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(The FASEB Journal. 1999;13:2214-2224.)
© 1999 FASEB

Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering

SHELLY E. SAKIYAMA*,{dagger}, JASON C. SCHENSE*,{dagger} and JEFFREY A. HUBBELL*1

* Department of Materials and Institute for Biomedical Engineering, ETH-Zurich and University of Zurich, Zurich, Switzerland; and
{dagger} Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA

1Correspondence: Institute for Biomedical Engineering, Moussonstrasse 18, CH-8044 Zurich, Switzerland. E-mail: hubbell{at}biomed.mat.ethz.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


Key Words: antithrombin III • neural cell adhesion molecule • platelet factor 4 • regeneration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DESPITE RECENT ADVANCES in the understanding of nerve regeneration and in surgical techniques, complete recovery of function in a damaged nerve is rare. The most common method of nerve repair is to directly suture the two severed nerve ends together; however, this is often not feasible if lacerations are present or if the gap between the two ends is too large and thus requires excessive tension to reconnect the two ends. In these cases, a nerve autograft is usually performed. The autograft provides a scaffold for the regenerating nerve and guides it to its proper target. This procedure has its limitations due to the defects created at the donor site for the autograft. Both of these procedures—direct anastomosis and nerve autografting—have poor results (1) .

One alternative method to autografting is to use nerve guide tubes to connect the proximal and distal ends of the severed nerve and guide the regeneration of axons back to the appropriate target. These tubes allow the micro-environment of regeneration to be controlled by manipulation of the contents of the nerve guide tube. 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. Aebischer et al. (4) have studied the effects of tube surface microgeometry on nerve regeneration and have found that the inner surface of tubes must be smooth for a longitudinally oriented fibrin matrix to form. In this study, we sought to modify fibrin, the natural biomaterial of nerve regeneration, by covalently incorporating exogenous adhesion peptides to enhance its ability to promote nerve regeneration. By such means we would hope to supply signals found in the extracellular matrix and on cell surfaces within the nerve bundle in order to endow the fibrin matrix with some of the characteristics of the natural micro-environment of the nerve.

Cell adhesion is mediated by many different types of interactions between cell-surface receptors and ligands in the extracellular matrix or on the surface of other cells. Neuronal cell attachment to substrates can occur via cell-surface receptor binding extracellular matrix proteins, including to receptor binding sequences found in proteins such as laminin and fibronectin. For example, several domains in laminin have been shown to support neuron adhesion: YIGSR of the laminin ß1 chain (5 6 7 8) , LRGDN of the laminin {alpha} (9 10 11) , SIKVAV of the laminin {alpha} chain (12) , and RNIAEIIKDI of the laminin {gamma} chain (13) . Neuronal adhesion can also occur through homophilic interactions between cell–cell adhesion molecules on the surfaces of adjacent cells, mediated through proteins such as neural cell adhesion molecule (NCAM) and N-cadherin. For example, the LRAHAVS sequence from N-cadherin has been shown to potentate neurite outgrowth by binding to N-cadherin (14) .

Another class of adhesion domains are the heparin-binding domains, which possess the ability to bind heparin and other sulfated glycosaminoglycans, typically components of cell-surface and extracellular matrix proteoglycans. It is with the role of heparin-binding domain peptides in promoting neurite extension, and possibly nerve regeneration, that this paper is concerned. Heparin-binding domains are found in a variety of proteins of different types including laminin, fibronectin, NCAM, midkine, heparin-binding growth-associated molecule (HB-GAM), and fibrinogen (15 16 17 18 19 20 21) . These domains can interact with cell-surface proteoglycans to promote cell adhesion and, in the case of neurons, to promote neurite extension. Heparin-binding domains have been found to enhance neurite extension from both peripheral and central neurons when studied as proteolytic cleavage fragments or peptides adsorbed to surfaces (15 , 16 , 22 , 23) . Previous work with the NCAM heparin-binding domain has suggested that membrane-bound proteoglycans, rather than soluble proteoglycans, mediate adhesion to heparin-binding domains (24) . The ubiquitous presence of heparin-binding domains in a variety of extracellular matrix and cell attachment proteins, as well as their general bioactivity in promoting neurite extension, suggests a potential role in both neuronal development and regeneration (22) .

Thus far, all work studying the effects of heparin-binding domains has focused on 2-dimensional neurite extension. However, in vivo neurite extension occurs in three dimensions, in which numerous phenomena compete and sometimes counteract. This distinction can be quite important. For example, inhibition of neurite-associated protease activity can have opposite effects in 3- vs. 2-dimensional cultures, decreasing neurite extension in 3-dimensional culture but increasing it in 2-dimensional cultures (25) . In the present study, 3-dimensional culture was performed within fibrin gels. A novel method for covalently attaching exogenous peptides to fibrin gels using the coagulation transglutaminase factor XIIIa has recently been developed (26) . In this method, a bi-domain peptide chimera is enzymatically coupled into a fibrin gel during coagulation, the amino-terminal domain of this peptide chimera being a factor XIIIa substrate and the carboxyl-terminal domain being a candidate peptide domain of study. This method allows the effects of bioactive factors, such as heparin-binding peptides, to be studied in 3-dimensional models of neurite extension that are realistic for nerve regeneration.

The experiments described below demonstrate the potent influence of heparin-binding domains on 3-dimensional neurite extension. The influence of heparin-binding affinity is also demonstrated—stronger binding peptides leading to more extensive neurite extension. Finally, the materials investigated in this study may be therapeutically useful in promoting peripheral nerve regeneration after transection due to trauma or surgery, providing within the fibrin matrix a biomimetic micro-environment using adhesion and signaling domains from nonfibrin proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All materials were obtained from Fluka (Buchs, Switzerland) unless otherwise specified.

Synthesis of peptides
Five bi-domain peptide chimeras were synthesized containing a heparin-binding domain at the carboxy terminus and the factor XIIIa substrate domain from {alpha}2-plasmin inhibitor at the amino terminus. The sequence of the factor XIIIa domain used was NQEQVSP (27) . The sequences of five bi-domain peptide chimeras are shown in Table 1 . For clarity, the heparin-binding domains are shown in italics. In the first peptide, denoted {alpha}2PI1–7-ATIII121–134, the peptide contains the functional factor XIIIa substrate, NQEQVSP, in its amino-terminal domain. The carboxy-terminal domain contains a modified sequence from the heparin-binding domain of antithrombin III (ATIII) (28) , as shown in Table 1 . A dansylated leucine residue at the amino terminus was used as a label to determine the extent of incorporation of the peptide chimera into the fibrin gel during coagulation (see below). The second peptide, denoted {alpha}2PI1–7, Q2->G-ATIII121–134, is similar but contains a nonfunctional factor XIIIa substrate obtained by substitution of glycine for glutamine in the transglutaminase cross-linking site. The third peptide, denoted {alpha}2PI1–7-NCAM133–146, contains a functional factor XIIIa substrate and the heparin-binding domain from NCAM (24) . The fourth peptide, denoted as {alpha}2PI1–7-PF460–67, contains a functional factor XIIIa substrate and the heparin-binding domain from platelet factor 4 (PF4) (29) . The fifth peptide, denoted as {alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R contains a functional factor XIIIa substrate and a modified heparin-binding domain from ATIII, where all the lysine residues in the Tyler-Cross domain have been replaced with arginine residues.


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  		Table 1. Sequences and notation for bi-domain peptide chimeras containing heparin-binding domains

Peptides were synthesized on solid amide resin (NovaSyn TGR, Novabiochem, Laüfelfingen, Switzerland) using an automated peptide synthesizer (Pioneer peptide synthesizer, PerSeptive Biosystems, Framingham, Mass.) with standard 9-fluorenyl-methyloxycarbonyl chemistry (30) . Peptides used in cross-linking quantification were labeled with a fluorescent probe by placing an {alpha}-dansyl leucine at the amino terminus of the sequence. All solvents for peptide synthesis were obtained from Paul Bucher Company (Basel, Switzerland). All other reagents for peptide synthesis were obtained from Novabiochem, unless noted. Peptides were cleaved using 88% trifluoroacetic acid (Paul Bucher Co.), 0.5% phenol, 0.5% water, and 0.2% triisopropylsilane for 2–3 h. After cleavage, the peptides were precipitated into 10 volumes of cold ethyl ether; the precipitate was recovered by filtration and washed twice with ethyl ether to remove hydrophobic protecting groups and scavengers. The peptides were dried for 4 h under vacuum and dissolved into 20 ml of deionized water. They were dialyzed against 4 l of deionized water for 24 h and lyophilized. Peptides were dissolved in Tris-buffered saline (TBS) (33 mM Tris, 8 g/l NaCl, 0.2 g/l KCl, pH 7.4) at a concentration of 0.01 M and syringe filtered (0.22 µm) prior to use.

Quantification of peptide incorporation
The amount of peptide cross-linked into fibrin gels through the action of factor XIIIa was determined by size exclusion chromatography, using methods exactly as previously reported (26) . Briefly, the peptide to be incorporated was added to the coagulation mixture and cross-linked to the fibrin gel during polymerization. Unincorporated peptide was washed from the gels with TBS and the gels were degraded with a minimal amount of plasmin. The degraded gel fragments were analyzed by size exclusion chromatography to determine whether the peptide was cross-linked to fibrinogen fragments or was present as free peptide. A decrease in the elution time of the fluorescence signal, indicative of an increase in molecular weight, would demonstrate coupling of the peptide to the fibrin gel. As controls, the peptide was analyzed by size exclusion chromatography (both pure and as added to the plasmin-degraded fibrin) to demonstrate that this apparent increase in molecular weight could only be explained by covalent binding of the chimeric peptide to the fibrin gel. The effect of heparin on peptide incorporation was analyzed by including heparin and peptide in the polymerization mixture, degrading the gels with plasmin and running the degradation products on high-performance liquid chromatography, as described.

Preparation of fibrin gels
Fibrinogen solutions were prepared as described previously, using plasminogen-free fibrinogen from pooled human plasma (26) . Dorsal root ganglia (DRGs) were dissected from day 8 White Leghorn chicken embryos (31) and placed in Hanks-buffered salt solution (HBSS) (Life Technologies, Inc., Basel, Switzerland). The DRGs were pipetted into the bottom of flat 24-well tissue culture plates (1 per well) and fibrin gels were polymerized around the ganglia so that they were 3-dimensionally embedded within the gel. Fibrin gels (400 µl per well) were made by mixing the components to obtain the following final solution concentrations: 3.5 mg/ml fibrinogen, 2.5 mM Ca2+, 2 NIH units/ml of thrombin, 0.25 mM peptide or 0.125 mM peptide (to obtain 8 mol of cross-linked peptide per mole fibrinogen or 4 mol of cross-linked peptide per mole fibrinogen, respectively), and 0.125 mM heparin (sodium salt from porcine intestinal mucosa, 176 USP U/mg, when used). The polymerization mixture was incubated for 60 min at 37°C, 95% relative humidity, and 5% CO2.

DRG culture and analysis
After polymerization to form the fibrin gel, 1 ml of modified neural basal medium was added to each well, consisting of insulin (5 µg/ml), transferrin (100 µg/ml), progesterone (6.4 ng/ml), putrescine (16.11 µg/ml), selenite (5.2 ng/ml) (all from Life Technologies, Inc.), 5 µg/ml fibronectin, 0.1% bovine serum albumin, 20 ng/ml mouse nerve growth factor, 0.5 mM L-glutamine, 25 µM L-glutamate (all from Fluka), and 1% antibiotic-anti-mycotic solution (Life Technologies, Inc.) added to neural basal medium (Life Technologies, Inc.). The medium was changed at 3, 6, 9, and 24 h to wash out uncross-linked peptide. DRGs were cultured within fibrin gels without peptide, and all measurements of neurite extension were normalized with respect to this level.

Enzymatic removal of glycosaminoglycans from cell surfaces was performed using heparitinase or chondroitinase ABC. Upon dissection, DRGs were incubated in HBSS containing 0.1 NIH units/ml of either heparitinase or chondroitinase ABC (Fluka). Culture medium for these ganglia contained 0.1 NIH units/ml of either heparitinase or chondroitinase ABC in the modified neural basal medium described above.

Bright-field images of the ganglia were taken at 24 and 48 h with 4.0x Achroplan (24 h) and 2.5x Plan (48 h) objectives (Zeiss, Zurich, Switzerland). The images were digitized with a Shimatsu chilled color 3-chip CCD camera, Matrox Meteor PCI frame grabber (Matrox Electronic Systems, Dorval, Quebec, Canada), and Leica Qwin software (both from Leica, Zurich, Switzerland). These images were then analyzed to determine the average length of neurite extension, which was calculated as the radius of an annulus between the DRG body and the outer halo of extending neurites, as described previously (25) . Neurite length for each experiment was normalized by the average neurite extension through unmodified fibrin gels from the same experiment at the same time point.

Confocal scanning laser microscopy of DRGs was performed at 48 h with a 10x/0.30 Plan Neofluor objective (Zeiss) using a MRC 600 confocal system (Bio-Rad, Glattbrugg, Switzerland). Two hundred microliter fibrin gels containing DRG were polymerized in the center of a 35 mm petri dish to allow imaging of the sample from above. DRGs were cultured as usual except that 2 ml of media per sample was used in order to cover the gel completely. Samples were stained at 48 h prior to imaging by adding 4 µl of a 5 mg/ml stock solution of fluorescein diacetate in acetone to 2 ml of TBS. Samples were placed in the incubator for 5 min and then washed with TBS prior to imaging. Approximately 50 to 100 images were taken at 7–10 µm intervals and a composite image was assembled using Imaris (Bitplane, Zurich, Switzerland) image processing software on an Indigo2 extreme Silicon Graphics Workstation (Silicon Graphics, Mountain View, Calif.).

Heparin-affinity chromatography
The relative affinity for heparin of the heparin-binding peptides used in these studies was determined by heparin-affinity chromatography, using a TSK-GEL Heparin-5PW (7.5 cm x 7.5 mm ID) column (TosoHass, Stuttgart, Germany). Samples of the bi-domain peptides were injected in 20 mM Tris, pH 7.4, 0.05 M NaCl. Elution was accomplished by running a gradient of NaCl from 0.05 M to 2.0 M over 40 min; the NaCl concentration at which elution was observed was taken as a measure of the heparin-binding affinity of the peptide.

Statistics
Statistical analysis was performed using Statview 4.5 (Abacus Concepts, Berkeley, Calif.). Comparative analyses were completed using the Scheffe’s F post-hoc test by analysis of variance at a 95% confidence level. Mean values and standard error of the mean are reported.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Quantification of peptide incorporation
Size exclusion chromatography was used to determine the amount of peptide cross-linked into fibrin gels using the previously developed incorporation method (Fig. 1 ). A bi-domain peptide ({alpha}2PI1–7-ATIII121–134) containing the factor XIIIa substrate from {alpha}2-plasmin inhibitor, the heparin-binding domain from ATIII, and a fluorescent label was incorporated into fibrin gels during coagulation. The free peptide was washed from the gels (as determined by measuring fluorescence in the wash fluids) and the fibrin gels were degraded with plasmin. The degradation products were analyzed by size exclusion high-performance liquid chromatography to determine the amount of peptide (by fluorescence from the dansyl label) incorporated per mole of fibrinogen (by UV absorbance). The fluorescence signal from peptide-modified gels appeared at an earlier elution time (24–36 min) than did the signal from free peptide added to the degraded fibrin or chromatographed alone (45 min), indicating that all peptide present in the modified gels was covalently attached to protein in the degraded fibrin and as such had been present cross-linked to the fibrin gel (Fig. 1) . When additional peptide was added free to degraded fibrin gel that also contained enzymatically incorporated peptide, the region of the peak corresponding to free peptide was observed to split, indicating the possibility of physiochemical interaction between the free peptide and the fibrin degradation products. This eluted at a much later time than did the covalently incorporated peptide, demonstrating unequivocally that the peptide eluting at times earlier than 40 min in the samples with enzymatic incorporation was indeed covalently coupled to the fibrin gel. Quantification based on standards of known concentration for both peptide and fibrin gels degraded with plasmin showed incorporation of 8.7 ± 0.2 mol of peptide per mole of fibrinogen (n=10), which is in close agreement with previously published results for a peptide containing the same factor XIIIa substrate domain but a vastly different carboxy-terminal sequence (26) .



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  		Figure 1. Fluorescence detection chromatograms of plasmin-degraded peptide-containing fibrin gels and free peptide. Size exclusion chromatography of a degraded fibrin gel with the {alpha}2PI1–7-ATIII121–134 peptide dLNQEQVSPK(ßA)FAKLAARLYRKA-NH2 incorporated (-) and with the same peptide free added to the degraded fibrin gel containing incorporated peptide (... . ), and free peptide alone (- -) are shown. The amino-terminal leucine residue was dansylated (abbreviated dL). The free peptide eluted at longer times (corresponding to a lower molecular weight) than did the peptide incorporated into the fibrin gel during coagulation, demonstrating covalent attachment to degraded fibrin and thus covalent incorporation via the action of factor XIIIa activity.

Neurite extension in fibrin gels
When DRGs were cultured in 3-dimensions within fibrin gels, they extended neurites in all directions. Extension of neurites continued until such time when the neurites degraded the fibrin gel so that it no longer provided enough mechanical support for neuronal adhesion. Pior to this point, neurons extended neurites vigorously through fibrin gels. During the first 2 days of culture, growth was generally more rapid on the second day than on the first, with an average neurite extension of 0.25 mm at 24 h and 0.74 mm at 48 h. The morphology of the neurites was generally more fasciculated near the ganglion body and less fasciculated near the corona of growth cones. Images of DRGs near the ganglion body and near the growth cones are shown in Fig. 2A, B ).



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  		Figure 2. Images of DRGs cultured within fibrin gels with and without heparin binding peptide. A) Unmodified fibrin near ganglion body. B) Unmodified fibrin near growth cones. C) Fibrin containing {alpha}2PI1–7-ATIII121–134 heparin-binding peptide near ganglion body. D) Fibrin containing {alpha}2PI1–7-ATIII121–134 peptide near growth cones. Confocal scanning laser microscopy of DRGS was performed using 10x magnification. The images shown are extended focus projections of ~50–100 images taken at 7–10 µm intervals. The scale bar represents 100 µm. Cells were stained with fluorescein diacetate prior to imaging.

Neurite extension through fibrin gels containing heparin-binding domains
The effect of incorporation of heparin-binding domains on 3-dimensional neurite extension through fibrin was determined by incorporating bi-domain peptides using factor XIIIa catalyzed cross-linking. These peptides contained one of four different heparin-binding domains, as shown in Table 1 . Each of the heparin-binding sequences tested promoted more vigorous neurite extension compared to unmodified fibrin gels when incorporated at 8 mol of peptide per mole fibrinogen (Fig. 3 ). At 48 h of culture, incorporation of {alpha}2PI1–7-ATIII121–134 induced an increase in neurite extension of 73 ± 7%, {alpha}2PI1–7-NCAM133–146 induced an increase of 25 ± 6%, and {alpha}2PI1–7-PF460–67 induced an increase of 20 ± 3% (all P<0.05) relative to native fibrin. A peptide with a variation on the heparin-binding domain of ATIII (in which all lysine residues were changed to arginine), denoted {alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R, induced an increase of 41 ± 6% (P<0.05) relative to native fibrin. The observation that all of the heparin-binding peptides tested promoted more vigorous neurite extension suggests that the observed enhancement in neurite extension is not specific to one particular sequence, but rather is a result of the affinity for heparin and heparan sulfate proteoglycans of the various peptide sequences tested.



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  		Figure 3. Effect of incorporated heparin-binding peptides on neurite extension within 3-dimensional fibrin gels. Neurite growth from dorsal root ganglia was normalized to growth in unmodified fibrin gels at 48 h culture time. Experiments with the {alpha}2PI1–7-ATIII121–134 peptide and {alpha}2PI1–7, Q2->G-ATIII121–134 peptide at 8 mol/mole fibrinogen were performed in quadruplicate; experiments with the {alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R, {alpha}2PI1–7-NCAM133–146 and {alpha}2PI1–7-PF460–67 peptides at 8 mol/mole fibrinogen and {alpha}2PI1–7, Q2->G-ATIII121–134 at 4 mol/mole fibrinogen were performed in duplicate, each with 6 ganglia per replication. Mean values and standard error of the mean are shown. *P<0.05 compared to unmodified fibrin. Normalized neurite extension of 1 corresponds to 0.74 mm. Incorporation of heparin-binding peptides into fibrin gels at 8 mol/mole fibrinogen increased neurite extension in all cases. When incorporation was prevented by using the inactive factor XIIIa substrate {alpha}2PI1–7, Q2->G, and the amount of the ATIII peptide incorporated was reduced to 4 mol/mole of fibrinogen, no such statistically significant increase was observed.

Images of DRGs cultured within fibrin gels containing the {alpha}2PI1–7-ATIII121–134 heparin-binding peptide are shown in Fig. 2C, D . DRGs cultured within fibrin containing heparin-binding peptides were observed to display more extensive fasciculation near the ganglia bodies relative to unmodified fibrin. In some cases, more glial cells appeared to migrate toward the growth cones relative to that in unmodified fibrin.

To ensure that the increase in neurite extension induced by heparin-binding peptides was due to cross-linked peptide and not free peptide in solution, which was not covalently incorporated during coagulation, a bi-domain peptide that contained the ATIII heparin-binding domain, but an {alpha}2-plasmin inhibitor factor XIIIa substrate in which the glutamine residue that is required for cross-linking had been replaced with glycine ({alpha}2PI1–7, Q2->G-ATIII121–134), was tested. This peptide was present during coagulation and was subjected to the standard washing and culture protocols described above. Gels thus treated with this peptide showed no significant increase in neurite extension over unmodified fibrin (P=0.78) (Fig. 3) . This result demonstrates that the enhancement of neurite extension by heparin-binding domains is due to peptide that is covalently cross-linked into the fibrin gel.

To test the effect of peptide concentration on the ability of the ATIII heparin-binding domain to promote neurite extension, lower concentrations of peptide were incorporated into fibrin gels. At 48 h of culture, incorporation of {alpha}2PI1–7-ATIII121–134 at a concentration of ~4 mol of peptide per mole fibrinogen did not induce an increase in neurite extension (10 ±3%, P=0.17) relative to unmodified fibrin. This observation demonstrates that there is some minimum concentration of the exogenous heparin-binding peptide required to significantly enhance neurite extension.

To test whether soluble proteins or cell-surface proteoglycans were responsible for the interaction leading to enhanced neurite extension, heparin was added during coagulation or in culture. The addition of heparin during cross-linking or in the culture medium inhibited the effect of the four heparin-binding domains tested to increase neurite length, such that outgrowth was not statistically different from the native fibrin control (P>0.75 for each) (Fig. 4 ). The addition of heparin during polymerization decreased cross-linking of the peptide to 7.1 ± 0.1 mol of peptide per mole fibrinogen, a difference that is statistically different (P<0.0001) but probably not particularly important compared to the value of 8.7 ± 0.2 for the peptide cross-linked in the absence of heparin.



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  		Figure 4. Effect of addition of heparin during coagulation and to cell culture medium on neurite extension within fibrin gels containing heparin-binding peptides at 8 mol/mole of fibrinogen. Neurite growth from dorsal root ganglia was normalized to growth in unmodified fibrin gels at 48 h culture time. Experiments with the {alpha}2PI1–7-ATIII121–134 peptide were performed in quadruplicate; experiments with the {alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R peptide, the {alpha}2PI1–7-NCAM133–146 peptide, the {alpha}2PI1–7-PF460–67 peptide, and the medium containing heparin were performed in duplicate with 6 ganglia per replication. Mean values and standard error of the mean are shown. *P<0.05 compared to unmodified fibrin. Normalized neurite extension of 1 corresponds to 0.74 mm. The addition of heparin either during coagulation or to the cell culture medium inhibited the ability of heparin-binding peptides to enhance neurite extension through fibrin gels.

To probe the role of direct interaction with cell-surface proteoglycans, enzymatic removal before and during culture was used. The enzymatic cleavage of cell-surface proteoglycans resulted in decreased neurite outgrowth. The addition of chondroitinase ABC or heparitinase to fibrin gels containing the peptide {alpha}2PI1–7-ATIII121–134 at 8 mol of peptide per mole fibrinogen resulted in increases in neurite extension of only 37 ± 4% and 35 ± 5%, respectively, relative to native fibrin (P<0.05), compared to 73 ± 7% in the absence of either enzyme (P<0.0001) (Fig. 5 ). Together, the results obtained with the addition of bound heparin during coagulation, free heparin during culture, and proteoglycan degrading enzymes before and during culture suggest that a proteoglycan located on the cell surface, rather than a soluble factor, is involved in the enhancement of neurite extension.



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  		Figure 5. Effect of enzymatic cleavage of glycosaminoglycans on neurite extension through fibrin gels containing heparin-binding peptides at 8 mol/mole of fibrinogen. Neurite growth from dorsal root ganglia was normalized to growth in unmodified fibrin gels at 48 h culture time. Experiments with the {alpha}2PI1–7-ATIII121–134 peptide were performed in quadruplicate, and experiments with heparitinase and chondroitinase ABC added to the culture medium were performed in duplicate with 6 ganglia per replication. Mean values and standard error of the mean are shown. *P<0.05 compared to unmodified fibrin. §P<0.05 compared with fibrin gels containing covalently attached {alpha}2PI1–7-ATIII121–134 peptide. Normalized neurite extension of 1 corresponds to 0.74 mm. Enzymatic cleavage of either glycosaminoglycan decreased the ability of the {alpha}2PI1–7-ATIII121–134 peptide to enhance neurite extension through fibrin gels, suggesting an adhesive role for cell-surface proteoglycans in interaction with the immobilized exogenous heparin-binding peptides.

Heparin-affinity chromatography
The relative affinity of the four bi-domain heparin-binding peptides was determined by heparin-affinity chromatography. Bi-domain peptides containing the {alpha}2-plasmin inhibitor factor XIIIa substrate domain at the aminoterminus and heparin-binding domains from PF4({alpha}2PI1–7-PF460–67), NCAM ({alpha}2PI1–7-NCAM133–146), modified ATIII ({alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R), and ATIII ({alpha}2PI1–7-ATIII121–134) are listed here from weakest to strongest heparin-binding affinity. The ATIII121–134 variant in which the three arginine residues were substituted for lysine was synthesized and tested to explore the affinity vs. activity of the most strongly binding and most neurite-promoting peptide, but with the minimum number of structural changes possible. The NaCl concentrations at whicheach peptide eluted are given in Table 2 . The observed influence on neurite extension was the same as the heparin-binding affinity, with the PF4 domain having the least influence and the ATIII domain having the greatest. Thus, the degree of neurite extension enhancement was observed to correlate closely with the relative heparin-binding affinity of the peptide incorporated into the fibrin gel, stronger binding leading to greater enhancement.


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  		Table 2. NaCl concentration required to elute heparin-binding peptides from a heparin-affinity column


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this work was to design biomaterials for use as fillers for nerve guide tubes to enhance peripheral nerve regeneration. Fibrin was chosen as the base material to be used for these studies 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 3-dimensional fibrin gel (25) . We sought to enhance the ability of fibrin to promote nerve regeneration by covalently attaching neurite outgrowth-promoting peptides—in this case, heparin-binding domains—thus endowing fibrin with the bioactivity of nonfibrin proteins.

Heparin-binding domains are found in a variety of proteins and can promote neurite outgrowth. Heparin-binding domains from a variety of adhesion proteins have been shown to promote neurite extension when adsorbed to 2-dimensional surfaces, including domains from the extracellular matrix proteins laminin, fibronectin, and HB-GAM and a domain from the cell–cell adhesion protein NCAM (15 , 16 , 32 , 33) . For example, the heparin-binding domain of NCAM has been shown to promote neurite extension when adsorbed to surfaces. Furthermore, the neurite outgrowth induced by this domain could be inhibited by the addition of heparin to the cell culture media, digestion with heparitinase, or inhibition of cell proteoglycan synthesis, suggesting that the proteoglycan interacting with this domain was located on the cell surface. Moreover, heparin-binding proteins that are not naturally involved in supporting cell adhesion have also been shown to influence neurite outgrowth, e.g., the chemokine PF4, the anticoagulant protein ATIII, and the chemokine midkine (20 , 34 , 35) . The heparin-binding domain of PF4 has been used as a mimic of the heparin-binding domain of fibronectin and shown to promote neurite extension from various types of neurons on 2-dimensional surfaces (22 , 34) . The heparin-binding domain from ATIII was mimicked by Borrajo et al. (35) , and in the soluble form was found to inhibit neurite extension on polylysine adsorbed to surfaces. However, when itself adsorbed to a surface, this ATIII domain mimic promoted neurite extension.

Based on reports in the literature demonstrating the ability of heparin-binding peptides to promote neurite extension in two dimensions, we decided to explore the ability of immobilized heparin-binding peptides to promote 3-dimensional neurite extension when enzymatically incorporated into fibrin gels. Such exploration might lead to a better understanding of neurite extension in a more realistic 3-dimensional context and also to the development of therapeutically useful materials. Incorporation of biofunctional peptides was accomplished by constructing bi-domain peptides containing a factor XIIIa substrate, {alpha}2PI1–7, at one end and a heparin-binding domain at the other end. The transglutaminase factor XIIIa then cross-links the bi-domain peptide, via the Q2 residue in {alpha}2PI1–7, to the fibrin gel during coagulation. The amount of peptide covalently incorporated into fibrin gels by factor XIIIa was quantified and found to be 8.7 ± 0.2 mol/ mole of fibrinogen without heparin and to be 7.1 ± 0.1 mol/mole of fibrinogen with heparin present, agreeing well with previous results for the same factor XIIIa substrate sequence but a vastly different carboxy terminus (26) . These results show that a method developed previously for the incorporation of bi-functional peptides into fibrin gels during coagulation can easily be adapted to attach peptides with very different carboxy termini and even large molecules complexed to the carboxy terminus. The incorporated peptides have been shown both here and earlier (26) to retain their bioactive function on cross-linking. The cross-linked peptides are displayed in a manner such that their active domain is accessible for interactions with cell-surface receptors.

Peptides containing the heparin-binding domains {alpha}2PI1–7-ATIII121–134, {alpha}2PI1–7-PF460–67, {alpha}2PI1–7-NCAM133–146,, and {alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R were incorporated within fibrin gels via a substrate for factor XIIIa. The quantitative degree of enhancement of neurite extension resulting from this incorporation was observed to increase with increasing heparin affinity, as determined by heparin-affinity chromatography. Of the three naturally occurring heparin-binding domain sequences studied, ATIII121–134 bound heparin most strongly and also induced the highest degree of neurite extension, an increase of 73 ± 7% at 48 h. This ATIII domain mimic is somewhat special in that Tyler-Cross et al. (28) engineered the peptide sequence, modifying it to mimic more closely the native structure of the heparin-binding domain in intact ATIII and to induce {alpha}-helix formation in the presence of heparin. This modified ATIII domain has substitutions at positions F121 and F122 for K121 and (ßA)122 to better mimic the effect of R47 in the native protein, and (ßA)122 was added to increase flexibility of K121. Substitutions at positions N127 and C128 for alanine at both positions were made to prevent di-sulfide bond formation between peptides and to potentially induce {alpha}-helix formation when interacting with heparin. Such modifications were not made with heparin-binding domain mimics from other proteins, and thus it is difficult to extrapolate results on the potency of neurite promotion of the peptides studied to the native intact proteins.

A close correlation between heparin affinity and enhancement of neurite extension was observed with the peptides explored. The PF460–67 and NCAM133–146 peptides eluted at lower salt concentrations than ATIII121–134 are similar to each other. Likewise, neurite extension was statistically lower than with ATIII121–134 and statistically similar between PF460–67 and NCAM133–146. As a more direct probe of the effect of binding affinity, a variant on ATIII121–134 was synthesized, with all of the lysine residues substituted with arginines {alpha}2PI1–7-ATIII121–134, K121, 125, 133 ->R. This modification resulted in a lower heparin-binding affinity and a proportionally lower potential to enhance neurite extension. This suggests that even greater enhancement of neurite outgrowth would be possible in gels containing a heparin-binding peptides with higher heparin-affinity than the {alpha}2PI1–7-ATIII121–134 peptide. The increase in neurite extension with increased ligand affinity is similar to results seen by Lauffenberger et al. (36) , who demonstrated that increasing ligand–receptor affinity could result in maximal cell migration at a lower ligand concentration. At some higher heparin-binding affinity, this effect may saturate or go through a maximum, so that beyond some point increased heparin-binding affinity at a constant ligand concentration may actually decrease neurite outgrowth.

The influence of the nature of the adhesive interactions between the heparin-binding domain peptide and the cell can also be seen in the influence of the amount of the peptide. When the concentration of the ATIII121–134 peptide incorporated was reduced by half to ~4 mol of peptide per mole of fibrinogen, the extent of neurite extension was reduced proportionally from 73 ± 7% to 10 ± 3%, the latter of which was not statistically different from neurite extension in native fibrin. Thus, it may also be the case that incorporation of a greater number of heparin-binding peptides can lead to greater enhancement in neurite extension.

Taken together, the results of this study are consistent with the primary nature of the interactions between the cell surfaces and the immobilized peptides being adhesive: addition of soluble inhibitor (heparin, either during or after coagulation), cleavage of the presumed receptors (with enzymatic treatment), and prevention of immobilization of the presumed ligand (with the use of a nonfunctional factor XIIIa substrate analog) all demonstrated results consistent with this hypothesis. Candidate receptors include phosphacan, a chondroitin sulfate proteoglycan that interacts with HB-GAM and can be inhibited by heparin and chondroitin sulfate C (37 , 38) , and N-syndecan, a heparin sulfate proteoglycan that in its soluble form can inhibit neurite outgrowth on HB-GAM (39 , 40) . Both of these proteoglycan receptors for heparin-binding domains play an important role in adhesion and associated signaling; on the basis on the results presented here, one cannot distinguish which of these effects is more important—that is, if they can be separated.

The materials described in this paper may have broad usefulness in tissue engineering. A variety of applications in tissue engineering could benefit from novel 3-dimensional matrices. These include the use of matrices for cell seeding to generate tissues in vitro for ultimate transplantation and the use of matrices for cell invasion to generate and regenerate tissue in vivo. Examples of tissue morphogenesis include generation of tissue-engineered skin, blood vessels, liver, nerve, and cartilage (41 42 43 44 45 46 47) . In these approaches, a matrix component plays a key role, and its interactions with cells either in vitro or in vivo assume an important part in the process of morphogenesis. Requirements of the characteristics of such matrices include 1) the ability of cells to infiltrate and remodel these matrices, 2) the ability to tailor the adhesive nature of the matrix, and 3) the ability to present other exogenous bioactive signals, such as polypeptide growth factors, from the matrix. Each of these characteristics potentially can be met in the context of engineered fibrin. 1) As to cell infiltration and remodeling, the activation of plasminogen to plasmin is localized to the surface of neurite growth cones via receptors for plasminogen activators (48) . Through this localization, an intact fibrin structure has been observed to within 100 nm of the growth cone surface as it penetrates the fibrin matrix in neurite extension (25) . 2) As to tailoring the adhesive character of fibrin matrices, Schense and Hubbell (26) provided one example of incorporation of the RGD sequence; the present paper provides a second example in which cell-surface proteoglycans serve as the targeted receptors. 3) As to incorporation of other bioactive species, such as polypeptide growth factors, one can incorporate heparin into fibrin gels via the peptides described here, then use these heparin chains to bind heparin-binding growth factors and release them biomimetically as occurs naturally in the extracellular matrix (S. E. Sakiyama and J. A. Hubbell, unpublished observations).


   ACKNOWLEDGMENTS
 
This work was funded by grant 31–52261 NFP 38 from the Swiss National Science Foundation. We would like to thank Dr. M. Höchli and Prof. T. Bächi of the Electronmicroscopical Laboratory (EMZ) at the University of Zurich for their assistance with confocal scanning laser microscopy.


   FOOTNOTES
 
Received for publication February 1, 1999. Revised for publication June 21, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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