No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Exogenous fibrin matrix precursors stimulate the temporal progress of nerve regeneration with a silicone chamber
Abstract
The silicone chamber model permits the investigation of the cellular and molecular events underlying successful regeneration of the rat sciatic nerve across a 10 mm gap. When 25 microliter chambers are implanted prefilled with phosphate-buffered saline (PBS), it takes 5-7 days before sufficient fibrin matrix (derived from plasma precursors) accumulates naturally to form a complete bridge across the chamber gap; at 1 week postimplantation, cellular migration into the matrix from the nerve stumps is just beginning. The temporal progress of regeneration might be stimulated if a fibrin matrix, conducive to cell migration, was provided to the nerve stumps at or shortly after the time of chamber implantation. To test this hypothesis, chambers were prefilled, at the time of implantation, with different preparations of homologous plasma. A solution of 90% platelet-free plasma dialyzed against PBS (DP) formed a fibrin matrix by 24 hours postimplantation that, like the naturally formed matrix, had a predominantly longitudinal orientation. The temporal progress of regeneration was stimulated in the DP-prefilled chambers; at 17 days postimplantation, the extents of Schwann cell migration and axonal elongation were significantly greater than in the control system. In contrast, prefilling chambers with either non-citrated plasma or DP + calcium resulted in the generation of a matrix within 8 minutes that was composed of randomly oriented fibrin polymers. These matrices significantly retarded the progress of regeneration.
... [9][10][11][12] The basic sequence of events leading to successful nerve regeneration (when gaps are shorter) through guidance tubes has been characterized and is shown here schematically (Fig. 1). [13,14] Within a day of the initial injury, the tube fills with plasma exudate, and precursor molecules from within this fluid gradually coalesce into an oriented fibrin matrix that physically bridges the nerve gap. Cell types including fibroblasts and Schwann cells migrate from both nerve stumps through the aligned fibrin matrix and begin to multiply and differentiate, enriching and transforming the surrounding matrix. ...
... The formation of a continuous fibrin cable/matrix across the severed axonal ends is a critical step in the regenerative sequence, as the initial influx of Schwann cells involved in regeneration is dependent upon the physical support and guidance that the oriented matrix provides. [14,15,17] In cases where this fibrin cable fails to form, for example across longer gaps, the cause is often attributed to improper or incomplete fibrin matrix formation. [15,18] Furthermore, the structure of the fibrin matrix determines the organization, alignment, and distribution of the infiltrating glial cells (largely Schwann cells, and fibroblasts to a lesser degree), and thus influences the morphology of the developing regeneration cable. ...
... [15,18] Furthermore, the structure of the fibrin matrix determines the organization, alignment, and distribution of the infiltrating glial cells (largely Schwann cells, and fibroblasts to a lesser degree), and thus influences the morphology of the developing regeneration cable. [14,17,19,20] A properly formed and located fibrin cable in the initial stages of regeneration thus sets into motion the sequence of events culminating in Schwann cell stimulated peripheral nerve regeneration. [21] 1. ...
... Conventional light microscopy was used to visualize the presence and orientation of the fibrin matrix. 51 Statistical analysis. The study was designed to allow comparison of the effects of polymers E10-0.5(1K) and PE in 520 EZRA ET AL. both a 2D and conduit fashion on nerve regeneration. ...
... [65][66][67] During this process, the fibrin matrix (derived from plasma precursors) accumulates naturally to form a complete bridge across the chamber gap providing a substrate for cellular migration. 51 The E10-0.5(1K) conduit may encourage the early orientation of regenerating components on its own, as opposed to filling the inner lumen with a synthetic material to act as a longitudinal cable. [68][69][70] This may occur by increasing infiltrating Schwann cells and fibroblasts across the nerve injury gap accelerating the temporal progress of regeneration. ...
... This matrix behaves as a physical scaffold for nonneuronal calls, including fibroblasts, Schwann cells, macrophages, and endothelial cells to influence axonal elongation and initiate the regenerative process. 51,71 Due to the critical role Schwann cells play in nerve regeneration, we used biochemical analysis techniques (Fig. 8C) to investigate the presence of proteins specific to Schwann cells within the nerve conduits post-implantation. 51,57,64,72,73 Results showed that these protein levels were higher in E10-0.5(1K) ...
Following complete nerve transection, entubulation of the nerve stumps helps guide axons to reconnect distally. In this study, a biodegradable and non-cytotoxic tyrosine-derived polycarbonate terpolymer composed of 89.5 mol % desaminotyrosyl tyrosine ethyl ester (DTE), 10 mol % desaminotyrosyl tyrosine (DT), and 0.5 mol % poly(ethylene glycol) (PEG, Mw= 1 kDa) (designated as E10-0.5(1K)) was used to fabricate conduits for peripheral nerve regeneration. These conduits were evaluated against commercially available non-porous polyethylene (PE) tubes. The two materials are characterized in vitro for differences in surface properties, and the conduits are then evaluated in vivo in a critical size nerve defect in the mouse femoral nerve model. Conduits were fabricated from E10-0.5(1K) in both porous (P-E10-0.5(1K)) and non-porous (NP-E10-0.5(1K)) configurations. The results illustrate that adsorption of laminin, fibronectin, and collagen type I was enhanced on E10-0.5(1K) compared to PE. In addition, in vivo the E10-0.5(1K) conduits improved functional recovery over PE conduits, producing regenerated nerves with a five-fold increase in the number of axons, and an eight-fold increase in the percentage of myelinated axons. These increases were observed for both P-E10-0.5(1K) and NP-E10-0.5(1K) after 15 weeks. When conduits were removed at 7 or 14 days following implantation, an increase in Schwann cell proteins and fibrin matrix formation was observed in E10-0.5(1K) conduits over PE conduits. These results indicate that E10-0.5(1K) is a pro-regenerative material for peripheral nerves and that the porosity of P-E10-0.5(1K) conduits was inconsequential in this model of nerve injury.
... [9][10][11][12] The basic sequence of events leading to successful nerve regeneration (when gaps are shorter) through guidance tubes has been characterized and is shown here schematically (Fig. 1). [13,14] Within a day of the initial injury, the tube fills with plasma exudate, and precursor molecules from within this fluid gradually coalesce into an oriented fibrin matrix that physically bridges the nerve gap. Cell types including fibroblasts and Schwann cells migrate from both nerve stumps through the aligned fibrin matrix and begin to multiply and differentiate, enriching and transforming the surrounding matrix. ...
... The formation of a continuous fibrin cable/matrix across the severed axonal ends is a critical step in the regenerative sequence, as the initial influx of Schwann cells involved in regeneration is dependent upon the physical support and guidance that the oriented matrix provides. [14,15,17] In cases where this fibrin cable fails to form, for example across longer gaps, the cause is often attributed to improper or incomplete fibrin matrix formation. [15,18] Furthermore, the structure of the fibrin matrix determines the organization, alignment, and distribution of the infiltrating glial cells (largely Schwann cells, and fibroblasts to a lesser degree), and thus influences the morphology of the developing regeneration cable. ...
... [15,18] Furthermore, the structure of the fibrin matrix determines the organization, alignment, and distribution of the infiltrating glial cells (largely Schwann cells, and fibroblasts to a lesser degree), and thus influences the morphology of the developing regeneration cable. [14,17,19,20] A properly formed and located fibrin cable in the initial stages of regeneration thus sets into motion the sequence of events culminating in Schwann cell stimulated peripheral nerve regeneration. [21] 1. ...
http://doi.wiley.com/10.1002/adma.v21:32/33
Bridging peripheral nerve gaps without the use of autografts has significant clinical importance. But in order to rationally design novel scaffolds, a good understanding of the nerve regeneration process is vital. Appropriate amount of structural and chemical cues are required to stimulate the endogenous mechanisms of repair and functional recovery. Synthetic and natural materials present various opportunities to induce the growth of supporting cells as well as promote axon regeneration. An overview of tissue engineering strategies currently being explored that stimulate the different steps of the regenerative sequence is presented.
... At day 3, the solid structure was present in all tubes examined (Fig. 3B). By microscopic examination, small threads distributed with a predominantly longitudinal orientation were observed (Fig. 3C), suggesting an accumulation of "brin matrices [18,19]. The structure had become "rmly connected with the stumps 5 days post-implantation. ...
... This suggests that tube integrity for a biodegradable NGC is probably required only at the very beginning of regeneration, during the formation of an extracellular matrix sca!old. This process starts with #uid exudation from the nerve stumps into the tube chamber and may be complete within 1 week [18,19]. When a fresh-cut nerve is repaired with our PPE conduits pre-"lled with PBS, a solid structure that bridges the two nerve stumps is formed within 3 days. ...
... When a fresh-cut nerve is repaired with our PPE conduits pre-"lled with PBS, a solid structure that bridges the two nerve stumps is formed within 3 days. This is much faster than that in a PBS-pre"lled silicone chamber, which takes 5}7 days before su$cient material accumulates to form a continuous bridge across a 10 mm gap [18]. It is not unlikely in our case that tube porosity [22] and increased permeability due to tube cracks had allowed the in#ux of nutrients and growth factors from the surrounding environment and enhanced the constitution of the matrix. ...
There is a resurgence of interest in the development of degradable and biocompatible polymers for fabrication of nerve guide conduits (NGCs) in recent years. Poly(phosphoester) (PPE) polymers are among the attractive candidates in this context, in view of their high biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under physiological conditions and a wide variety of physicochemical properties. The feasibility of using a biodegradable PPE, P(BHET-EOP/TC), as a novel NGC material was investigated. Two types of conduits were fabricated by using two batches of P(BHET-EOP/TC) with different weight-average molecular weights (Mw) and polydispersity indexes (PI). The polymers as well as conduits were non-toxic to all six types of cells tested, including primary neurones and neuronally differentiated PC12 cells. After in situ implantation in the sciatic nerve of the rat, two types of conduits triggered a similar tissue response, inducing the formation of a thin tissue capsule composed of approximately eight layers of fibroblasts surrounding the conduits at 3 months. Biological performances of the conduits were examined in the rat sciatic nerve model with a 10 mm gap. Although tube fragmentation, even tube breakage, was observed within less than 5 days post-implantation, successful regeneration through the gap occurred in both types of conduits, with four out of 10 in the Type I conduits (Mw 14,900 and PI 2.57) and 11 out of 12 in the Type II conduits (Mw 18,900 and PI 1.72). The degradation of conduits was further evidenced by increased roughness on the tube surface in vivo under scanning electron microscope and a mass decrease in a time-dependent manner in vitro. The Mw of the polymers dropped 33 and 24% in the Type I and II conduits, respectively, in vitro within 3 months. Among their advantages over other biodegradable NGCs, the PPE conduits showed negligible swelling and no crystallisation after implantation. Thus, these PPE conduits can be effective aids for nerve regeneration with potential to be further developed into more sophisticated NGCs that have better control of the conduit micro-environment for improved nerve regeneration.
... Conventional light microscopy was used to visualize the presence and orientation of the fibrin matrix. 51 Statistical analysis. The study was designed to allow comparison of the effects of polymers E10-0.5(1K) and PE in 520 EZRA ET AL. both a 2D and conduit fashion on nerve regeneration. ...
... [65][66][67] During this process, the fibrin matrix (derived from plasma precursors) accumulates naturally to form a complete bridge across the chamber gap providing a substrate for cellular migration. 51 The E10-0.5(1K) conduit may encourage the early orientation of regenerating components on its own, as opposed to filling the inner lumen with a synthetic material to act as a longitudinal cable. [68][69][70] This may occur by increasing infiltrating Schwann cells and fibroblasts across the nerve injury gap accelerating the temporal progress of regeneration. ...
... This matrix behaves as a physical scaffold for nonneuronal calls, including fibroblasts, Schwann cells, macrophages, and endothelial cells to influence axonal elongation and initiate the regenerative process. 51,71 Due to the critical role Schwann cells play in nerve regeneration, we used biochemical analysis techniques (Fig. 8C) to investigate the presence of proteins specific to Schwann cells within the nerve conduits post-implantation. 51,57,64,72,73 Results showed that these protein levels were higher in E10-0.5(1K) ...
In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.
... In the rat sciatic nerve, this critical distance measures > 10 mm, and in the primate ulnar nerve it is > 3 cm. 23,31,[47][48][49]52,[81][82][83] Critical distances can be overcome by adding exogenous growth factors and/or tissues to the tube environment. In the rat, a 13-mm gap can be successfully bridged using silicone chambers only when exogenous fibrin matrix precursors or a small segment of degenerated peripheral nerve is added to the conduit. ...
... In the rat, a 13-mm gap can be successfully bridged using silicone chambers only when exogenous fibrin matrix precursors or a small segment of degenerated peripheral nerve is added to the conduit. 23,40,47,48,[81][82][83] More specifically, some authors have demonstrated no evidence of regeneration within an empty or saline-filled 15-mm silicone tube (13-mm nerve gap) at 16 weeks, but the addition of a short, interposed, 2-mm nerve segment resulted in significant regeneration similar to that of an autograft. In the monkey, a 3-cm gap can be repaired with a bioabsorbable polyglycolic acid tube. ...
Object:
Segmental nerve defects pose a daunting clinical challenge, as peripheral nerve injury studies have established that there is a critical nerve gap length for which the distance cannot be successfully bridged with current techniques. Construction of a neural prosthesis filled with Schwann cells (SCs) could provide an alternative treatment to successfully repair these long segmental gaps in the peripheral nervous system. The object of this study was to evaluate the ability of autologous SCs to increase the length at which segmental nerve defects can be bridged using a collagen tube.
Methods:
The authors studied the use of absorbable collagen conduits in combination with autologous SCs (200,000 cells/μl) to promote axonal growth across a critical size defect (13 mm) in the sciatic nerve of male Fischer rats. Control groups were treated with serum only-filled conduits of reversed sciatic nerve autografts. Animals were assessed for survival of the transplanted SCs as well as the quantity of myelinated axons in the proximal, middle, and distal portions of the channel.
Results:
Schwann cell survival was confirmed at 4 and 16 weeks postsurgery by the presence of prelabeled green fluorescent protein-positive SCs within the regenerated cable. The addition of SCs to the nerve guide significantly enhanced the regeneration of myelinated axons from the nerve stump into the proximal (p < 0.001) and middle points (p < 0.01) of the tube at 4 weeks. The regeneration of myelinated axons at 16 weeks was significantly enhanced throughout the entire length of the nerve guide (p < 0.001) as compared with their number in a serum-only filled tube and was similar in number compared with the reversed autograft. Autotomy scores were significantly lower in the animals whose sciatic nerve was repaired with a collagen conduit either without (p < 0.01) or with SCs (p < 0.001) when compared with a reversed autograft.
Conclusions:
The technique of adding SCs to a guidance channel significantly enhanced the gap distance that can be repaired after peripheral nerve injury with long segmental defects and holds promise in humans. Most importantly, this study represents some of the first essential steps in bringing autologous SC-based therapies to the domain of peripheral nerve injuries with long segmental defects.
... During endogenous nerve regeneration, plasma-derived precursor proteins create an oriented fibrin matrix along the longitudinal direction between the proximal and distal stumps, and the Schwann cells migrate following the fibrin matrix. [46][47][48][49] Then, the Schwann cells proliferate, form the band of Bü ngner, and guide axonal regrowth. 46,[50][51][52][53] It is well known that the morphology of the fibrin matrix created at the initial stage and the distribution of Schwann cells within NGC govern the final morphology of the regenerated nerve fibers. ...
... The NGFimmobilized NGCs (Nanopore NGC/NGF and Micropore NGC/NGF) showed more dense nerve fibers than the NGCs without NGF (Nanopore NGC and Micropore NGC); however, their nerve fiber growth patterns were not highly different (not shown the data of the NGC groups without NGF). Many previous studies have reported that NGCs with a smooth surface 46,48,54,[56][57][58] provide an appropriate environment for an aligned fibrin matrix and, thus, allow for the formation of a neural structure in the NGCs. More recently, it was also demonstrated that the oriented topography in the NGC can enhance cellular alignment, and, therefore, accelerate nerve regeneration. ...
Polycaprolactone (PCL)/Pluronic F127 nerve guide conduits (NGCs) with different surface pore structures (nano-porous inner surface vs. micro-porous inner surface) but similar physical and chemical properties were fabricated by rolling the opposite side of asymmetrically porous PCL/F127 membranes. The effect of the pore structure on peripheral nerve regeneration through the NGCs was investigated using a sciatic nerve defect model of rats. The nerve fibers and tissues were shown to have regenerated along the longitudinal direction through the NGC with a nano-porous inner surface (Nanopore NGC), while they grew toward the porous wall of the NGC with a micro-porous inner surface (Micropore NGC) and, thus, their growth was restricted when compared with the Nanopore NGC, as investigated by immunohistochemical evaluations (by fluorescence microscopy with anti-neurofilament staining and Hoechst staining for growth pattern of nerve fibers), histological evaluations (by light microscopy with Meyer's modified trichrome staining and Toluidine blue staining and transmission electron microscopy for the regeneration of axon and myelin sheath), and FluoroGold retrograde tracing (for reconnection between proximal and distal stumps). The effect of nerve growth factor (NGF) immobilized on the pore surfaces of the NGCs on nerve regeneration was not so significant when compared with NGCs not containing immobilized NGF. The NGC system with different surface pore structures but the same chemical/physical properties seems to be a good tool that is used for elucidating the surface pore effect of NGCs on nerve regeneration.
... An exogenous fibrin matrix is thus a rational choice for an engineered NGC component because it is a natural biomaterial that acts in alignment with the endogenous processes that lead to regeneration. 9 Because of fibrin's importance, nerve regeneration outcomes may benefit from repair solutions that are informed by an improved understanding of the cell-driven accumulation, remodeling, and degradation of fibrin. One way to learn about the fate of fibrin and fibrinogen or other dynamic ECM components in vivo is to infuse the tissue with labeled ECM and longitudinally image it. ...
Significance:
Exogenous extracellular matrix (ECM) proteins, such as fibrinogen and the thrombin-polymerized scaffold fibrin, are used in surgical repair of severe nerve injuries to supplement ECM produced via the injury response. Monitoring the dynamic changes of fibrin during nerve regeneration may shed light on the frequent failure of grafts in the repair of long nerve gaps.
Aim:
We explored whether monitoring of fibrin dynamics can be carried out using nerve guidance conduits (NGCs) containing fibrin tagged with covalently bound fluorophores.
Approach:
Fibrinogen was conjugated to a near-infrared (NIR) fluorescent dye. NGCs consisting of silicone tubes filled with the fluorescent fibrin were used to repair a 5-mm gap injury in rat sciatic nerve ( n = 6 ).
Results:
Axonal regeneration in fluorescent fibrin-filled NGCs was confirmed at 14 days after implantation. Intraoperative fluorescence imaging after implantation showed that the exogenous fibrin was embedded in the early stage regenerative tissue. The fluorescent signal temporarily highlighted a cable-like structure within the conduit and gradually degraded over two weeks.
Conclusions:
This study, for the first time, visualized in vivo intraneural fibrin degradation, potentially a useful prospective indicator of regeneration success, and showed that fluorescent ECM, in this case fibrin, can facilitate imaging of regeneration in peripheral nerve conduits without significantly affecting the regeneration process.
... Williams and colleagues extensively studied the spatiotemporal process of peripheral nerve regeneration within a silicon chamber (Williams et al. 1983). After a nerve is cut, a clot mainly consisting of fibrin and fibronectin is formed to connect the two severed nerve ends (Williams and Varon 1985;Williams 1987;Le Beau et al. 1988;Liu 1992;Hoffman-Kim et al. 2010). Some authors also refer to this clot as a "cable" as it can cover distances of several centimeters depending on the length of the nerve injury. ...
... Regenerating axons then grow from the proximal nerve, directed by the Schwann cells in the intratubular connective cable. In vivo studies that used components of the naturally formed ECM, such as fibrin, fibronectin, hyaluronate, collagen and laminin-containing gels to prefill the lumen of the tube guide, have reported enhancement of peripheral nerve regeneration with respect to empty guides (Williams, 1987;Bailey et al., 1993;Chamberlain et al., 1998;Madison et al., 1988;Labrador et al., 1998). On the contrary, application of anti-laminin antibody or anti-laminin/collagen receptor to grafts or tubes inhibits axonal regeneration, whereas anti-fibronectin antibody has no effect (Wang et al., 1992). ...
Peripheral nerve injuries result in the loss of the motor, sensory and autonomic functions of the denervated segments of the body. Neurons can regenerate after peripheral axotomy, but inaccuracy in reinnervation causes a permanent loss of function that impairs complete recovery. Thus, understanding how regenerating axons respond to their environment and direct their growth is essential to improve the functional outcome of patients with nerve lesions. Schwann cells (SCs) play a crucial role in the regeneration process, but little is known about their contribution to specific reinnervation. Here, we review the mechanisms by which SCs can differentially influence the regeneration of motor and sensory axons. Mature SCs express modality-specific phenotypes that have been associated with the promotion of selective regeneration. These include molecular markers, such as L2/HNK-1 carbohydrate, which is differentially expressed in motor and sensory SCs, or the neurotrophic profile after denervation, which differs remarkably between SC modalities. Other important factors include several molecules implicated in axon-SC interaction. This cell-cell communication through adhesion (e.g., polysialic acid) and inhibitory molecules (e.g., MAG) contributes to guiding growing axons to their targets. As many of these factors can be modulated, further research will allow the design of new strategies to improve functional recovery after peripheral nerve injuries.
... While several biochemical approaches, such as the addition of growth factors, [38] fibrin, [39] and collagen [40] to a scaffold have shown to enhance neurite growth, the presence of SCs also demonstrated an improved neuronal growth likely due to their ability to express neurite promoting factors, such as neurotrophins. [35,41] A previous study has shown that SCs migrating ahead of neurite outgrowth may be establishing a pathway for neurite extension on uncoated smooth fibers, whereas on fibers coated with LN or FN, the neurites grew past the SCs suggesting that at the presence of ECM support is sufficient to promote neurite growth. ...
Surface topographies at micro- and nanoscales can influence different cellular behavior, such as their growth rate and directionality. While different techniques have been established to fabricate 2-dimensional flat substrates with nano- and microscale topographies, most of them are prone to high costs and long preparation times. The 2.5-dimensional fiber platform presented here provides knowledge on the effect of the combination of fiber alignment, inter-fiber distance (IFD), and fiber surface topography on contact guidance to direct neurite behavior from dorsal root ganglia (DRGs) or dissociated primary neurons. For the first time, the interplay of the micro-/nanoscale topography and IFD is studied to induce linear nerve growth, while controlling branching. The results demonstrate that grooved fibers promote a higher percentage of aligned neurite extension, compensating the adverse effect of increased IFD. Accordingly, maximum neurite extension from primary neurons is achieved on grooved fibers separated by an IFD of 30 μm, with a higher percentage of aligned neurons on grooved fibers at a large IFD compared to porous fibers with the smallest IFD of 10 µm. We further demonstrate that the neurite “decision-making” behavior on whether to cross a fiber or grow along it is not only dependent on the IFD but also on the fiber surface topography. In addition, axons growing in between the fibers seem to have a memory after leaving grooved fibers, resulting in higher linear growth and higher IFDs lead to more branching. Such information is of great importance for new material development for several tissue engineering applications.
Statement of Significance
One of the key aspects of tissue engineering is controlling cell behavior using hierarchical structures. Compared to 2D surfaces, fibers are an important class of materials, which can emulate the native ECM architecture of tissues. Despite the importance of both fiber surface topography and alignment to direct growing neurons, the current state of the art did not yet study the synergy between both scales of guidance. To achieve this, we established a solvent assisted spinning process to combine these two crucial features and control neuron growth, alignment, and branching. Rational design of new platforms for various tissue engineering and drug discovery applications can benefit from such information as it allows for fabrication of functional materials, which selectively influence neurite behavior.
... The repair of nerve defects using a pseudosynovial sheath demonstrated that plasma exudates from the proximal and distal nerve stumps fill the empty tube volume, and provide a deposition of extracellular matrix (ECM), including a fibrin matrix, that allows for innate immune system cell migration. [49][50][51][52][53][54][55][56] While the earliest (<4 days) macrophages are derived from tissue resident macrophages, subsequent macrophages are primarily hematogenous-derived. 40 Due to the hypoxic nature of this environment, macrophages support a substantial amount of angiogenesis enabling endothelial cell recruitment and vessel formation 56 ( Figure 1A). These blood vessels become polarized allowing for the migration of SCs, 56 which have a distinct phenotype similar to a stem cell in this, 26,57 and in turn form cellular cords through their interactions with fibroblasts 58 ( Figure 1B). ...
Despite advances in surgery, the reconstruction of segmental nerve injuries continues to pose challenges. In this review, current neurobiology regarding regeneration across a nerve defect is discussed in detail. Recent findings include the complex roles of non‐neuronal cells in nerve defect regeneration, such as the role of the innate immune system in angiogenesis and how Schwann cells migrate within the defect. Clinically, the repair of nerve defects is still best served by using nerve autografts with the exception of small, non‐critical sensory nerve defects, which can be repaired using autograft alternatives, such as processed or acellular nerve allografts. Given current clinical limits for when alternatives can be utilized, advanced solutions to repair nerve defects demonstrated in animals are highlighted. These highlights include alternatives designed with novel topology and materials, delivery of drugs specifically known to accelerate axon growth, and greater attention to the role of the immune system. This article is protected by copyright. All rights reserved.
... [131][132][133] As such, fibrin has been trialed as an interlumen material by prefilling the conduit with fibrin in lieu of endogenous filling. A series of earlier experiments by Williams found that presenting the regenerative nerve with such a gel-filled lumen had generally positive effects compared to an empty tube; [134] this was performed for only a 10 mm gap defect in rat sciatic nerve, just at the critical gap length for this animal model. It was observed from this work that performance was dependent on the gelation and composition of the fibrin matrix, with innervation increasing with lower matrix density and increased longitudinal arrangement of polymerized fibrin fibers; these factors were later confirmed in vitro. ...
Biofabrication strategies have made significant steps toward overcoming the clinical challenges of peripheral nerve repair. Many activities have focused on emulating the structural features of native nerve tissue which promote nerve regeneration, resulting in the development of a variety of fabrication techniques. In article number 1701164, Lorenzo Moroni and co‐workers discuss the most relevant approaches, highlight their biomimetic character, and assess performance in achieving nerve repair.
... [131][132][133] As such, fibrin has been trialed as an interlumen material by prefilling the conduit with fibrin in lieu of endogenous filling. A series of earlier experiments by Williams found that presenting the regenerative nerve with such a gel-filled lumen had generally positive effects compared to an empty tube; [134] this was performed for only a 10 mm gap defect in rat sciatic nerve, just at the critical gap length for this animal model. It was observed from this work that performance was dependent on the gelation and composition of the fibrin matrix, with innervation increasing with lower matrix density and increased longitudinal arrangement of polymerized fibrin fibers; these factors were later confirmed in vitro. ...
Biofabrication techniques have endeavored to improve the regeneration of the peripheral nervous system (PNS), but nothing has surpassed the performance of current clinical practices. However, these current approaches have intrinsic limitations that compromise patient care. The “gold standard” autograft provides the best outcomes but requires suitable donor material, while implantable hollow nerve guide conduits (NGCs) can only repair small nerve defects. This review places emphasis on approaches that create structural cues within a hollow NGC lumen in order to match or exceed the regenerative performance of the autograft. An overview of the PNS and nerve regeneration is provided. This is followed by an assessment of reported devices, divided into three major categories: isotropic hydrogel fillers, acting as unstructured interluminal support for regenerating nerves; fibrous interluminal fillers, presenting neurites with topographical guidance within the lumen; and patterned interluminal scaffolds, providing 3D support for nerve growth via structures that mimic native PNS tissue. Also presented is a critical framework to evaluate the impact of reported outcomes. While a universal and versatile nerve repair strategy remains elusive, outlined here is a roadmap of past, present, and emerging fabrication techniques to inform and motivate new developments in the field of peripheral nerve regeneration.
... When porous conduits are filled with a hydrogel, a balance must be achieved between the conduit porosity and the bioactivity of the filler matrix to encourage faster axonal regeneration than fibrotic tissue infiltration. 10,[39][40][41][42] As longer conduits are required in more severe nerve injuries, a greater porosity will be necessary to overcome limitations of nutrient and waste exchange. In these cases, the cell-friendly filler may become more detrimental to nerve regeneration by increasing the risk of fibrous tissue occluding the conduit lumen. ...
Nerve conduits pre-filled with hydrogels are frequently explored in an attempt to promote nerve regeneration. This study examines the interplay between the porosity of the conduit wall and the level of bioactivity of the hydrogel used to fill the conduit. Nerve regeneration in porous (P) or non-porous (NP) conduits that were filled with either collagen-only or collagen enhanced with a covalently attached neurite-promoting peptide mimic of the glycan Human Natural Killer Cell Antigen-1 (m-HNK) were compared in a 5 mm critical size defect in the mouse femoral nerve repair model. While collagen is a cell-friendly matrix that does not differentiate between neural and non-neural cells, the m-HNK-enhanced collagen specifically promotes axon growth and appropriate motor neuron targeting. In this study, animals treated with non-porous (NP) conduits filled with collagen grafted with m-HNK (CollagenHNK) had the best functional recovery, commensurate with a substantial improvement in all tested histomorphometric parameters (number of axons, degree of raw tissue area, percentage of myelinated nerve fibers, cross-sectional area of myelinated nerve fibers) relative to all other conduit conditions. Our data indicate that under some conditions, the use of generally cell friendly fillers such as collagen, may limit nerve regeneration. This finding is significant, considering the frequent use of collagen based hydrogels as fillers of nerve conduits.
... Fibrin was chosen as the base material for the delivery system because it is the natural biomaterial of nerve regeneration and is readily penetrated by the proteolytic activity of the neurite growth cone as it extends through the three-dimensional fibrin matrix (16). Others have previously demonstrated that the addition of fibrin matrices to nerve guide tubes enhanced peripheral nerve regeneration (17,18). Our goal was to enhance the ability of fibrin matrices to promote nerve regeneration by incorporating a drug delivery system into the matrix, thus allowing the fibrin matrix to serve not only as a wound-healing scaffold but also as vehicle for controlled drug release. ...
The goal of this research was to develop an approach to growth factor delivery that would allow the stable incorporation of growth factors within a cell in‐growth matrix in a manner such that local enzymatic activity associated with tissue regeneration could trigger growth factor release. We investigated this approach in the context of peripheral nerve regeneration by designing modified beta‐nerve growth factor (β‐NGF) fusion proteins and testing their ability to promote neurite extension. Fibrin was selected as the cell in‐growth matrix, and the transglutaminase activity of factor XIIIa was utilized to covalently incorporate β‐NGF fusion proteins within fibrin matrices. Novel β‐NGF fusion proteins, which contained an exogenous factor XIIIa substrate to allow incorporation into fibrin matrices, were expressed recombinantly. An intervening plasmin substrate domain was placed between the factor XIIIa substrate and the NGF domain to allow cell‐mediated growth factor release in response to plasmin, which is generated by invading cells. Immobilized NGF fusion protein with an intervening functional plasmin cleavage sequence enhanced neurite extension from embryonic chick dorsal root ganglia by 50% relative to soluble native β‐NGF and by 350% relative to the absence of NGF. These results suggest that this novel approach to growth factor delivery, in which the factor is delivered upon cellular demand, could enhance nerve regeneration and may be useful in tissue engineering.
... Numerous studies have been performed to determine some of the fundamental mechanisms of regeneration by varying the conditions within such tubes. Williams et al. (2,3) have observed the spontaneous formation of oriented fibrin matrices during the first week of regeneration within nerve guide tubes and postulated that this fibrin bridge might play a role in nerve regeneration. They found that the addition of dialyzed plasma led to the formation of an oriented fibrin matrix within 24 h and that the earlier formation of a fibrin matrix allowed regeneration to occur more quickly. ...
The goal of this work was to improve the potential of fibrin to promote nerve regeneration by enzymatically incorporating exogenous neurite-promoting heparin-binding peptides. The effects on neurite extension of four different heparin-binding peptides, derived from the heparin-binding domains of antithrombin III, neural cell adhesion molecule and platelet factor 4, were determined. These exogenous peptides were synthesized as bi-domain peptide chimeras, with the second domain being a substrate for factor XIIIa. This coagulation transglutaminase covalently bound the peptides within the fibrin gel during coagulation. The heparin-binding peptides enhanced the degree of neurite extension from embryonic chick dorsal root ganglia through 3-dimensional fibrin gels, and the extent of enhancement was found to correlate positively with the heparin-binding affinity of the individual domains. The enhancement could be inhibited by competition with soluble heparin, by degradation of cell-surface proteoglycans, and by inhibition of the covalent immobilization of the peptide. These results demonstrate an important potential role for proteoglycan-binding components of the extracellular matrix in neurite extension and suggest that fibrin gels modified with covalently bound heparin-binding peptides could serve as a therapeutic agent to enhance peripheral nerve regeneration through nerve guide tubes. More generally, the results demonstrate that the biological responses to fibrin, the body's natural wound healing matrix, can be dramatically improved by the addition of exogenous bioactive peptides in a manner such that they become immobilized during coagulation.-Sakiyama, S. E., Schense, J. C., Hubbell, J. A. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. [on SciFinder (R)]
... Mesenchymal stem cells also promote enhanced axon regeneration by promoting Schwann cells within the central and distal nerve stumps to migrate into the nerve gap where they proliferate and release additional axon regenerationpromoting factors [254,[262][263][264][265][266][267][268][269][270][271]. These and other mesenchymal stem cell-released factors simultaneously act directly on axons to induce and promote their regeneration [28,221]. ...
Platelet-rich plasma (PRP) has been tested in vitro, in animal models, and clinically for its efficacy in enhancing the rate of wound healing, reducing pain associated with injuries, and promoting axon regeneration. Although extensive data indicate that PRP-released factors induce these effects, the claims are often weakened because many studies were not rigorous or controlled, the data were limited, and other studies yielded contrary results. Critical to assessing whether PRP is effective are the large number of variables in these studies, including the method of PRP preparation, which influences the composition of PRP; type of application; type of wounds; target tissues; and diverse animal models and clinical studies. All these variables raise the question of whether one can anticipate consistent influences and raise the possibility that most of the results are correct under the circumstances where PRP was tested. This review examines evidence on the potential influences of PRP and whether PRP-released factors could induce the reported influences and concludes that the preponderance of evidence suggests that PRP has the capacity to induce all the claimed influences, although this position cannot be definitively argued. Well-defined and rigorously controlled studies of the potential influences of PRP are required in which PRP is isolated and applied using consistent techniques, protocols, and models. Finally, it is concluded that, because of the purported benefits of PRP administration and the lack of adverse events, further animal and clinical studies should be performed to explore the potential influences of PRP.
... Previous studies demonstrate the effectiveness of the fibrin as intraluminal filler. In these studies, silicone-based NC were filled with exogenous fibrin hydrogel promoting an increase of the nerve regeneration across 1 cm [121] and 2 cm [122] of nerve gap. Similarly, fibrin was used to fill synthetic permeable and biodegradable NC, showing a significant increase of the nerve regeneration and the mechanical properties of these NC [123,124]. ...
The structure and function of peripheral nerves can be affected by a range of conditions with severe consequences in these patients. Currently, there are several surgical techniques available to treat peripheral nerve defects. Direct repair is the preferred treatment for short nerve gaps, and nerve autografting is the gold standard in critical nerve defects. The autografting is not always available, and the use of allograft, decellularized allograft and nerve conduits are often used with variable success. During the recent years, several outcomes were achieved in peripheral nerve tissue engineering. Promising experimental results have been demonstrated with this novel generation of nerve conduits, mainly composed by biodegradables materials in combination with intraluminal fillers, growth factors and different cell sources.
... The luminal matrix assists SC migration into the conduits and leads to the increase in regeneration [48]. Fibrin gels or glue have also been employed as a matrix and have demonstrated positive regenerative effects [49][50][51][52][53]. ...
Tissue engineering has been defined as "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ". Traumatic peripheral nerve injury resulting in significant tissue loss at the zone of injury necessitates the need for a bridge or scaffold for regenerating axons from the proximal stump to reach the distal stump.
A review of the literature was used to provide information on the components necessary for the development of a tissue engineered peripheral nerve substitute. Then, a comprehensive review of the literature is presented composed of the studies devoted to this goal.
Extensive research has been directed toward the development of a tissue engineered peripheral nerve substitute to act as a bridge for regenerating axons from the proximal nerve stump seeking the distal nerve. Ideally this nerve substitute would consist of a scaffold component that mimics the extracellular matrix of the peripheral nerve and a cellular component that serves to stimulate and support regenerating peripheral nerve axons.
The field of tissue engineering should consider its challenge to not only meet the autograft "gold standard" but also to understand what drives and inhibits nerve regeneration in order to surpass the results of an autograft.
... Other studies focussing on hydrogel-mediated axonal growth, using DRG explants, have shown similarly poor neurite penetration into the hydrogel in vitro [23,24]. In vivo experiments using hollow conduits filled with fibrin hydrogels have demonstrated a slightly better performance, with some studies reporting axon regeneration and myelination beyond the coaptation site as early as 1-week after transplantation [25,26]. Such in vivo axonal penetration into fibrin hydrogels has been suggested to be due to the extensive migration of host cells into the fibrin hydrogel where they degraded and reshaped the matrix, rather than to direct penetration of axons into the hydrogel [25]. ...
Peripheral nerve injuries can be surgically repaired by suturing the transected nerve stumps or, in case of larger lesions, by the transplantation of an autologous nerve graft. To avoid donor site morbidity, the development of artificial implants is desired. Clinically, hollow conduits have been used for this purpose but are inferior to the autograft because they lack internal guidance cues for Schwann cells and regenerating axons. In this article, we describe the design of a three-dimensional (3D) scaffold consisting of parallel fibers embedded in a collagen matrix. For this purpose, an electrospinning device was developed to produce and manipulate a 3D array of aligned poly(ɛ-caprolactone) (PCL) microfibers. This fiber array was then incorporated into biodegradable PCL tubes to serve as artificial nerve bridges. Using primary cultures of embryonic chicken dorsal root ganglia, we show that PCL microfibers in the 3D matrix of our composite scaffold guide the direction of Schwann cell migration and axonal growth. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.
... Ainsi, les polymères synthétiques ont été principalement utilisés pour étudier les facteurs pouvant influencer la régénération axonale de fibres nerveuses périphériques lésées (Lundborg, 1982a(Lundborg, , 1982b(Lundborg, , 1982cLundborg et al., 1982aLundborg et al., , 1982bLundborg et al., , 1982cHall, 1986 ;Berry et al., 1992 ;Guenard et al., 1992 ;Hall et al., 1992 ;Liu, 1992aLiu, , 1992bLabrador et al., 1995 ;Liu et al., 1995 ;Buti et al., 1996). Ces polymères synthétiques peuvent être remplis de composants non neuronaux tels que ceux de la matrice intratubulaire (fibrine, laminine et collagène) ou des cellules dissociées (fibroblastes, astrocytes et cellules de Schwann) (Jenq et Coggeshall, 1985a, 1985b, 1985c, 1985dWilliams, 1987 ;Williams et al., 1987 ;Montgomery et Robson, 1993). L'objectif des chercheurs et des cliniciens est d'obtenir des conduits biocompatibles imitant l'anatomie du nerf (Heath et Rutkowski, 1998). ...
Introduction
Nerve injury compromises sensory and motor functions. Techniques of peripheral nerve repair are based on our knowledge regarding regeneration. Microsurgical techniques introduced in the late 1950s and widely developed for the past 20 years have improved repairs. However, functional recovery following a peripheral mixed nerve injury is still incomplete.
State of art
Good motor and sensory function after nerve injury depends on the reinnervation of the motor end plates and sensory receptors. Nerve regeneration does not begin if the cell body has not survived the initial injury or if it is unable to initiate regeneration. The regenerated axons must reach and reinnervate the appropriate target end-organs in a timely fashion. Recovery of motor function requires a critical number of motor axons reinnervating the muscle fibers. Sensory recovery is possible if the delay in reinnervation is short. Many additional factors influence the success of nerve repair or reconstruction. The timing of the repair, the level of injury, the extent of the zone of injury, the technical skill of the surgeon, and the method of repair and reconstruction contribute to the functional outcome after nerve injury.
Conclusion
This review presents the recent advances in understanding of neural regeneration and their application to the management of primary repairs and nerve gaps.
... Regenerating axons then grow from the proximal nerve, directed by the Schwann cells in the intratubular connective cable. In vivo studies that used components of the naturally formed ECM, such as fibrin, fibronectin, hyaluronate, collagen and laminin-containing gels to prefill the lumen of the tube guide, have reported enhancement of peripheral nerve regeneration with respect to empty guides (Williams, 1987;Bailey et al., 1993;Chamberlain et al., 1998;Madison et al., 1988;Labrador et al., 1998). On the contrary, application of anti-laminin antibody or anti-laminin/collagen receptor to grafts or tubes inhibits axonal regeneration, whereas anti-fibronectin antibody has no effect (Wang et al., 1992). ...
Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. The significance of axonal regeneration is to replace the degenerated distal nerve segment, and achieve reinnervation of target organs and restitution of their functions. However, axonal regeneration does not always allows for adequate functional recovery, so that after a peripheral nerve injury, patients do not recover normal motor control and fine sensibility. The lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. Key factors for successful axonal regeneration include the intrinsic changes that neurons suffer to switch their transmitter state to a pro-regenerative state and the environment that the axons find distal to the lesion site. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. The aim of this review is to look at those interactions, trying to understand if some of these molecular factors are specific for motor and sensory neuron growth, and provide the basic knowledge for potential strategies to enhance and guide axonal regeneration and reinnervation of adequate target organs.
... Fibrin has been successfully utilized in repair strategies for a variety of in vivo neuronal injury models (e.g. (Patist, et al., 2004) (Iwaya, et al., 1999, Stokols, et al., 2006, Taylor, et al., 2006, Tsai, et al., 2006, Williams, 1987). In spite of these advantages, mammalian fibrin gels degrade rapidly (Bensaid, et al., 2003, Novikova, et al., 2003, Sieminski and Gooch, 2004) and may be contaminated with blood-borne pathogens such as HIV, hepatitis C, and prion proteins (Fischer, et al., 2000). ...
The neural degeneration caused by spinal cord injury leaves a cavity at the injury site that greatly inhibits repair. One approach to promoting repair is to fill the cavity with a scaffold to limit further damage and encourage regrowth. Injectable materials are advantageous scaffolds because they can be placed as a liquid in the lesion site then form a solid in vivo that precisely matches the contours of the lesion. Fibrin is one type of injectable scaffold, but risk of infection from blood borne pathogens has limited its use. We investigated the potential utility of salmon fibrin as an injectable scaffold to treat spinal cord injury since it lacks mammalian infectious agents and encourages greater neuronal extension in vitro than mammalian fibrin or Matrigel®, another injectable material. Female rats received a T9 dorsal hemisection injury and were treated with either salmon or human fibrin at the time of injury while a third group served as untreated controls. Locomotor function was assessed using the BBB scale, bladder function was analyzed by measuring residual urine, and sensory responses were tested by mechanical stimulation (von Frey hairs). Histological analyses quantified the glial scar, lesion volume, and serotonergic fiber density. Rats that received salmon fibrin exhibited significantly improved recovery of both locomotor and bladder function and a greater density of serotonergic innervation caudal to the lesion site without exacerbation of pain. Rats treated with salmon fibrin also exhibited less autophagia than those treated with human fibrin, potentially pointing to amelioration of sensory dysfunction. Glial scar formation and lesion size did not differ significantly among groups. The pattern and timing of salmon fibrin's effects suggest that it acts on neuronal populations but not by stimulating long tract regeneration. Salmon fibrin clearly has properties distinct from those of mammalian fibrin and is a beneficial injectable scaffold for treatment of spinal cord injury.
... Across short gaps (< 8 mm), these conduits support provisional fibrin cable formation which acts as a substrate for Schwann cell (SC) and fibroblast (FB) migration into the nerve gap from proximal and distal nerve stumps[9]. These cells help reorganize the extracellular matrix (ECM) and provide the trophic support to the regenerating axons enabling bridging of the nerve gap [10]. However, nerve conduits have not been effective in bridging critically sized nerve gaps that are typically (greater than 3 cm in humans and greater than 1.3-1.5 cm in rats), and functional recovery is rarely attained [4]. ...
Bridging of long peripheral nerve gaps remains a significant clinical challenge. Electrospun nanofibers have been used to direct and enhance neurite extension in vitro and in vivo. While it is well established that oriented fibers influence neurite outgrowth and Schwann cell migration, the mechanisms by which they influence these cells are still unclear. In this study, thin films consisting of aligned poly-acrylonitrile methylacrylate (PAN-MA) fibers or solvent casted smooth, PAN-MA films were fabricated to investigate the potential role of differential protein adsorption on topography-dependent neural cell responses. Aligned nanofiber films promoted enhanced adsorption of fibronectin compared to smooth films. Studies employing function-blocking antibodies against cell adhesion motifs suggest that fibronectin plays an important role in modulating Schwann cell migration and neurite outgrowth from dorsal root ganglion (DRG) cultures. Atomic Force Microscopy demonstrated that aligned PAN-MA fibers influenced fibronectin distribution, and promoted aligned fibronectin network formation compared to smooth PAN-MA films. In the presence of topographical cues, Schwann cell-generated fibronectin matrix was also organized in a topographically sensitive manner. Together these results suggest that fibronectin adsorption mediated the ability of topographical cues to influence Schwann cell migration and neurite outgrowth. These insights are significant to the development of rational approaches to scaffold designs to bridge long peripheral nerve gaps.
Peripheral nerve injuries can be debilitating to motor and sensory function, with severe cases often resulting in complete limb amputation. Over the past two decades, prosthetic limb technology has rapidly advanced to provide users with crude motor control of up to 20° of freedom; however, the nerve-interfacing technology required to provide high movement selectivity has not progressed at the same rate. The work presented here focuses on the development of a magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI) that combines polyimide “threads” encapsulated within a magnetically aligned hydrogel scaffold. The technology exploits tissue-engineered strategies to address concerns over traditional peripheral nerve interfaces including poor axonal sampling through the nerve and rigid substrates. A magnetically templated hydrogel is used to physically support the polyimide threads while also promoting regeneration in close proximity to the electrode sites on the polyimide. This work demonstrates the utility of magnetic templating for use in tuning the mechanical properties of hydrogel scaffolds to match the stiffness of native nerve tissue while providing an aligned substrate for Schwann cell migration in vitro. MARTEENI devices were fabricated and implanted within a 5-mm-long rat sciatic-nerve transection model to assess regeneration at 6 and 12 weeks. MARTEENI devices do not disrupt tissue remodeling and show axon densities equivalent to fresh tissue controls around the polyimide substrates. Devices are observed to have attenuated foreign-body responses around the polyimide threads. It is expected that future studies with functional MARTEENI devices will be able to record and stimulate single axons with high selectivity and low stimulation regimes.
Significance
Cell contact guidance in aligned fibers is, in many ways, a final frontier in directed cell migration. Despite its ubiquitous importance in normal/pathological processes, notably metastasis from solid tumors, and scaffold design for tissue engineering/regenerative medicine, the signal that induces contact guidance in an aligned fiber network has defied elucidation. We report definitive demonstration of a biophysical signal sensed by fibroblasts cultured within aligned fibrils: mechanical resistance anisotropy, the resistance resulting from viscous and elastic properties of fibril network to pseudopod protrusion/retraction and how it differs in different directions (anisotropy). This discovery is of fundamental importance to understanding contact guidance, providing a rationale, currently lacking, for future studies of the intracellular signal transduction/response pathways for physiologically relevant contact guidance.
The treatment of peripheral nerve injuries remains a major problem worldwide despite the availability of a number of Food and Drug Administration (FDA) approved devices which fail to match the efficacy of autografts. Different strategies are used to improve regeneration and functional recovery using biomaterial nerve conduits. However, there is little investigation of the transcriptomic and proteomic changes which occur as a result of these interventions, particularly regarding transection injuries. This study explores differences between autograft-mediated repair and conduit-material-mediated repair of peripheral nerve injuries to understand fundamental differences in their repair mechanisms at the proteomics level at the proximal, middle, and distal components in the early stages of repair. Pathway analysis demonstrates that each material selectively activates different regenerative pathways and alters different biological functions spatially throughout the biomaterial conduits. The analysis highlights some of the deficiencies in conduit-mediated repair in comparison to autograft (e.g., recycling of myelin and cholesterol, reduction in reactive oxygen species, and higher expression of regenerative proteins). These findings thus suggest that by supplementing the expression of these proteins on the biomaterial of choice, this study can potentially attain regeneration equivalent to autograft. This approach paves the way for incorporating future biomaterial-specific functionalities in nerve guidance conduits.
The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. The present investigation has studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied), however, cell behaviour in the hydrogel was somewhat different to that observed on two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres where they extended long processes that followed the longitudinal orientation of the nanofibres. Process length along nanofibre-containing fibrin hydrogel reached near maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Biodegradable polymers of poly(lactic acid) (PLLA) and synthesized in‐house poly(trimethylene carbonate) (PTMC) with admixture of water‐soluble methyl cellulose (MC) were used for development of nerve guidance tubes for peripheral nervous system regeneration after injury. Fabrication method involved phase separation of viscous dixane solution of polymers mixture seat on a rod in a proper nonsolvent, which resulted in tubular structure of large porosity. Influence of electron beam sterilization on molecular weight, thermal properties of the polymers, and mechanical performance of the tubes was evaluated. Admixture of hydrophilic MC to synthetic polymers resulted in modification of mechanical properties of the channels. Extraction of MC showed potential of the tubes for releasing water‐soluble bioactive molecules, such as for instance growth factors. Basic in vitro MTT and LDH assays showed no cytotoxic effect of manufactured tubes, therefore, animal experimentations may be considered. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013
Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7-9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon-substrate interactions. By 7-14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.
Although autogenous nerve grafting remains the gold standard for repair of peripheral nerve defects, the use of various conduits can be a substitute provided these conduits meet the above-mentioned prerequisites. For the moment, autogenous vein grafts or denatured muscle grafts can be used to bridge short defects, especially in distal sensory nerves. Incorporation of muscle into a vein graft expands its application to longer defects in bigger nerves. PGA conduits have also been clinically proven to be reliable in reconstruction of digital nerve defects. Although nonabsorbable conduits cause irritation and nerve compression that necessitates secondary surgery removal, silicone tubes or Goretex tubes can be used in selected cases until absorbable conduits large enough for major peripheral nerves are available. To date, 3 cm seems to be the barrier for conduits. Incorporation of trophic factors and Schwann cells into the conduits will make their way into the clinic if problems like controlled release of trophic factors, obtaining and sustenance of an appropriate number of viable Schwann cells, are solved.
In the United States more than 200 000 people per year are treated for severe peripheral nerve injuries that require surgical intervention. Functional recovery of motor and sensory capability is limited after autografting, the most common surgical intervention for severe peripheral nerve injuries. The process of peripheral nerve regeneration has been studied extensively in a variety of animal models using a tubular conduit. This model has been used to generate a large base of data from a wide variety of experimental devices; however, this data has not been analyzed comparatively due to a lack of standardization of experimental conditions, assays, and reported measures of the quality of regeneration. As a result, progress in understanding conditions for optimal nerve regeneration has been stunted and the optimal characteristics for such an implant have not been identified. So while tubulation repair of a transected peripheral nerve presents an attractive alternative to autograft, it has not yet shown the ability to satisfactorily restore lost function. In this article, we provide an overview of mammalian wound healing following severe injury, the physiology of the peripheral nervous system, the standardized wound models used to study peripheral nerve regeneration, and the critical axon elongation criteria and how it can be used to directly compare results from dissimilar studies. We complete this review article with a description of the critical features of tubular implants used to induce peripheral nerve regeneration that can be optimized in order to improve the quality of regeneration.
With increasing fetal development, the mechanism of mammalian wound healing transitions from regeneration to a repair process characterized by organized wound contraction and scar synthesis. Recently, a variety of tissue-engineering constructs have been developed to block the contraction and scar formation mechanisms of repair, and to induce regeneration following injury with similar mechanisms of action observed for both the skin and peripheral nervous system (PNS). Such constructs, mostly scaffolds that are analogs of extracellular matrix and possess specific biological activity, have become the basis of studies of in vivo synthesis of tissues and organs. In this article, we provide an overview of mammalian wound healing processes following severe injury as well as a description of the tissue triad and the regenerative capacity of the three distinct tissue types that comprise the triad. We also discuss the critical structural elements of an active extracellular matrix analog that induces regeneration, and describe the use of standardized wound models for study of in vivo regeneration processes. We conclude this review describing recent data from studies utilizing active extracellular matrix analogs (scaffolds) that have shown regenerative activity.
The mechanism of neurite penetration of three-dimensional fibrin matrices was investigated by culturing embryonic chick dorsal root ganglia (DRGs) within fibrin gels, upon fibrin gels, and upon laminin. The length of neurites within three-dimensional matrices of fibrin was decreased in a concentration-dependent manner by agents that inhibited plasmin, e.g., aprotinin, or that inhibited plasminogen activation, e.g., ϵ-aminocaproic acid (EACA), or plasminogen antiserum. In contrast, such agents increased the length of neurites growing out from DRGs cultured upon two-dimensional substrates of fibrin and had no effect on the length of neurites growing out from DRGs cultured upon laminin. Visualization of neurites within three-dimensional fibrin matrices demonstrated that the distance between fibrin strands was much smaller than the diameter of neurites. All these data were consistent with the hypothesis that fibrinolysis localized to the region of the neurite tip is an important mechanism for neurite penetration of a physical barrier of fibrin strands arranged in a three-dimensional matrix. © 1996 Wiley-Liss, Inc.
The nerve chamber model has dominated the experimental study of peripheral nerve (PN) regeneration with animal models as well as in several clinical applications, such as the treatment of paralysis of limbs following severe trauma. The two stumps resulting from nerve transection are inserted inside a tubular chamber made from one of several materials, occasionally filled with various substances, and the quality of the reconnected nerve is assayed. Recent use of methods for data reduction has led to generation of a large normalized database from independent investigations. Methods for data normalization (reduction) are based on systematic use of the critical axon elongation, Lc, the gap length between the transected stumps at which the frequency of reconnection is just 50% for a given configuration. Four theories are compared for their ability to explain the normalized data. Although the neurotrophic and contact guidance theories explain some of the data, combined use of the more recent microtube theory and pressure cuff theory appears capable of explaining a much larger data set. PN regeneration appears to be upregulated by chamber configurations that facilitate formation of basement membrane microtubes about 10–20 m in diameter, comprising linear columns of Schwann cells surrounded by basement membrane, into which axons elongate and eventually become myelinated. Regeneration is downregulated by experimental configurations that permit formation of a contractile cell (myofibroblast) capsule around the regenerating nerve that appears to restrict growth of a nerve trunk by application of circumferential mechanical forces. These two processes work competitively to regulate nerve regeneration in the chamber model.
An injury to neural tissue that disrupts axonal continuity causes disconnection of pre- and postsynaptic elements of a neuronal
circuit. The axon also transports to the cell body neurotrophic factors from the innervation territory, and axotomy, by interrupting
this trophic supply, may lead to degeneration or death of the neuron unless supplementary trophic factors are available. Neural
regeneration could be broadly defined as a process leading to the restitution of function in the damaged circuit. Such a process
would involve, among other aspects, the maintenance of afferents to the presynaptic element, the regrowth of an axon, selection
of and connection to the correct postsynaptic partner, and the promotion or restoration of downstream circuits to which this
postsynaptic element contributes. Axonal regeneration, the topic of this chapter, is only concerned with the new growth of
an axon in the direction of a putative postsynaptic partner.
Introduction:
Previously, we introduced the biogenic conduit (BC) as a novel autologous nerve conduit for bridging peripheral nerve defects and tested its regenerative capacity in a short- and long-term setting. The aim of the present study was to clarify whether intraluminal application of regeneration-promoting glial cells, including Schwann cells (SC) and olfactory ensheathing cells (OEC), displayed differential effects after sciatic nerve gap bridging.
Material and methods:
BCs were generated as previously described. The conduits filled with fibrin/SC (n = 8) and fibrin/OEC (n = 8) were compared to autologous nerve transplants (NT; n = 8) in the 15-mm sciatic nerve gap lesion model of the rat. The sciatic functional index was evaluated every 4 weeks. After 16 weeks, histological evaluation followed regarding nerve area, axon number, myelination index and N ratio.
Results:
Common to all groups was a continual improvement in motor function during the observation period. Recovery was significantly better after SC transplantation compared to OEC (p < 0.01). Both cell transplantation groups showed significantly worse function than the NT group (p < 0.01). Whereas nerve area and axon number were correlated to function, being significantly lowest in the OEC group (p < 0.001), both cell groups showed lowered myelination (p < 0.001) and lower N ratio compared to the NT group.
Discussion:
SC-filled BCs led to improved regeneration compared to OEC-filled BCs in a 15-mm-long nerve gap model of the rat.
Studies on axonal regeneration in Y-chambers over the past decade have consistantly provided evidence of preferential growth toward a distal nerve piece. However, these findings are in contrast to the observations by Weiss and Taylor (1944. J. Exp. Zool. 95: 233–257), indicating that in fresh aortic Y-chambers regenerating axons had no tendency to grow into a channel containing a distal nerve piece as compared to an open channel. The discrepancy between Weiss and Taylor's findings and those of later authors remains unexplained. In the present study we repeated the investigations by Weiss and Taylor, using both isogenic and allogenic aortic Y-chambers. In control groups the aorta graft was frozen and treated chemically to kill all cells and to deactivate possible growth factors of protein nature. In these groups preferential growth toward the distal nerve was pronounced although such specificity in growth was evident also in the nontreated types of aortic chambers. The findings do not support the results presented by Weiss and Taylor.
Bioresorbable collagen nerve guides filled with either magnetically aligned type I collagen gel or control collagen gel were implanted into 4- or 6-mm surgical gaps created in the sciatic nerve of mice and explanted 30 and 60 days postoperation (dpo) for histological and immunohistochemical evaluation. The hypothesis was that contact guidance of regenerating axons and/or invading nonneuronal cells to the longitudinally aligned collagen fibrils would improve nerve regeneration. The criterion for regeneration was observation of regenerating myelinated fibers distal to the nerve guide. Consistent with previous studies showing poor regeneration in 6-mm gaps at 60 dpo with entubulation repair, only one of six mice exhibited regeneration with control collagen gel. In contrast, four of four mice exhibited regeneration with magnetically aligned collagen gel, including the appearance of nerve fascicle formation. The numbers of myelinated fibers were less than the uninjured nerve in all groups, however, which may have been due to rapid resorption of the nerve guides. An attempt to increase the stability of the collagen gel, and thereby the directional information presented by the aligned collagen fibrils, by crosslinking the collagen with ribose before implantation proved detrimental for regeneration.
In order to assess the usefulness of collagen and laminin gels prefilling nerve chambers to enhance nerve regeneration, we compared reinnervation of target organs after sciatic nerve resection leaving gaps of 4 or 6 mm followed by repair with silicone tubes in different groups of mice. Tubes were prefilled with saline solution, collagen gels, or laminin-containing gels at different concentrations. Functional reinnervation was assessed by noninvasive methods to quantitate recovery of sweating, nociceptive, sensory, and motor functions in the hindpaw repeatedly during 4–5 months postoperation. The increase in gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved. Reinnervation started earlier and achieved slightly higher levels with collagen gel diluted at 1.28 mg/ml than with more concentrated (1.92 and 2.56 mg/ml) collagen gels and with saline-prefilled tubes bridging a 4-mm gap. Recovery was also better with diluted (4 mg/ml) than with concentrated (12 mg/ml) laminin-containing gel, although lower than with collagen gels and saline. By prefilling silicone tubes bridging a 6-mm gap, a length considered limiting for regeneration in the mouse sciatic nerve, with diluted collagen or laminin gels, both matrices allowed for higher levels of recovery and for successful regeneration in a higher proportion of mice than saline solution. The laminin gel performed slightly better than the collagen gel.
The aim of this study was to evaluate long-term regenerative capacity over a 15-mm nerve gap of an autologous nerve conduit, the biogenic conduit (BC), 16 weeks after sciatic nerve transection in the rat.
A 19-mm long polyvinyl chloride (PVC) tube was implanted parallely to the sciatic nerve. After implantation, a connective tissue cover developed around the PVC-tube, the so-called BC. After removal of the PVC-tube the BCs filled with fibrin (n = 8) were compared to autologous nerve grafts (n = 8). Sciatic functional index (SFI) was evaluated every 4 weeks, histological evaluation was performed at 16 weeks postimplantation. Regenerating axons were visualized by retrograde labelling.
SFI revealed no significant differences. Nerve area and axon number in the BC group were significantly lower than in the autologous nerve group (P < 0.05; P < 0.01). Analysis of myelin formation showed no significant difference in both groups. Analysis of N-ratio revealed lower values in the BC group (P < 0.001).
This study reveals the suitability of BC for nerve gap bridging over a period of 16 weeks with functional recovery to comparable extent as the autologous nerve graft despite impaired histomorphometric parameters.
Fibrin is a promising matrix for use in promoting nerve repair given its natural occurrence in peripheral nerve injuries, and the biophysical properties of this matrix can be regulated to modulate tissue regeneration. In this study, we examined the effect of physical and mechanical properties of fibrin gels on dorsal root ganglia (DRG) neurite extension. Increases in fibrinogen concentration increased the number of fibrin strands, resulting in decreased pore size and increased stiffness. Neurite extension was reduced when DRG explants were cultured within fibrin gels of increasing fibrinogen concentrations (from 9.5 to 141 mg/mL). The addition of NaCl also increased the number of fibrin strands, reducing fiber diameter and porosity, while increasing mechanical strength, and reductions in neurite extension correlated with increases in NaCl content. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and matrix metalloproteinases by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction. Taken together, the results of this study provide new insight into the effect of fibrin gel biophysical properties on neurite extension and suggest new opportunities to improve the efficacy of these materials when used as nerve guidance conduits.
Biomaterial nerve cuffs are a clinical alternative to autografts and allografts as a means to repair segmental peripheral nerve defects. However, existing clinical biomaterial constructs lack true incorporation of physical guidance cues into their design. In both two- and three-dimensional systems, it is known that substrate geometry directly affects rates of axon migration. However, the ability to incorporate these cues into biomaterial scaffolds of sufficient porosity to promote robust nerve regeneration in three-dimensional systems is a challenge. We have developed fibrin constructs fabricated by a sacrificial templating approach, yielding scaffolds with multiple 10-250 μm diameter conduits depending on the diameter of the template fibers. The resulting scaffolds contained numerous, highly aligned conduits, had porosity of ∼ 80%, and showed mechanical properties comparable to native nerve (150-300 kPa Young's modulus). We studied the effects of the conduit diameters on the rate of axon migration through the scaffold to investigate if manipulation of this geometry could be used to ultimately promote more rapid bridging of the scaffold. All diameters studied led to axon migration, but in contrast to effects of fiber diameters in other systems, the rate of axon migration was independent of conduit diameter in these templated scaffolds. However, aligned conduits did support more rapid axon migration than non-aligned, tortuous controls.
Damage to the peripheral nervous system is surprisingly common and occurs primarily from trauma or a complication of surgery. Although recovery of nerve function occurs in many mild injuries, outcomes are often unsatisfactory following severe trauma. Nerve repair and regeneration presents unique clinical challenges and opportunities, and substantial contributions can be made through the informed application of biomedical engineering strategies. This article reviews the clinical presentations and classification of nerve injuries, in addition to the state of the art for surgical decision-making and repair strategies. This discussion presents specific challenges that must be addressed to realistically improve the treatment of nerve injuries and promote widespread recovery. In particular, nerve defects a few centimeters in length use a sensory nerve autograft as the standard technique; however, this approach is limited by the availability of donor nerve and comorbidity associated with additional surgery. Moreover, we currently have an inadequate ability to noninvasively assess the degree of nerve injury and to track axonal regeneration. As a result, wait-and-see surgical decisions can lead to undesirable and less successful "delayed" repair procedures. In this fight for time, degeneration of the distal nerve support structure and target progresses, ultimately blunting complete functional recovery. Thus, the most pressing challenges in peripheral nerve repair include the development of tissue-engineered nerve grafts that match or exceed the performance of autografts, the ability to noninvasively assess nerve damage and track axonal regeneration, and approaches to maintain the efficacy of the distal pathway and targets during the regenerative process. Biomedical engineering strategies can address these issues to substantially contribute at both the basic and applied levels, improving surgical management and functional recovery following severe peripheral nerve injury.
Entubulation of transected nerves using bioabsorbable conduits is a promising alternative to sural nerve autografting, but full functional recovery is rarely achieved. Numerous studies have suggested that scaffold-based conduit fillers may promote axon regeneration, but no neuroinductive biomaterial filler has been identified. We previously showed that a nerve guide filled with keratin hydrogel actively stimulates regeneration in a mouse model, and results in functional outcomes superior to empty conduits at early time points. The goal of the present study was to develop a peripheral nerve defect model in a rabbit and assess the effectiveness of a keratin hydrogel filler. Although repairs with keratin-filled conduits were not as consistently successful as autograft overall, the use of keratin resulted in a significant improvement in conduction delay compared to both empty conduits and autograft, as well as a significant improvement in amplitude recovery compared to empty conduits when measurable regeneration did occur. Taking into account all study animals (i.e., regenerated and nonregenerated), histological assessment showed that keratin-treated nerves had significantly greater myelin thickness than empty conduits. These data support the findings of our earlier study and suggest that keratin hydrogel fillers have the potential to be used clinically to improve conduit repair.
Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.
Using stopped flow light scattering, we show that assembly of fibrin following activation with non-rate-limiting amounts of thrombin or reptilase occurs in two steps, of which the first is end-to-end polymerization of fibrin monomers to protofibrils and the second is lateral association of protofibrils to fibers, in agreement with Ferry's original proposal. Polymerization is found to proceed as a bimolecular association of bifunctional monomers; the overall rate varies as the inverse first power of the concentration; end-to-end association of two monomers, of a monomer and an oligomer, and of two oligomers occurs with the same rate constant. The value of the rate constant is 8.2 C 10(5) M-1 s-1 in 0.5 M NaCl, is three times larger in 0.1 M NaCl (0.05 M Tris, pH 7.4), and is the same following activation by reptilase and by thrombin. The onset of growth of fibers from protofibrils takes 12 times longer in 0.5 than in 0.1 M salt, i.e. thick fibers ("coarse" gels) form from short protofibrils, and thin fibers ("fine" gels) form from longer protofibrils. Jumps of salt concentration at times when protofibrils, but not fibers, have formed result in immediate growth of thick fibers at low salt from long protofibrils formed at high salt. The rate of fiber growth in these experiments varies as the inverse first power of the concentration. 3the instant of gelation (formation of a network of fibers) falls in the later half of the time during which the scattering rises due to fiber growth; the rise of gel rigidity after gelation is found to continue beyond the end of this period. Jumps from low to high salt result in retention of whatever fibers have formed at low salt and a very small additional increase of the scattering due to further fiber growth at high salt. From a variety of evidence, we conclude that the properties of fibrin are determined by kinetics and not equilibria of assembly steps. Results obtained here agree with the following scheme of fibrin assembly: monomers polymerize to protofibrils; long protofibrils associate laterally to fibers; occasionally a long protofibril associates with two different fibers to form an interfiber connection; fiber growth does not reverse to yield stabler, more compact, structures and terminates in formation of a network of fibers. The typical delay of fiber growth is the time during which protofibrils form from monomers. Measurements at rate-limiting concentrations of thrombin have allowed estimation of turnover rates of fibrinopeptides that agree with kinetic parameters obtained with direct assay of fibrinopeptide. Release of fibrinopeptide B causes more rapid fiber formation. Addition of thrombin after activation by reptilase, at a time when protofibrils, but not fibers, have formed, is followed rapidly by fiber formation; this proves that thrombin readily removes fibrinopeptide B from protofibrils. On the basis of these new results and earlier work (in particular, Blombäck, B., Hessel, B., Hogg, D., and Therkildsen, L...
Cold-insoluble globulin (CI globulin) was purified from human plasma and identified on the basis of its sedimentation coefficient, electrophoretic mobility, and concentration in normal plasma. CI globulin was distinguished from antihemophilic factor (AHF) by amino acid analysis, position of elution from 4% agarose, and electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate without prior reduction. CI globulin and AHF could not be distinguished by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction and probably have very similar subunit molecular weights. CI globulin apparently consists of two polypeptide chains, each of molecular weight 2.0 x 10(5), held together by disulfide bonds. CI globulin was a substrate for activated fibrin-stabilizing factor (FSF, blood coagulation factor XIII). FSF catalyzed the incorporation of a fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, into CI globulin and also catalyzed the cross-linking of CI globulin into multimers, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction. In the presence of fibrin, cross-linking of CI globulin by FSF occurred without the formation of CI globulin multimers. Instead, polypeptides with apparent molecular weights of 2.6 x 10(5) and 3.0 x 10(5) were seen. The formation of these polypeptides coincided with the loss of the alpha chain of fibrin and CI globulin. The polypeptides were not seen when fibrin alone was cross-linked. The formation of the polypeptides was greater in fine clots than in coarse clots, and greater in clots incubated at 0 degrees than in clots incubated at 37 degrees. In clots made from purified fibrinogen, CI globulin, and FSF, the concentration of CI globulin in the clot liquor was greater if either FSF or calcium ion was omitted and cross-linking did not take place. These observations suggest that CI globulin is enzymically cross-linked to one of the chains of fibrin, most likely the alpha chain, and is thus covalently incorporated into the fibrin clot. CI globulin is very similar to a protein in the plasma membrane of fibroblasts. The cross-linking of CI globulin to itself and to fibrin may typify reactions also involving the fibroblast membrane protein.
Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.
It has been suggested that fibrinogen (fg) or its physiological derivatives influence the motility and growth of endothelial cells (ECs), but direct support for this concept is still lacking. In the present study, the capacity of fg to interact with ECs and induce the migration of ECs was examined. The capacity of fg to induce EC migration was studied by means of a modification of the Boyden chamber technique. fg in the lower compartment of the chamber caused a time- and concentration-dependent migration of ECs across filters. fg present in equal concentrations above and below the filter increased EC migration, but the maximal effect invariably occurred in the presence of a gradient between the lower and the upper compartments. Trypsin or plasmin digestion of fg and preincubation of fg with Fab fragments from specific antibody completely abolished fg-induced EC migration. Dialysis of fg to eliminate small peptides that might contaminate the preparation did not modify fg-induced migration. Plasma obtained from healthy donors induced EC migration, but plasma from an afibrinogenemic patient was completely ineffective. The addition of purified fg to afibrinogenemic plasma restored plasma-induced EC migration. Plasmin degradation fragments D and E, of 100,000 and 50,000 mol wt, respectively, did not induce EC migration. However, fragment E caused dose-related inhibition of fg-induced EC migration Direct interaction of highly purified radioiodinated human fg with cultured human and bovine Ecs was observed. The binding was time dependent and plateaued at 10 min. Nonlabeled fg in a large molar excess inhibited the interaction, but unrelated proteins, including fibronectin, ovalbumin, and myoglobin, did not. Monospecific Fab fragments directed to fg inhibited binding by 38% at a 50 to 1 molar ratio whereas nonimmune Fab caused only 2% inhibition at a similar concentration. The binding of 125I-fg with ECs was saturable, and an apparent dissociation constant of 0.23 x 10(-6) M was estimated from binding isotherms. After 30 min of incubation the interaction between 125I-fg and the cells was completely reversible and displaceable by a large molar excess of unlabeled fg. Autoradiography of the display of EC-bound 125I on polyacrylamide gel showed the constitutive B beta- and gamma-chains of the fg molecule, with a partial loss of the A alpha-chain. Purified fragment E and E were tested for their capacity to inhibit fg binding. At a 1 to 400 125I-fg-to-fragment molar ratio, fragment E, which also inhibited migration, competed for binding by 44%, but fragment D was completely ineffective. These data show that fg may specifically associate with ECs and induce migration of these cells; it also appears that the structural requirement of this activity is located in the N-terminal part of the molecule.
Addition of gamma-globulin, serum albumin, hemoglobin, or ovalbumin in concentrations of 1-10 g/dl to solutions of purified fibrinogen results in a substantial (up to six-fold) decrease in the lag time preceding appearance of a firm fibrin gel following addition of thrombin at 24 degrees C. The effect does not appear to be due to a protein-induced enhancement in the enzymatic activity of thrombin, nor does it appear to be due to the co-condensation of the added protein with fibrin/fibrinogen. It is suggested that the observed effect is primarily due to nonspecific volume exclusion arising from increased fractional occupancy of solution volume by macromolecules.
Brain cells and glioma cells in culture release a protein which induces neurite outgrowth in neuroblastoma cells. This neurite-promoting factor (NPF), which has been purified from serum-free glioma conditioned medium, has an apparent mol. wt. of 43 000. NPF inhibits urokinase as well as plasminogen activator-dependent caseinolysis or fibrinolysis. NPF and urokinase form an SDS-resistant complex. The fact that this glia-derived NPF is a potent protease inhibitor indicates that glial cells modulate the proteolytic activity associated with neuronal cells and suggests that this phenomenon is one of the biochemical events involved in the regulation of neurite growth.
During blood coagulation alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked with fibrin by an activated fibrin-stabilizing factor (FSFa) plasma transglutaminase, activated coagulation factor XIII). When alpha 2PI was treated with FSFa in the absence of acceptor amino groups, the inhibitor lost more than 90% of its capacity to be cross-linked to fibrin because of hydrolysis of the gamma-carboxamides of FSFa-susceptible glutamine residues. Chemical modifications of the inhibitor's lysine epsilon-amino groups did not affect the cross-linking capacity of the inhibitor with fibrin, whereas the same chemical modifications in fibrinogen resulted in a remarkable loss of cross-linking capacity. These observations suggest that alpha 2PI plays a role as an acyl donor with its FSFa-susceptible glutamine residues in the cross-linking reaction with fibrin, and fibrin serves as an acyl acceptor with its lysine residues. The number of FSFa-susceptible glutamine residues/molecule of the inhibitor was estimated by measuring the maximum incorporation of [3H]histamine into the inhibitor and by analyzing the distribution of radioactivity in a tryptic digest of [14C]histamine-incorporated alpha 2PI.l It was found that each inhibitor molecule has one glutamine residue that is most susceptible to FSFa. When the radioactive histamine-incorporated inhibitor was reacted with excess amounts of plasmin, a small fragment carrying all the released radioactivity was rapidly released from the NH2-terminal part of the inhibitor moiety of the complex. The NH2-terminal amino acid sequence of the inhibitor was analyzed before and after treatment with FSFa or before and after incorporation of radioactive histamine. The glutamine residue at the second position from the NH2-terminal end was converted to a glutamic acid residue when the inhibitor was treated with FSFa. When the radioactive histamine-incorporated inhibitor ws analyzed, the radioactivity was found predominantly at the second position from the NH2-terminal end. These results indicate that the glutamine residue susceptible to FSFa in alpha 2PI is located next to the NH2-terminal residue.
The secretion of the protease plasminogen activator (PA) by cells of developing peripheral nerve was demonstrated. Fetal and early postnatal dorsal root ganglia were established in culture as explants or as individual neurons and Schwann cells. A fibrin overlay assay was used to visualize the locations of PA secretion. Fibrinolytic zones formed around the somata of explants and were skewed in the direction of maximal fiber outgrowth. Individual growth cones at the tips of long fasiculated fiber bundles also released PA. Approximately 50% of individual neurons showed PA secretion; especially pronounced release occurred at some growth cones. Culture of nerve growth factor-independent adult neurons showed that PA expression was independent of effects of this growth hormone. A subpopulation of Schwann cells was also active in PA secretion, which could be detected at the soma, at the bipolar processes, or along the entire cell length. Possible functions of neural PA in development and regeneration are discussed.
Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5-10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor.
Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.
Highly porous collagen-glycosaminoglycan (CG) membranes have been useful in the treatment of skin injuries including full-thickness burns in humans. It has been recently shown that by seeding these CG membranes with autologous epidermal cells, large full-thickness wounds of guinea pigs can be closed in less than two weeks. Studies with similar CG polymeric materials were used to bridge gaps of 15 mm in the rat sciatic nerve. Silicone tubes 25 mm length were either filled with a porous CG polymer (test grafts) or were left empty (control grafts). The CG polymer used in the sciatic nerve study differed from that used for full-thickness skin wounds in that the pore structure was oriented parallel to the axis of the tube (rather than random) and the degree of crosslinking was decreased. These grafts were vascularized along their length and were encased in dense multiple wrappings of perineurial tissue.
Fibrin clots, freshly formed from recalcified plasma, displayed high susceptibility to lysis by the plasminogen activator, urokinase, or the proteolytic enzymes, plasmin or trypsin; later these clots became insoluble in 5 m urea or 1% monochloroacetic acid and developed increased resistance to enzymic lysis (5- to 10-fold that of the unstabilized clot). In contrast, clots formed from EDTA plasma and clotted with thrombin usually remained soluble in solvents capable of rupturing hydrogen bonds, and did not develop resistance to enzymic lysis. Transamidation inhibitors (glycine amide, methylester, and glycyl derivative) and sulfhydryl inhibitors (iodoacetamide, p-chloromercuribenzoic acid, etc.) inhibited fibrin stabilization and the development of clot resistance to enzymic lysis. Only limited correlation existed between conventional solubility measures of clot stabilization and clot resistance to enzymic lysis, the latter increasing after solubility criteria demonstrated fibrin stabilization. It is suggested that clot resistance to enzymic lysis may constitute an additional valuable measure and criterion of fibrin stabilization.
Fibronectin is a cell-surface and blood glycoprotein that apparently mediates adhesion of cells to the extracellular matrix. Malignant cells tend to lack cell surface fibronectin, and this may contribute to their capacity for invasive and metastatic growth. Early isolation methods for plasma fibronectin made use of its propensity to precipitate with fibrinogen and some other proteins when plasma is allowed to stand in the cold. A large fraction of the plasma cryoprecipitate is fibronectin, and it can be purified from such precipitate by using a combination of differential precipitation steps and ion exchange chromatography. Fibronectin from the surface of cultured fibroblasts can be isolated by extraction with low concentrations of urea. Antifibronectin columns can also be used to isolate fibronectin from both plasma and cell cultures. These methods have now largely been replaced by affinity chromatography on gelatin-Sepharose. The methods used in immunochemical quantitation of fibronectin include radioimmunoassay, enzyme immunoassay, and rocket immunoelectrophoresis. Of these methods, enzyme linked immunosorbent assay is probably the most useful one.
The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 μl. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefill-ing affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2,6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.
The presence of neuronotrophic factors (NTFs) in noninjured sciatic nerve extract and the course of their accumulation from 3 h to 30 days after nerve transection was examined. Rat sciatic nerves were transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers leaving a 10-mm interstump gap. Previous studies had shown that regeneration occurs in chambers containing both stumps but is absent in chambers lacking the distal stump. Chambers became completely filled with fluid 10 to 12 h after implantation. Fluid from chambers without nerve stumps (open-ended) implanted adjacent to nerve-containing chambers had markedly lower trophic activities than those containing one or both stumps. In fluid collected from chambers containing both proximal and distal nerve stumps, the highest titers of NTFs directed to sensory neurons were measured at 3 h posttransection whereas the highest titers of NTFs directed to sympathetic and spinal cord neurons were detected at 1 and 3 days, respectively. Chambers containing only the proximal or only the distal stumps showed similar temporal dynamics for sensory and sympathetic NTFs. Sensory and sympathetic neuronotrophic activity in extracts of proximal and distal stumps followed a similar temporal course to those in chamber fluid. Extracts of nonlesion nerve segments 5 mm from the transection site contained higher sensory and lower sympathetic trophic activity than extracts including the transection site. Spinal cord activity was undetectable in all extracts. Antiserum to nerve growth factor had no effect on fluid or extracts containing high sensory or sympathetic activities. These observations suggested that (i) some NTFs may be present in normal nerves and others may be synthesized or accumulated in response to nerve injury, (ii) sensory, sympathetic, and spinal cord NTFs are separate agents and immunochemically distinct from nerve growth factor, (iii) NTFs predominantly originate from nerve stumps rather than from surrounding fluid, and (iv) proximal and distal nerve stumps accumulate and release NTFs at similar rates.
The light-scattering method was applied to study the clotting of purified bovine fibrinogen. The molecular weight of fibrinogen was found to be 540,000 and the length 840 A.Fibrin dissolved in urea and guanidine gave the same molecular weight as fibrinogen under similar conditions.It was found that during clotting of fibrinogen larger and larger particles were built up through side-by-side and end-to-end associations of the apparently original fibrinogen particles. The relative importance of the two kinds of associations depends on pH and ionic strength. The arrest of the polymerization and a subsequent depolymerization of the polymerized particles revealed that the end-to-end associations are weaker than the side-by-side assocations.The clotting of fibrinogen by papain was found to be similar to the clotting brought about by thrombin.
Mass–length ratios of fibers in fibrin gels were obtained from measurements of the angular dependence of the intensity of light scattered by dilute gels and from the permeability of more concentrated gels. The permeability was determined by measuring the forced flow of buffer through a short column of gel held in a glass tube. The results obtained with the two methods are consistent. At high pH and high ionic strength the mass–length ratio is found to be that calculated for a protofibril, i.e., a double strand of fibrin molecules laid end to end at a separation of 450 Å. This same value is found under conditions where the polymerized fibrin is not gelled (pH 10.25) and where it is just gelled (pH 10.0). At pH 7.4, ionic strength 0.35, the fibers are found to have a higher mass–length ratio, the average fiber consisting of some three protofibrils associated laterally. At pH 7.4 and low ionic strength (0.10 M) the fibers are up to a hundred times more massive.
The effect of Factor XIII-induced crosslinking of fibrin on its subsequent lysis by plasmin has been further investigated due, in part, to conflicting reports in the literature on this issue. The test system used involved 125I labelled plasma fibrin clots and the release of the radiolabel to measure lysis rate. It was found that, while the fibrin γ-γ crosslinks do not affect the rate of fibrin lysis, the α chain crosslinkage has a significant effect. Indeed, the increased complexity of the α chain crosslinkage endows fibrin with increased resistance to lysis. 125I labelled fibrin clots containing fibrin with and without crosslinked α chains, allowed the preferential removal of the non-crosslinked material. Discrepancies were noted when measuring the lysis of crosslinked fibrin only by chemical means (Lowry method) and by 125I labelled peptide release. Both methods of measurement are prone to criticism; however, it was found that the 125I release method amply reflected the release of D dimer, the major soluble structural entity in crosslinked fibrin and probably the most reliable yardstick of its lysis.
The clotting of plasma in vertebrate blood centers around the fibrinogen molecule, a large protein (MW 340,000 in the human species) with a disulfide bridged doublet structure comprising three different constituent chains: (aβy)2. Another substrate, the so-called “cold insoluble globulin” (MW 400,000) may also be involved by helping to anchor the fibrin network to the surface of fibroblasts. The clotting of fibrinogen is a par excellence example of the enzyme-triggered higher protein associations, and it is controlled in a consecutive manner by two different enzymes: thrombin, a serine-OH protease, and fibrinoligase, a transamidase with a cysteine-SH active center. This chapter aims at indicating the interrelationship of components, in clotting and lysis in blood plasma. It also examines the cascade process of blood coagulation.
The elastic properties of fibrin gels prepared under a variety of conditions were studied using a Couette elastometer. Fibrin gels formed either by addition of thrombin to fibrinogen or by dilution of previously prepared fibrin monomer were seen to be dependent on calcium ion for the expression of maximum elasticity. By dialyzing a fibrinogen solution it was shown that two calcium ions per mole are tightly bound. Factor XIIIa was shown to have an effect on the elastic modulus by means of its crosslinking the α-chains to form α-polymer. γ-γ crosslinking caused no additional increase over calcium in elasticity of fibrin gels. The relative effects of calcium concentration on the rates of crosslinking of α-chains and γ-chains to form α-polymers and γ-γ dimers were determined by means of SDS gel electrophoresis in the presence of dithiothreitol. At calcium concentrations below 2×10−4 M α- and γ-chain crosslinking proceed at the same rate which depends strongly on the calcium concentration, while at higher concentrations the rate of α-crosslinking remains constant and the γ-chain crosslinking rate is dependent on calcium concentration. Factor XIIIa was shown also to lower the gel point of dilute solutions of fibrinogen clotted with thrombin in the presence of calcium. This demonstrates that Factor XIIIa promotes gelation in very dilute fibrin solution, probably by irreversibly crosslinking intermediate polymers of fibrin.
The regeneration of transected peripheral nerves of mice was studied using autoradiographical and electron microscopical techniques. In general, maximal proliferation occurred between the 5th and 7th day after dissection and stopped when the cells emigrating from the proximal and distal stumps of the nerve started to contact one another. Special attention was paid to the reaction of the connective tissue cells of the endo-, epi- and perineurium. The perineurial cells seemed to dedifferentiate between the 3rd and 5th day after the transection and then started to proliferate into the defect. Labelled perineurial cells were completely absent, when the minifascicles were fully developed in the neuroma. The epineurial fibroblasts started to proliferate during the 1st day. Even 6 weeks after transection the multiplication rate was about ten fold that of the controls. The results are discussed with special reference to clinical nerve repair.
A glia-derived neurite promoting factor (GdNPF) has serine protease inhibitory activity and in addition regulates the migration of neuronal cells. cDNA cloning of GdNPF is necessary for studying the physiological relevance and the mode of action of this protein and similar cell-derived protease inhibitors. Xenopus oocytes injected with rat glioma cells mRNA release this inhibitor. A rat cDNA clone coding for the previously purified glia-derived neurite promoting factor (GdNPF) was isolated upon hybridization-selected translation, followed by immunoprecipitation. The correct identity of this cDNA is proven by the presence of a sequence coding for a tryptic fragment from pure GdNPF. Northern analysis indicates that GdNPF mRNA is found almost exclusively in brain tissue and could be developmentally regulated. The same cDNA clone has been used to isolate full-length rat and human GdNPF cDNA. The deduced human GdNPF amino acid sequence indicates that the protein is a member of a family of cell-derived protease inhibitors named protease nexins.
Cultures of neurons from neonatal rat superior cervical, dorsal root, and trigeminal ganglia were grown in the absence of nonneuronal cells in serum-free defined medium. Proteins metabolically labeled with radioactive amino acids and spontaneously released into the culture medium were studied using two-dimensional gel electrophoresis and photofluorography. All three populations of neurons released 12-15 major proteins into the culture medium. Four proteins were released selectively by sympathetic neurons and two proteins were consistently released by both populations of sensory neurons but not by sympathetic neurons. Enzymatic activities are associated with at least two of the released proteins. One is a calcium-dependent metalloprotease, and the other a plasminogen activator. The calcium-dependent metalloprotease has a MW of 62 kDa, requires millimolar calcium for maximum activity, and has a restricted substrate specificity. It degraded native and denatured collagen more readily than casein, albumin, or fibronectin and denatured collagen (gelatin) was a better substrate than native collagen. The plasminogen activator released by neurons has a MW of 51 kDa and is converted to an active 32 kDa form. Its physiochemical properties are similar to urokinase and it was precipitated by a rabbit antiserum produced against human urokinase. A large fraction of both proteases was released by distal processes and/or growth cones suggesting that these proteases could be involved in growth cone functions.
Clot lysis induced by tissue plasminogen activator and urokinase has been studied and the influences of pH, ionic environment and reactant concentrations have been determined. Both pH and ionic strength strongly affect the rate of clot lysis and distinctly different dependency profiles are obtained for tissue plasminogen activator and urokinase. Variations in concentration of plasminogen also profoundly affect the rate of clot lysis, maxima being obtained at different plasminogen concentrations depending on the concentration of fibrinogen. For urokinase, these maxima occurred at about a tenfold higher concentration of plasminogen than for the tissue plasminogen activator. The lysis times are directly dependent on the concentration of fibrinogen. Variation in thrombin concentration did not significantly affect the lysis times. Suitable conditions for the assays of tissue plasminogen activator and urokinase are suggested.
Networks were developed in fibrinogen solution under pathophysiological conditions of clotting and their permeability and turbidity were measured. Mass-Length ratio of fibrin fibres (a measure of fibre thickness) was derived from two independent methods based on turbidity (mu T) and permeability (mu P). Both mu T and mu P increased as thrombin concentration was decreased. Data are in agreement with the concept that the fibrin network develops initially as a mesh of protofibrils. Fibrin monomer generated subsequently is incorporated into the existing network making individual fibres thicker. Consequently, slow fibrin monomer generation promotes thicker fibres. When the concentration of thrombin or fibrinogen was altered systematically, mu T and mup were found to mirror each other except when the fibrinogen concentration was increased at low thrombin concentrations. This breakdown between the two derivations of mass-length ratio is discussed in terms of the two network model.
One pathway of blood coagulation, the extrinsic system, is initiated by a specific interaction between tissue factor, which is a membrane lipoprotein, and factor VII, one of the plasma coagulation factors. Factor VII was prepared from bovine plasma by adsorption onto and elution from BaSO(4). The eluate was chromatographed on DEAE-Sephadex and purified by preparative disc-gel electrophoresis. Factor VII complexed with purified bovine-brain tissue factor and, when eluted from the complex, factor VII had a greater mobility in acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate, i.e., it had a reduced molecular weight. Factor VII was also cleaved in the presence of orthophenanthroline, an inhibitor of the peptidase activity of tissue factor. Prior treatment of factor VII with diisopropylphosphorofluoridate, however, completely blocked its cleavage and the development of coagulant activity, although factor VII treated with diisopropylphosphorofluoridates complexed equally well as the native protein with tissue factor. Factor VII in whole bovine plasma was also inhibited by the drug. Factor VII labeled with [(32)P]diisopropylphosphorofluoridate and radioautographed after electrophoresis in gels showed two major components, only one of which was labeled. We conclude that tissue factor initiates blood coagulation by facilitating a proteolytic attack within the factor VII "complex." The most likely mechanism is proteolysis of one form of factor VII by the diisopropylphosphorofluoridate-sensitive enzyme, although additional intramolecular proteolysis may be involved.
Liquid permeation studies of fibrin gels, which were formed in the presence of thrombin (Fibrin II) and in the presence of Batroxobin (Fibrin I), showed that those gels have distinctly different flow properties. For both gels the permeability coefficient (Ks) and average pore size varied depending on the conditions for gel formation. Low pH and ionic strength favour high Ks and large pore sizes, whereas high pH and ionic strength produce gels with low Ks and small pore sizes. Parallel turbidity studies showed correlations between the optical properties of the gels and permeation data. Of particular importance was the finding that the clotting time (Ct) is directly related to Ks of the final gels. Thus events preceding gel formation determine the gel structure. It is proposed that the average lengths of polymers formed prior to gelatin varies directly with Ct. Shorter polymers give infinite net works (gels) which are tight and longer polymers provide for formation of infinite net works which are more porous. At the same Ct, the net work formed with thrombin is, over a wide range of Ct, tighter than that formed with Batroxobin. It was also found that Ks is inversely related to the fibrin concentration (C) in the gels. The ratio Ct/C (permeability index) is thus an important determinant for the gel structure. The applicability of the permeability index to whole blood as a predictive test for thromboembolic and bleeding conditions is discussed.
To test the validity of a proposed two step fibrin assembly mechanism and directly visualize the molecular species present at various stages of fibrin formation, we have carried out an electron microscopic investigation. Assembly conditions duplicated those of a recent light scattering study and specimens were prepared at different time points with the use of a negative staining technique recently employed to visualize the trinodular structure of fibrinogen. Under near-physiological buffer conditions, protofibrils structurally similar to those postulated by Ferry have been found at early stages of fibrin assembly. In parallel with the light scattering results, a dramatic increase in fiber diameter was found in specimens prepared during the postulated lateral association stage of gelation. Light scattering and electron microscopic results both showed that high ionic strength reduces the rate and extent of fiber formation. Reptilase cleavage is shown to result in typical cross striated fibrin.
Neuroontogenesis results from a synchronized series of elementary events including cellular proliferation, migration, differentiation, recognition and death. Neuronal migration is a key step in neural morphogenesis since inadequately located neurones may not establish the appropriate connections and this may lead to neuronal death or to functional deficit of synaptic circuits. Impairment of neuronal migration has been implicated in human pathology1 and well documented in animal pathology, such as the weaver mutation in mice2. The cerebellum of small rodents is particularly well suited for the study of neuronal migration because a subpial neuronogenesis occurs postnatally. The subsequent inward migration of postmitotic neurones (mostly granule cells) has been studied using classical neuroanatomical methods, such as the Golgi stain3, and autoradiography after systemic injection of 3H-thymidine4. Systematic ultrastructural investigation of neuronal migration at different levels of the central nervous system and in different species led Rakic to propose the radial glia hypothesis-that neurones migrate along radial glia cells which serve as guides during migration5. The observation that neurones migrate inside a densely packed neuropile has prompted us to consider the possible role of extracellular neutral proteolysis during neuronal migration. We have focused on the plasminogen activator (PA) serine proteases because these enzymes are known to be involved in several phenomena that involve cell migration or tissue remodelling6. The usual substrate for PA is plasminogen, which is converted to plasmin, although other substrates may exist7. We report here that both PA and plasmin are released by cultived 7-day-old rat paraflocculus, but not by 1-month-old or adult rat paraflocculus (that is, after granule cell migration), and that inward migration of cerebellar granule neurones, which account for 95% of the cells in adult cerebellum, can be inhibited by inhibitors of the PA-plasmin system.
LamininPlasminogenPlasminogen activatorProteolysis
The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)
The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.
The role of the serum proteolytic system plasminogen/plasminogen activator as a biochemical tool used by the glia or neurons, or both, for maintaining their temporary and flexible cellular interactions during histogenesis of the nervous system is under study. The present report identifies a glia cell type, the Schwann cell, as one of the cellular components of the nervous system that uses extracellular proteolysis at the time of the tissue construction. Purified dividing mouse Schwann cells in culture produce extracellular plasminogen activator. The levels of extracellular plasminogen-activator activity, as measured by the biochemical fibrinolytic assay, were directly related to the proliferation rates of the Schwann cells. The cellular plasminogen-activator specific activity at the maximal rate of cell proliferation was 3-4 times higher than that of the cells at low rate of mitosis. It is concluded that plasminogen-activator activity is expressed predominantly by the proliferating Schwann cell populations, suggesting that the extracellular proteolysis is used by the tissue at those stages when the cells divide.
The spatial-temporal progress of peripheral nerve regeneration across a 10-mm gap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7-14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1-5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.
A new peripheral nerve forms across a 10 mm gap within a silicone chamber regeneration model when the distal segment of a transected sciatic nerve, connected to its end organs, is sutured into the distal end of the chamber. We have tested the ability of other tissue inserts to support axonal regeneration in the chamber. When an isolated 2 mm piece of sciatic nerve was sutured into the distal end, fibrin matrix formation, cell immigration and axonal regeneration were identical to those occurring in the control. When the distal nerve insert was replaced with a 2 mm piece of skin or a ligation, a matrix did not form and subsequent cell immigration and axonal regeneration did not occur. When a 2 mm piece of tendon was inserted, a matrix did form at 1 week, but a structure across the gap was observed at later time periods in only 2 out of 7 chambers. The matrix either dissolved before cells could enter the chamber or did not promote cellular immigration and subsequent axonal regeneration. When the distal end was left open, a matrix formed and cells from the reactive tissue outside the chamber entered the matrix and formed a granulation tissue bridge across the gap. This tissue failed to support axonal regeneration; at 3 weeks, axons stopped 1 mm beyond the proximal stump at the interface with the granulation tissue. Thus, matrix formation and a cellular bridge are necessary but not sufficient to ensure regeneration. Successful regeneration across the silicone chamber gap requires humoral and/or cellular contributions available from peripheral nervous tissue and not from the other tested tissues.
The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.
Endothelial cells are known to migrate and come into contact with fibrin during numerous physiological processes, such as in wound healing and in tumor growth. The present study was initiated to investigate the effect of fibrin on endothelial cell migration in vitro. Endothelial cell migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Wound-induced proliferation of endothelial cells was inhibited by mitomycin C-treatment without affecting endothelial cell migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro was inhibited by fibrin in a concentration dependent manner. Endothelial cell migration under fibrin was further reduced by plasminogen depletion of the serum, and fibrin still inhibited the migration of mitomycin C-treated endothelial cells. Kadish et al. (Tissue and Cell, 11, 99, 1979) previously reported that fibrin did not affect EC migration in vitro. The inability to inhibit EC migration with fibrin appears to be due to their assay system which employed agarose, since pre-treating the wounded monolayer with agarose eliminated the inhibition of EC migration by fibrin. The present results indicate that EC migration in vitro can be used as a model system for studying the interaction of fibrin with EC.
When blood is clotted, alpha(2)-plasmin inhibitor (alpha(2)PI) is cross-linked to fibrin by activated fibrin-stabilizing factor (activated coagulation Factor XIII, plasma transglutaminase). The amount of cross-linked alpha(2)-PI is proportional to the amount of alpha(2)PI present at the time of clotting. Plasma from a patient with congenital deficiency of alpha(2)PI was supplemented with various amounts of purified alpha(2)PI. Clots were prepared from these plasmas and were suspended in plasma containing a normal concentration of alpha(2)PI, and spontaneous clot lysis was observed. When the clot was formed in the presence of calcium ions and thereby allowing cross-linking to occur, the rate and extent of fibrinolysis were found to be inversely proportional to the concentrations of alpha(2)PI present in the clot at the time of clotting. When the clot was formed in the absence of calcium ions so that no cross-linking occurred, the clot underwent fibrinolysis at similar rates, regardless of the concentrations of alpha(2)PI in the clot. When the clot formed in the presence of calcium ions was squeezed and washed to remove unbound proteins before being suspended in plasma, the extent of fibrinolysis was also inversely proportional to the amount of alpha(2)PI cross-linked to fibrin. Similar results were obtained when the clot was suspended in buffered saline instead of plasma. These observations suggest that spontaneous fibrinolysis is mainly carried out by plasminogen/plasminogen activator bound to fibrin, and this fibrinolysis caused by fibrin-associated activation of plasminogen was mainly inhibited by alpha(2)PI cross-linked to fibrin. To further support this concept, alpha(2)PI treated with activated fibrin-stabilizing factor and that had lost most of its cross-linking capacity was used in similar experiments. This modified alpha(2)PI had the same inhibitory activity on plasmin as the native inhibitor, but gave significantly less inhibition of fibrinolysis in every experiment, particularly when the clot was compacted by platelet-mediated clot retraction or by squeezing. Thus, it was concluded that alpha(2)PI cross-linked to fibrin plays a significant role in inhibition of physiologically occurring fibrinolysis. It is further suggested that the absence of cross-linked alpha(2)PI contributes to accelerated fibrinolysis and hemorrhagic tendency in patients with congenital deficiency of fibrin-stabilizing factor.
Shear moduli and creep compliances have been measured for four types of clots of human fibrin (about 7 mg/ml) clotted with and without human plasma fibronectin (usually 1.2 mg/ml). Fine clots (with little lateral aggregation of the fibrin protofibrils) were found at pH 8.5, ionic strength 0.45; coarse clots (with substantial lateral aggregation) were formed at pH 7.5, ionic strength 0.15; in both cases with and without ligation by fibrinoligase. In fine clots, the addition of fibronectin without ligation scarcely affected the shear modulus; with ligation, the modulus was decreased by a factor of 0.48. In coarse clots, the shear modulus was increased by addition of fibronectin. The increase was by a factor of 2.0 without ligation and by a factor of 2.4 with ligation. Creep and creep recovery in clots formed with and without fibronectin were similar except for the scale factor represented by the change in modulus.
We carried out experiments to determine conditions for fibroblast adhesion to fibrinogen and fibrin substrata. Baby hamster kidney (BHK) cells did not attach to substrata composed of purified fibrinogen or fibrin. When cold-insoluble globulin (CIG) (plasma fibronectin) was bound to fibrinogen or fibrin substrata, adhesion of BHK sells was observed and the extent of adhesion was dependent upon the CIG conecntration. Binding of CIG to fibrinogen or fibrin substrata in the presence of Factor XIII (factor) under covalent crosslinking conditions resulted in a marked increased in the ability of the substrata to support cell adhesion. Control experiments indicated that CIG formed the sites on the fibrinogen and fibrin substrata to which the cells were attaching. In addition, the effect of factor XIII was shown to require covalent crossliking of CIG to the fibrinogen or fibrin, which involved a glutamine residue on the CIG molecule and could be prevented by prior crosslinking of CIG with putrescine or with itself. The enhanced ability of Factor XIII-crosslinked CIG substrata to support cell adhesion could not be accounted for by the absolute amount of CIG bound to the substrata. We present in this paper the possibility that the orientation of CIG on the substrata is the critical factor.
Competence of nerve tissue as distal insert pro-293
- L R Williams
- H C Powell
- G Lundborg
- S Varon
Williams, L. R., Powell, H. C., Lundborg, G., and Varon, S. 1984. Competence of nerve tissue as distal insert pro-293:201-211.
Fibrinogen and Fibrin. Pages 163?191
- R F Doolittle
- R. F. Doolittle
Modification of the microenvironment allows axonal regeneration across a 20 mm nerve gap using entubulation repair
- R Madison
- C F Da Silva
- P Dikkes
- R. Madison