacity of neonatal neurons in allografts of ganglia in normal and immunologically tolerant rats

Previous studies have demonstrated that neurons in Ag-B histoincompatible allografts of adult ganglia are rejected unless the recipients have been rendered immunologically tolerant. Because neonatal grafts sometimes survive in situations where adult grafts of the same genotype are rejected, the fate and functional capacity of neurons in Ag-B histoincompatible allografts of neonatal ganglia was investigated. Sensory or sympathetic ganglia from neonatal Lewis rats were transplanted to the anterior chamber of the eye or into sternomastoid muscle of normal adult Brown Norway (BN) rats or to adult BN rats previously made immunologically tolerant to Lewis antigens. Normal BN rats rejected neonatal Lewis sensory ganglia (i.e., no neurons were present) at 70 days, whereas tolerant animals accepted these grafts for the 120 days they were followed. Furthermore, surviving Lewis neurons in tolerant BN rats were functional in that they regenerated nerve fibers into BN isografts of tongue and induced the development of taste buds. On the other hand, no neurons survived in neonatal sympathetic allografts to normal or tolerant BN rats and none or very few survived for even 2 weeks in sympathetic isografts. These results demonstrate that immunological factors (Ag-B antigens) are responsible for the ultimate loss of sensory neurons in neonatal allografts of ganglia whereas nonimmunological factors (i.e., transection of nerve fibers, separation from end-organ, nerve growth factor dependency) cause the loss of neurons in neonatal sympathetic ganglia. We conclude that both immunological and nonimmunological factors are important in determining the survival of neonatal neurons after transplantation.     

Neurofibromatosis xenografts. Contribution to pathogenesis

We transplanted Schwann cells of 3 patients with neurofibromatosis from neurofibromas, sural nerve, and from a malignant schwannoma into sciatic nerves of immunoincompetent mice. Three and six months later, the grafts and distal nerve segments contained normal myelinated fibers. After rendering host animals immune competent again, neurofibroma and malignant schwannoma Schwann cells were rejected, but grafts retained normally myelinated fibers indicating that these were of mouse origin. Sural nerve Schwann cells from a neurofibromatosis patient were rejected also leaving naked axons in the grafted segments showing that human Schwann cells from the sural nerve of one patient had invested and myelinated the regenerating mouse axons. The nature of putative signals passing between axons and Schwann cells might be elucidated by the combination of human and animal cells in immunoincompetent host nerves. Hypothetical signals for myelination of mouse axons were normally received by sural nerve Schwann cells of a patient with neurofibromatosis, but not by Schwann cells from neurofibromas or malignant schwannomas.     

End-Organ reinnervation does not prevent axonal degeneration in nerve allografts following immunosuppression withdrawal

Previous work indicated that appropriate end-organ reinnervation fails to influence axonal degeneration in nerve allografts following immunosuppression withdrawal. In the present study, we examined if differences existed in axonal degeneration when axons regenerated across nerve allografts are allowed or completely denied end-organ reinnervation. Two ACI rat nerve allografts (3 cm long) were sutured into gaps created in both peroneal nerves in Lewis rats. In the right leg, the distal end of the graft was connected to the distal host nerve stump to allow end-organ reinnervation. In the left leg, the distal end was turned back and double ligated (unconnected) to prevent end-organ reinnervation. Rats received Cyclosporin A daily for 12 weeks to allow for regeneration and were sacrificed at 16 (n = 5) or 18 (n = 5) weeks following engraftment to assess axonal degeneration following immunosuppression withdrawal. Five Lewis rats receiving autografts served as control and were sacrificed at 12 weeks. Morphometric analysis was performed. In the control group (autografts) the cross-sectional area of and the number of myelinated fibres in the unconnected grafts was double that of the connected grafts, suggesting a sprouting effect. There was a tenfold reduction in the mean number of fibres at weeks 16 and 18 in the allografts compared to controls, without any significant differences in the connected versus unconnected sides. End-organ reinnervation decreases sprouting of axons within the graft but does not protect axons from degeneration following immunosuppression withdrawal.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Abstract

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently,the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Long term evaluation of experimental median nerve repair by frozen and fresh nerve autografts,allografts and allografts repopulated by autologous Schwann cells

The authors used different kinds of peripheral nerve grafts to reconstruct a terminal branch of the brachial plexus (the median nerve) gap of adult Sprague-Dawley rats, including fresh or frozen autografts and allografts from Norway rats. They also performed acellular allograft repopulation by autogenous Schwann cells, to improve the environment for nerve regeneration. Three, six, nine and twelve months after grafting, rats underwent histological assessment (muscle, nerve and spinal cord) and simple functional assessment by the grasping test. Initially, the functional recovery of frozen grafts was lower than fresh graft recovery, but twelve months after surgery it was similar for both types of graft.     

Immunopathological factors in peripheral nerve allograft rejection: quantification of lymphocyte invasion and major histocompatibility complex expression

The numbers of helper T and cytotoxic T lymphocytes and macrophages were quantified, and the expression of major histocompatibility complex (MHC) class I and class II molecules was examined in rat peripheral nerve allografts from 1 to 14 days after implantation, using the indirect immunoperoxidase method for light and electron microscopy. Two centimetre segments of peripheral nerve freshly obtained from inbred Dark Agouti strain rats were inserted in a gap created in n. fibularis or n. tibialis of young adult inbred Wistar strain rats, using fascicular nerve repair techniques under general anaesthesia. There was a gradual increase in the number of helper T and cytotoxic/suppressor T cells from day 2 with peak numbers of both types of T cells observed around day 7. The results suggest that the critical time for T cell proliferation is between day 6 and day 7 post-operatively. The number of macrophages increased over 10 days, with peak numbers being observed at day 10 post-operatively. This is in accord with the pattern of rejection observed in allografts of other tissue. Schwann cells were found to express MHC class I and class II molecules by day 2 post-operatively, which is well before there is any substantial T cell and macrophage infiltration. It may be that the donor Schwann cells act as antigen presenting cells, triggering the immune response and finally becoming a target of the rejection process.     

Allograft pretreatment for the repair of sciatic nerve defects: green tea polyphenols versus radiation

Pretreatment of nerve allografts by exposure to irradiation or green tea polyphenols can eliminate neuroimmunogenicity, inhibit early immunological rejection, encourage nerve regeneration and functional recovery, improve tissue preservation, and minimize postoperative infection. In the present study, we investigate which intervention achieves better results. We produced a 1.0 cm sciatic nerve defect in rats, and divided the rats into four treatment groups: autograft, fresh nerve allograft, green tea polyphenol-pretreated(1 mg/m L, 4°C) nerve allograft, and irradiation-pretreated nerve allograft(26.39 Gy/min for 12 hours; total 19 k Gy). The animals were observed, and sciatic nerve electrophysiology, histology, and transmission electron microscopy were carried out at 6 and 12 weeks after grafting. The circumference and structure of the transplanted nerve in rats that received autografts or green tea polyphenol-pretreated nerve allografts were similar to those of the host sciatic nerve. Compared with the groups that received fresh or irradiation-pretreated nerve allografts, motor nerve conduction velocity in the autograft and fresh nerve allograft groups was greater, more neurites grew into the allografts, Schwann cell proliferation was evident, and a large number of new blood vessels was observed; in addition, massive myelinated nerve fibers formed, and abundant microfilaments and microtubules were present in the axoplasm. Our findings indicate that nerve allografts pretreated by green tea polyphenols are equivalent to transplanting autologous nerves in the repair of sciatic nerve defects, and promote nerve regeneration. Pretreatment using green tea polyphenols is better than pretreatment with irradiation.     

Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with autologous mesenchymal stem cells

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult in humans to achieve full functional recovery following extended peripheral nerve lesions. Optimizing repair of peripheral nerve injuries has been hindered by the lack of viable and reliable biologic or artificial nerve conduits for bridging extended gaps. In this study, we utilized chemically extracted acellular allogenic nerve segments implanted with autologous non-hematopoietic mesenchymal stem cells (MSCs) to repair a 40 mm defect in the rhesus monkey ulnar nerve. We found that severely damaged ulnar nerves were structurally and functionally repaired within 6 months following placement of the MSC seeded allografts in all animals studied (6 of 6, 100%). Furthermore, recovery with the MSC seeded allografts was similar to that observed with Schwann cell seeded allografts and autologous nerve grafts. The findings presented here are the first demonstration of the successful use of autologous MSCs, expanded in culture and implanted in a biological conduit, to repair a peripheral nerve gap in primates. Given the difficulty in isolating and purifying sufficient quantities of Schwann cells for peripheral nerve regeneration, the use of MSCs to seed acellular allogenic nerve grafts may prove to be a novel and promising therapeutic approach for repairing severe peripheral nerve injuries in humans.     

Tissue-engineered rhesus monkey nerve grafts for the repair of long ulnar nerve defects:similar outcomes to autologous nerve grafts

Acellular nerve allografts can help preserve normal nerve structure and extracellular matrix composition.These allografts have low immunogenicity and are more readily available than autologous nerves for the repair of long-segment peripheral nerve defects.In this study,we repaired a 40-mm ulnar nerve defect in rhesus monkeys with tissue-engineered peripheral nerve,and compared the outcome with that of autograft.The graft was prepared using a chemical extract from adult rhesus monkeys and seeded with allogeneic Schwann cells.Pathomorphology,electromyogram and immunohistochemistry findings revealed the absence of palmar erosion or ulcers,and that the morphology and elasticity of the hypothenar eminence were normal 5 months postoperatively.There were no significant differences in the mean peak compound muscle action potential,the mean nerve conduction velocity,or the number of neurofilaments between the experimental and control groups.However,outcome was significantly better in the experimental group than in the blank group.These findings suggest that chemically extracted allogeneic nerve seeded with autologous Schwann cells can repair 40-mm ulnar nerve defects in the rhesus monkey.The outcomes are similar to those obtained with autologous nerve graft.     

Dorsal root ganglion-derived Schwann cells combined with poly（lactic-co-glycolic acid）/chitosan conduits for the repair of sciatic nerve defects in rats

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and puriifcation of Schwann cells are complicated by contamination with ifbroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly puriifed Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells （〉95%） using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly（lactic-co-glycolic acid）/chi-tosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were signiifcantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental ifndings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly（lactic-co-glycolic acid）/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.