METHODS OF PRODUCTION OF FIBROUS FIBRINOGEN SCAFFOLDS AND PRODUCTS THEREOF

Abstract

The present invention relates to methods for producing fibrous fibrinogen biomaterials that can be used as three-dimensional scaffolds. The methods of this invention enable the controlled detachment of fibrous fibrinogen scaffolds in vitro. The fibrous fibrinogen biomaterials generated by the methods of this invention can be detached in a solution. Alternatively, the fibrous fibrinogen scaffolds of this invention can be immobilized on a surface. The fibrous fibrinogen biomaterial can be used in medicine, such as in wound healing, regenerative medicine, dermal reconstruction, skin repair, bone vessel repair, blood vessel regeneration, tissue engineering, and implant coatings. The biomaterials can be generated on-demand and can be transferred to a site of injury, such as to a wound.

Claims

1. A method of generating a fibrous fibrinogen biomaterial, comprising a) Providing a substrate, b) Optionally, cleaning said substrate, c) Optionally, modifying of at least one surface of said substrate, d) Adding a fibrinogen solution to said substrate, or submerging said substrate in said fibrinogen solution, e) Adding a salt buffer and/or an aqueous buffer and/or water to said substrate, or submerging said substrate in said salt buffer and/or said aqueous buffer and/or said water, f) Optionally, drying said substrate, g) Optionally, adding a salt buffer and/or an aqueous buffer and/or water to said substrate, or submerging said substrate in said salt buffer and/or said aqueous buffer and/or said water, h) Optionally, drying said substrate, i) Optionally, performing one or more repetitions of steps g) to h), j) Fixating said substrate, and k) Washing said substrate, thereby generating said fibrous fibrinogen biomaterial.

2. The method according to claim 1, wherein said generated fibrous fibrinogen biomaterial is composed of three-dimensional fibrinogen fibers, optionally wherein said fibrous fibrinogen biomaterial can be used as a scaffold.

3. The method according to claim 2, wherein said fibrinogen fibers have a diameter of between 0.1 nm and 1000 nm.

4. The method according to claim 1, wherein said fibrous fibrinogen biomaterial to be generated has a hydrodynamic radius of at least 0.00001 mm.

5. The method according to claim 1, wherein said substrate as provided in a) is composed of a solid material, optionally wherein said substrate comprises at least one surface composed of a metal or a polymer.

6. The method according to claim 1, wherein said modifying step c) is selected from silanization, esterification, phosphorization, hydrosilyation, physisorption, aldehyde modification, carboxylate modification, amine modification, epoxy modification, and mixtures thereof.

7. The method according to claim 6, wherein said silanization is performed by immersing the substrate in a silanization agent selected from 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)-dimethyl-ethoxysilane (APDMES), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS), 3-aldehydepropyltrimethoxysilane (APMS), mercaptopropyltrimethoxysilane (MPTMS), mercaptopropyltriethoxysilane (MPTES), biotin 4-nitrophenyl ester (BNPE), 11-hydroxyundecyl-phosphonate (HUP), organopolysiloxane, trimethylchlorosilane, and mixtures thereof.

8. The method according to claim 1, wherein said fibrinogen solution as added in step d) has a fibrinogen concentration of between 0.01 and 1000 mg/ml.

9. The method according to claim 1, wherein said fibrinogen solution as added in step d) is an aqueous fibrinogen solution.

10. The method according to claim 1, wherein said salt buffer comprises at least one component selected from sodium phosphate, sodium chloride, acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium chloride, potassium citrate, potassium metaphosphate, potassium phosphate (monobasic), sodium acetate, sodium citrate, sodium lactate, sodium phosphate (dibasic), sodium phosphate (monobasic), and mixtures thereof.

11. The method according to claim 1, wherein said salt buffer is phosphate-buffered-saline (PBS), optionally wherein said PBS comprises sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate.

12. The method according to claim 1, wherein fibrous fibrinogen nanofibers are generated when said optional steps g), h), and i) of said method are not performed.

13. The method according to claim 1, wherein fibrous fibrinogen microfibers are generated when steps g), h), and i) of said method are performed.

14. The method according to claim 1, wherein said fixating step j) is performed by immersing said substrate in a fixation agent selected from paraformaldehyde, formaldehyde, glutaraldehyde, mercury oxide, lead oxide, osmium oxide, trichloroacetic acid, acetic acid, and mixtures thereof.

15. The method according to claim 1, wherein said washing step k) is performed in an aqueous solution.

16. The method according to claim 1, wherein when said modifying step c) is performed prior to addition of the fibrinogen solution to the substrate in step d), and wherein said modifying is silanization of at least one surface of said substrate, said fibrous fibrinogen biomaterial to be generated will be immobilized on said silanized surface of said substrate.

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21. A method of treating a subject suffering from a degenerative disease, a wound, a dermal disease, a vessel damage, and/or a skin damage comprising: generating a fibrous fibrinogen biomaterial according to claim 1; and administering to the subject the fibrous fibrinogen biomaterial or a composition comprising said fibrous fibrinogen biomaterial.

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23. A method for inhibiting bleeding at a target site in a patient's body, said method comprising; generating a fibrous fibrinogen biomaterial according to claim 1; and delivering the fibrous fibrinogen biomaterial or a composition comprising said fibrous fibrinogen biomaterial to a target site, a bleeding tissue, an abraded tissue surface, and/or a damaged tissue surface in an amount sufficient to inhibit said bleeding.

24. A method for delivering a bioactive substance to a target site in a patient's body, said method comprising generating a fibrous fibrinogen biomaterial according to claim 1; and delivering the fibrous fibrinogen biomaterial or a composition comprising said fibrous fibrinogen biomaterial in combination with a bioactive substance to the target site.

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Description

DESCRIPTION OF THE DRAWINGS

[0091] In the figures, the figures show the following as described below.

[0092] FIG. 1: Schematic of the procedure to prepare free-floating nanofibrous fibrinogen scaffolds using salt-induced self-assembly. Piranha-cleaned glasses are used as substrates for fiber formation. Fixation in formaldehyde vapor then leads to controlled scaffold detachment upon subsequent washing.

[0093] FIG. 2: Preparation of fibrinogen microfibers by rehydration. a) Schematic of the procedure to prepare fibrinogen microfibers by rehydration. b) Piranha-cleaned glasses are used as substrates for fiber formation. A 5 mg/ml in 10 mM NH.sub.4CO.sub.3 solution is added to the glass substrate. A 24 h drying step is followed by a rehydration step using 10 mM NH.sub.4CO.sub.3 as a rehydration liquid. The latter rehydration step can be repeated multiple times. Optionally, the fibrinogen fibers that have been generated can be fixated using a fixation agent, such as 2% glutaraldehyde.

[0094] FIG. 3: Schematic of the preparation of immobilized nanofibrous fibrinogen scaffolds using salt-induced self-assembly. An APTES-modification is introduced on the glass slide prior to fiber assembly. After fixation in formaldehyde vapor and subsequent washing, the fibrous fibrinogen scaffolds are specifically immobilized on the APTES layer. Alternatively, immobilized nanofibrous fibrinogen scaffolds can be generated on a substrate that comprises at least one surface composed of a metal, such as of gold, or that comprises at least one surface composed of a polymer matrix, such as polylactide (PLA).

[0095] FIG. 4. SEM images of nanofibrous fibrinogen scaffolds, which were prepared using varying protein concentrations and adding either 25 mM NaPO.sub.4 buffer (A to D) or 2.5PBS (F to I) at pH 7.4. Below fibrinogen concentrations of 2 mg/ml nanofibers could not be assembled (A, B and F, G). With concentrations of 2 mg/ml fibrinogen in the sample volume or higher protein concentrations fiber formation was observed (C, D and H, I). With increasing protein concentration the fibrous scaffolds were found to be more dense. Cross-sections of scaffolds prepared with 5 mg/ml fibrinogen were additionally imaged for 25 mM NaPO.sub.4 buffer (E) and 2.5PBS (J). Both cross-sections show a scaffold thickness of several micrometers. Scale bars represent 2 m.

[0096] FIG. 5. SEM images of self-assembled fibrinogen scaffolds prepared with varying salt concentrations. Fibrinogen nanofibers were assembled by drying a 5 mg/ml fibrinogen solution in the presence of either NaPO.sub.4 buffer (B to E) or PBS (G to J), which were both at pH 7.4. Without the presence of any salt buffer no fiber formation was observed (A and F). For all other salt concentrations fibrinogen reproducibly assembled into nanofibers. Scale bars represent 2 m.

[0097] FIG. 6. Representative FTIR spectra of different fibrinogen samples prepared on gold surfaces: (A) planar fibrinogen prepared with 5 mg/mL fibrinogen solution, (B) fibrinogen nanofibers prepared via self assembly of 5 mg/ml fibrinogen solution with 2.5PBS (pH 7.4). Both spectra show the amide I and amide II regions, which are sensitive to protein superstructure. With nanofiber formation in the presence of PBS the peak positions were shifted towards lower wave numbers as compared to the spectra of planar fibrinogen.

[0098] FIG. 7. Coverage of 15 mm glass slides with fibrinogen nanofibers increases with increasing salt concentration. Fibrinogen nanofibers were prepared by drying a 5 mg/ml fibrinogen solution in the presence of different concentrations of either (A) NaPO.sub.4 buffer or (B) PBS. The coverage of the 15 mm glass substrates was measured using a USB microscope. Data represent means and standard deviations of three independently prepared samples for each salt concentration. The average coverages of the samples with the highest concentration were compared to the other conditions by ANOVA and are indicated by asterisks (****p<0.0001, **p<0.001, ns: not significant). Insets show overview images of the 15 mm glass slides with 5 mg/ml fibrinogen dried in the presence of 25 mM NaPO.sub.4 buffer or 2.5PBS, respectively. Scale bars in insets represent 5 mm.

[0099] FIG. 8. SEM images of fibrinogen nanofibers, which were assembled using different PBS components. 5 mg/ml fibrinogen were dried in the presence of (A) 375 mM NaCl, (B) 10 mM KCl, (C) 375 mM KCl or (D) 25 mM KPO.sub.4 buffer. Nanofibers could be assembled in all buffers, if the salt concentration was at least 25 mM. No fibrinogen fibers formed with 10 mM KCl. Fiber morphology was less defined when single PBS components were used. Scale bars represent 2 m.

[0100] FIG. 9. SEM images of fibrinogen nanofibers, which were assembled by drying a 5 mg/ml fibrinogen solution in either 25 mM NaPO.sub.4 buffer (A to E) or 2.5PBS (F to J) under varying pH conditions. In both buffers the pH was varied from 5 to 9. Fiber formation was only induced for pH 7 to 9 whereas more acidic pH ranges did not yield any nanofibers. Scale bars represent 2 m.

[0101] FIG. 10. Controlled detachment or immobilization of fibrinogen nanofiber scaffolds depends on the underlying substrate material. Scaffolds were prepared using 5 mg/ml fibrinogen and 2.5PBS. After fixation in formaldehyde vapor the samples were washed with water. On glass substrates the scaffolds detached immediately (A, B) whereas on APTES-modified substrates fibrinogen scaffolds stayed immobilized (C, D). Scale bars represent 1 cm.

[0102] FIG. 11. SEM images of self-assembled fibrinogen scaffolds prepared in the presence of AEBSF. Fibrinogen nanofibers were assembled by drying a 5 mg/ml fibrinogen solution in the presence 2.5PBS and 0.1 mM (A) or 1 mM (B) AEBSF. Even in the presence of the thrombin inhibitor AEBSF fiber formation was observed. Scale bars represent 2 m.

[0103] FIG. 12. Self-assembled fibrinogen scaffold with a total surface area of approximately 6 cm2. Fibrinogen nanofibers were assembled by drying a 5 mg/ml fibrinogen solution in the presence of 2.5PBS on a glass slide. Scale bar represents 10 mm.

[0104] FIG. 13. SEM images of self-assembled fibrinogen scaffolds prepared on different surfaces. Fibrinogen nanofibers were assembled by drying a 5 mg/ml fibrinogen solution in the presence of 2.5PBS on (A) an APTES-modified surface, (B) a sputtered gold surface or (C) a polystyrene cell culture dish. Fibrinogen fibers formed on both substrates when PBS was added. Scale bars represent 2 m.

[0105] FIG. 14. Fibrinogen scaffold preparation at different time points. Fibrinogen nanofibers were assembled by drying a 5 mg/ml fibrinogen solution in the presence of 2.5PBS. After 60 min a change in turbidity was observed during drying, after 90 min macroscopic salt crystals were visible. Scale bar represents 5 mm.

DETAILED DESCRIPTION

Examples

Preparation of Substrates and Protein Solutions

[0106] Round glass coverslips with a diameter of 15 mm (VWR, Darmstadt, Germany) and square glass slides (Gerhard Menzel GmbH, Braunschweig, Germany) were cleaned by 5 min immersion into H.sub.2SO.sub.5 (piranha solution). Piranha solution was freshly prepared by mixing 95% sulfuric acid (VWR) with 30% hydrogen peroxide solution (VWR) in a 3:1 ratio. After washing with deionized water from a TKA water purification system (Thermo Fisher Scientific, Schwerte, Germany) the activated glass coverslips were stored in deionized water and dried under nitrogen flow directly before further use. Moreover, the inventors studied the self assembly of fibrinogen on polystyrene petri dishes (Sarstedt, Numbrecht, Germany) and on glass, which was sputter-coated with a 25 nm gold layer in a Bal-Tec SCD 005 system (Leica Microsystems, Wetzlar, Germany). The inventors also analyzed fibrillogenesis of fibrinogen on glass slides, which were modified with (3-Aminopropyl)triethoxysilane (APTES) by immersion in an ethanol solution (VWR) containing 5% APTES (Sigma) for 16 h at room temperature. Fibrinogen stock solutions were prepared by dissolving 10 mg/ml fibrinogen (100% clottable Merck, Darmstadt, Germany) in a 10 mM ammonium carbonate solution (Roth, Karlsruhe, Germany). The fibrinogen stock solution was dialyzed against 10 mM ammonium carbonate solution overnight using cellulose membrane dialysis tubing with 14 kDa cut-off (Sigma, Steinheim, Germany) to remove low molecular weight compounds. In control experiments varying concentrations of the thrombin inhibitor 4-(2-Aminoethyl)benzensulfonylfluoride (AEBSF, Sigma) were added to the buffer system to inactivate any potential thrombin residues in the fibrinogen stock.

Self Assembly of Fibrinogen Nanofibers

[0107] The standard procedure to prepare nanofibrous fibrinogen scaffolds was to deposit 100 l of the 10 mg/ml fibrinogen stock solution on piranha-cleaned glass slides. Subsequently, fiber formation was induced by adding 100 l of either 5PBS solution (Thermo Fisher) or 50 mM sodium phosphate (NaPO.sub.4) buffer (Roth, Karlsruhe, Germany), which were both at pH 7.4. The final concentration in the samples was 5 mg/ml fibrinogen and 5 mM NH.sub.4HCO.sub.3 in either 2.5PBS or 25 mM NaPO.sub.4 buffer. For planar reference samples 100 l of deionized water was added to the fibrinogen instead of PBS or NaPO.sub.4 buffer. All samples were subsequently dried overnight in ambient conditions. To analyze the influence of varying buffer conditions on the fibrillogenesis process the inventors later used variations of this standard procedure by adjusting the fibrinogen or buffer concentration. The inventors also studied the dependence of fibrillogenesis on varying pH in PBS or NaPO.sub.4 buffers, which were prepared by adding different ratios of NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4 (Roth). NaCl (AppliChem, Darmstadt, Germany) was added to the NaPO.sub.4 buffers to obtain PBS with varying pH. To investigate the influence of different salt ions on the fiber formation the inventors also prepared samples with 375 mM NaCl (AppliChem), 10 and 375 mM KCl (Roth, Karlsruhe, Germany) or 25 mM KPO.sub.4 buffer (pH 7.4, AppliChem).

Free-Standing and Immobilized Fibrinogen Scaffolds

[0108] For the preparation of free-standing scaffolds the inventors crosslinked self-assembled fibrinogen nanofibers on glass slides overnight using a sealed chamber, which contained 200 l of 37% formaldehyde (AppliChem). Afterwards, fixated scaffolds were dried for 1 hour, followed by a washing step in deionized water. To fabricate immobilized fibrinogen scaffolds the inventors carried out the same procedure on APTES-coated glass, gold and polystyrene petri dishes.

Microscopy Analysis of Nanofibrous Fibrinogen Scaffolds

[0109] The coverage of glass slides with dried fibrinogen nanofibers was analyzed using a USB universal microscope (Meade Instruments, Rhede, Germany) using an 20 magnification and brightfield imaging. For morphological analysis dried fibrinogen samples were sputter-coated with 7 nm of gold using a Bal-Tec SCD 005 sputter system (Leica Microsystems). Scanning electron microscopy (SEM) analysis was carried out with a Zeiss Auriga field emission device (Carl Zeiss, Oberkochen, Germany) at acceleration voltages of 3 kV. Surface coverage and fiber diameters were analyzed using the open source software ImageJ. Differences in surface coverage were analyzed by ANOVA followed by the Tukey post hoc-test.

Fourier-Transform Infrared Reflective Absorption Spectroscopy

[0110] Fourier-transform infrared reflective absorption spectroscopy (FTIR) analysis of fibrinogen scaffolds on AU-coated glasses was conducted using a Bruker Vertex 70 with IR Scope II (Bruker, Ettlingen, Germany). First, round glass coverslips with a diameter of 15 mm (VWR, Darmstadt, Germany) were sputter-coated with an adhesion layer of 5 nm of chrome, followed by 50 nm of gold using an EM ACE600 high vacuum sputter coater (Leica Microsystems, Wetzlar, Germany). Subsequently, fibrinogen scaffolds were prepared on the Au layers using a final protein concentration of 5 mg/ml and either 2.5PBS with 5 mM NH.sub.4HCO.sub.3 for fibrous scaffolds or 5 mM NH.sub.4HCO.sub.3 for planar fibrinogen samples. Each sample was dried under ambient conditions and measured in 15 different points in a zig-zag pattern. Spectra were recorded at 4 cm-1 resolution and 60 scans were averaged per measurement. The reference spectrum was measured against air and subtracted from the obtained spectra. The spectra were processed and evaluated by the Bruker software package OPUS. After subtraction of water vapor absorbencies (atmosphere compensation) the resulting spectra were smoothed by a 7-17 point Savitsky-Golay function, depending on the quality of the data and baseline corrected by rubber band baseline correction. Amide I and amide II peak positions were determined by peak integration using the Origin 9.0 software (OriginLab Northampton, USA).

Effect of Fibrinogen Concentration on Nanofiber Assembly

[0111] When 25 mM NaPO.sub.4 buffer or 2.5PBS were added to fibrinogen dissolved in NH.sub.4HCO.sub.3 and samples were dried under ambient conditions for SEM analysis the inventors observed the formation of nanofibers in dependence of the fibrinogen concentration. Only salt crystals were found when no protein was present in the respective buffer. With fibrinogen concentrations of 1 mg/ml and lower only globular aggregates were found in both buffer systems. For both buffers the inventors observed that a minimum concentration of 2 mg/ml fibrinogen in solution was necessary to induce nanofiber assembly upon drying. The nanofibrous scaffolds became more dense when the fibrinogen concentration was increased further up to 5 mg/ml. The density of the self-assembled fibrous fibrinogen networks was comparable to previous electrospun fibrinogen mats.

[0112] To exclude the possible involvement of residual thrombin in the fibrinogen stock solution, the inventors added 0.1 and 1 mM of the thrombin inhibitor AEBSF to the fibrinogen solution and dried it in 2.5PBS. It can be seen clearly that fibrinogen fibers also formed in 2.5PBS when AEBSF was present. Cross sections of dense nanofiber scaffolds, which were prepared with the highest concentration of 5 mg/ml fibrinogen, revealed that the nanofibrous scaffolds had a thickness of 3 to 5 m.

Influence of Varying Buffer Concentration on Fibrinogen Nanofiber Assembly

[0113] To study the mechanism of salt-induced fiber formation further the inventors varied the concentration of NaPO.sub.4 buffer and PBS, in which the protein was dried. In a control experiment without the presence of any salts no fibers formed and only planar films of fibrinogen were obtained. With low salt concentrations of only 5 mM NaPO.sub.4 buffer or 0.5PBS fibrinogen already assembled into nanofibers upon drying. When salt concentrations were increased even further from 10 mM to 50 mM NaP4 buffer and from 1 to 5PBS fibrinogen also assembled into nanofibers. In all fibrous assemblies the diameter of single fibers varied between 100 and 300 nm and was independent of the respective buffer or salt concentration.

Structural Analysis of Fibrinogen Fibrillogenesis

[0114] To study the observed morphological changes upon salt-induced fiber assembly in more detail the inventors carried out FTIR analysis of planar and fibrous fibrinogen scaffolds on gold. The FTIR spectra were characterized within a range of vibration frequencies from 2000 to 1000 cm-1 as proteins exhibit characteristic bands in this spectral range. FIG. 6 shows representatives FTIR spectra of planar fibrinogen samples and of fibrinogen nanofibers. Two well defined and typical bands between 1600-1700 cm-1 (amide I) and between 1500-1600 cm-1 (amide II) were observed in both samples. For the planar samples the amide I band was centered at 1663 cm-1 while for fibers the peak position shifted to 1648 cm-1. The amide II band also shifted to lower wave number values from 1551 cm-1 for planar fibrinogen to 1543 cm-1 for fibrinogen nanofibers. Furthermore, it can be seen in FIG. 6 that the peak shape of planar fibrinogen differed from the peaks obtained for fibrinogen nanofibers. The peaks of planar fibrinogen exhibited a more narrow shape and an amide I/amide II ratio of 2.45. In contrast, fibrinogen nanofibers led to broader peaks and a lower amide I/amide II ratio of 1.50.

[0115] When comparing planar fibrinogen samples to nanofibrous fibrinogen biomaterial, it is apparent that planar fibrinogen samples appear to be white, while nanofibrous fibrinogen biomaterial as generated by salt-induced assembly (such as by addition of PBS) appears to have a whitish color. When analyzing a cross-section of a planar fibrinogen sample prepared according to this invention, the planar fibrinogen layers are very thin and smooth, even at a thickness of the entire scaffold of about 1.3 m. Accordingly, the transparency of the planar fibrinogen samples can be explained by their smooth structure. This smoothness of the planar fibrinogen samples prepared according to this invention can be observed for scales of up to multiple centimeters.

[0116] Interestingly, when the inventors increased the salt concentration in both buffer systems the inventors observed an increase in the overall coverage of fibrinogen nanofibers on the glass substrates as tested. ANOVA analysis confirmed that the lower buffer concentrations, i.e. 5 mM to 25 mM NaPO.sub.4 and 0.5 to 1PBS, resulted in a significantly lower coverage with fibrinogen nanofibers (FIG. 7). For NaPO.sub.4 concentrations up to 50 mM a maximum coverage of 80% was achieved on 15 mm large coverslips (FIG. 7A). With PBS the inventors even obtained a maximum coverage of 95% with concentrations of 2.5 and 5PBS, respectively (FIG. 7B). Hence, PBS-induced fibrillogenesis on glass slides resulted in fibrinogen scaffolds with a size of approximately 1.75 cm2. On larger glass substrates the inventors could also fabricate nanofibrous scaffolds with a surface area of approximately 6 cm2 (FIG. 12). Moreover, using the inventors' self assembly procedure the inventors could prepare nanofibrous fibrinogen scaffolds on other biocompatible surfaces like APTES, gold and polystyrene (FIG. 13). The fiber morphology on these substrates was comparable to fibers assembled on glass.

Effect of Individual PBS Components on Fibrinogen Nanofiber Assembly

[0117] So far, fiber formation was only observed when fibrinogen was dried in the presence of NaPO.sub.4 buffer or PBS. The 2.5PBS used in the inventors' initial experiments contained 25 mM NaPO.sub.4 buffer, 350 mM NaCl and 6.7 mM KCl. To elucidate whether any of these PBS components induce fiber formation on their own the inventors analyzed fiber assembly using 375 mM NaCl and 10 mM KCl, respectively (FIG. 8). Fiber formation was observed with 375 mM NaCl while the presence of 10 mM KCl did not yield any nanofibers. However, when the KCl concentration was increased to 375 mM, fiber formation could also be induced. Overall, in these buffer systems the fiber morphology was less defined and fibers were unevenly distributed as compared to fibers assembled in NaPO.sub.4 buffer or PBS. In comparison to the inventors' standard routine with 25 mM NaPO.sub.4-buffer the inventors carried out an additional experiment in the presence of 25 mM KPO.sub.4 buffer. FIG. 8D clearly shows that this buffer also induced self-assembly of fibrinogen into nanofibers.

Nanofiber Assembly Under Varying pH Conditions

[0118] Since the addition of NaPO.sub.4 buffer or PBS (both at pH 7.4) to the inventors' fibrinogen stock in ammonium carbonate solution (pH 8.6) resulted in a pH shift from the slightly alkaline to physiological pH the inventors were interested in the influence of different pH values on the self assembly process. When 5 mg/ml fibrinogen were used and the pH of 25 mM NaPO.sub.4 buffer and 2.5PBS was adjusted to 5 or 6, respectively, no fiber formation could be induced (FIG. 9). Nevertheless, when NaP4 buffer and PBS with a pH between 7 and 9 were added to the fibrinogen solution the inventors observed fiber assembly in all samples.

Free-Standing and Immobilized Fibrinogen Scaffolds

[0119] When fibrinogen nanofibers were assembled on glass slides an additional fixation step in formaldehyde vapor was introduced followed by washing in water. After this treatment the inventors observed immediate scaffold detachment from the underlying glass surface. When APTES was used as substrate for self-assembled fibrinogen nanofibers the scaffolds stayed immobilized on the silane surface upon cross-linking and washing. Immobilized fibrinogen scaffolds were also obtained when fibrinogen fibers were assembled on gold and polystyrene surfaces prior to fixation and washing. SEM analysis of fixated fibrinogen scaffolds after the final washing step revealed that the nanofiber morphology was preserved during this procedure for bothimmobilized and free-floatingfibrinogen assemblies. Interestingly, without a fixation in formaldehyde vapor the inventors observed that the inventors' self-assembled fibrinogen fibers dissolved again upon washing.

TABLE-US-00001 TABLE 1 Ionic strength (mM) pH 25 mM NaPO.sub.4 2.5 PBS 5 26 376 6 32 382 7 62 412 8 72 422 9 74 424

[0120] The novel self assembly process of this invention to prepare nanofibrous fibrinogen scaffolds only requires the addition of salt solutions like NaPO.sub.4 buffer or PBS to induce fibrillogenesis in vitro. Interestingly, when the protease inhibitor AEBSF was added to the self assembly system, the inventors could also observe nanofiber formation. This result clearly indicates that nanofibrous fibrinogen scaffolds did not assemble due to possible thrombin residues in the purchased fibrinogen. Hence, the inventors assume that the formation of self-assembled fibrinogen fibers was solely induced by the presence of salt buffer in combination with a drying step. Various enzyme-independent methods have previously been used to induce fibrillogenesis of fibrinogen in vitroalso in the presence of NaPO.sub.4 buffer or PBS at pH 7.4.

[0121] The inventors observed that even salt concentrations as low as 5 mM NaPO.sub.4 were sufficient to induce self assembly of fibrinogen into nanofibers without any nanostructured substrates or acidic buffer conditions. The presence of single PBS components also induced successful fiber formation. Therefore, the inventors' results strengthen the hypothesis that an increase in salt ion concentration and particularly the presence of Na+ or K+ ions are responsible for in vitro fibrillogenesis of fibrinogen. Interestingly, the inventors could achieve salt-induced assembly of fibrinogen fibers over a broad pH range even in alkaline pH. Previously, an acidic pH of 2 was used to induce fibrillogenesis. It was suggested that this pH change induced a conformational change, which resulted in aggregation of fibrinogen molecules and in fiber formation. Furthermore, pH changes are known as the major driving force behind the well-understood self assembly mechanism of the ECM protein collagen into nanofibers. However, the pH change in the experimental system was comparably small or not present at all. The inventors therefore conclude that the small pH shift from 8.6 to 7.4, which was used in most of the inventors' experiments, was not the major driving force of fibrinogen fiber formation since fibers were also formed when both buffer systems were at pH 7.4. It had previously been reported that fibrinogen at pH 7.4 has a negative net charge. This finding supports the hypothesis of positively charged Na+ or K+ ions interacting with negatively charged fibrinogen molecules. Interestingly, no fiber formation was observed at pH lower than 7 for both buffer systems, which could be a result of a decreased net charge of the fibrinogen. NaPO.sub.4 buffers with low pH have a lower ionic strength compared to NaPO.sub.4 buffers with the same concentration at a higher pH (Table 1). However, the inventors did not observe any fiber formation when PBS with pH 5 or 6 was used, which had a much higher ionic strength than NaPO.sub.4 buffer at the same pH. Hence, the inventors conclude that lack of fiber formation is caused by the pH itself and not due to a difference in the ionic strength. When the inventors characterized planar and fibrous fibrinogen scaffolds with FTIR spectroscopy the inventors observed differences in the amide I and II bands. Both amide peaks of fibrous fibrinogen were shifted towards lower wave numbers as compared to the spectra of planar fibrinogen. Since the amide I vibration arises mainly from the CO stretching vibration it is very sensitive to changes in the secondary structure. Hence, the pronounced amide I band shift of 15 cm-1 indicated that fibrinogen might be arranged in two different conformations as previously described for adsorbed fibrinogen films by Liedberg et al. Moreover, the overall peak shape, intensity and the ratio between amide I and amide II bands indicated that changes in the protein conformation occurred between planar and nanofibrous fibrinogen scaffolds, i.e. without and with the presence of salt in the fibrinogen solution. The peaks of planar fibrinogen were very narrow with an amide I/amide II ratio of 2.45. In contrast, fibrinogen fibers exhibited a broader peak shape with a lower amide I/amide II ratio of 1.50. These clear differences between the spectra suggest that planar and nanofibrous fibrinogen scaffolds possess different protein conformations as it was previously shown for fibrinogen adsorption on self assembled monolayers.

[0122] The inventors also observed fiber assembly of fibrinogen on hydrophilic surfaces like piranha cleaned glass, APTES or gold. Hence, the surface energy of the underlying substrate did not pose any limitation for the successful fiber formation in the salt-induced self assembly process of this invention as it had been previously reported for other methods. When fibrinogen solutions were monitored while drying in 2.5PBS the inventors noticed a change from initially transparent to more turbid solutions towards the later stage of the drying process (FIG. 14). No change in turbidity was observed when planar fibrinogen samples were prepared by adding deionized water to fibrinogen dissolved in NH.sub.4HCO.sub.3.

[0123] Interestingly, the drying step influences the fibrinogen fibrillogenesis. When drying becomes the driving factor in fiber assembly one also has to consider the local increase in protein concentration and ionic strength in the solution during fibrillogenesis. Since the inventors observed that fibrinogen only assembled into nanofibers with protein concentrations of 2 mg/ml or higher, this finding strengthens the hypothesis that drying is crucial in salt-induced fibrillogenesis of fibrinogen. Based on observations the inventors assume that the detected change in turbidity occurred due to the lower solubility threshold of fibrinogen in comparison to the surrounding salt buffer. Hence, the inventors now suggest the following mechanism to be responsible for fiber assembly in salt buffers: during drying in the presence of salt ions fibrinogen molecules change their conformation and are oriented. Due to the gradual increase in protein concentration upon drying oriented molecules assemble into fibrils, and fibrillogenesis is induced. Interestingly, when dense nanofibrous assemblies were formed by salt-induced fibrillogenesis with 5 mg/ml fibrinogen the inventors mostly observed a characteristic star shape morphology where the fibers reticulated from individual center points. Therefore, the inventors assume that short fibrils function as individual nucleation sites, which subsequently trigger the assembly of further oriented fibrinogen molecules into nanometer-sized fibers upon drying.

[0124] The novel biofabrication process of this invention of salt-induced self assembly reproducibly yielded dense fibrinogen scaffolds with a very high nanofiber yield. Fiber diameters ranged from 100 to 300 nm, which is much larger in comparison to fibers, which were previously formed on hydrophobic surfaces with diameters between 2 and 20 nm. The dimensions of the self-assembled fibrinogen fibers were rather in the range of electrospun fibrinogen fibers, which exhibited diameters between 80 and 700 nm. With regard to future tissue engineering applications it will be advantageous that the dimensions of the self-assembled fibrinogen fibers are well comparable to nanofibers in the native ECM, which vary from 50 to 500 nm, and with the dimensions of natural fibrin fibers, which range from 20 to 200 nm. With overall diameters of several centimeters the scaffold size of the self-assembled fibrinogen networks is also comparable with the dimensions of electrospun fibrinogen mats. Nevertheless, electrospinning required fibrinogen concentrations of 100 to 200 mg/ml to prepare nanofibrous scaffolds with overall dimensions in the centimeter range. On the contrary, the self assembly approach of this invention only required extremely low fibrinogen concentrations starting from 2 mg/ml. With such low protein concentrations the new self assembly routine of this invention will be a very cost-efficient procedure to prepare nanofibrous fibrinogen scaffolds on a large scale. Moreover, self-assembled scaffolds prepared from only 5 mg/ml fibrinogen were 3 to 5 m thick whereas electrospun fibrinogen scaffolds prepared with much higher protein concentrations were approximately 0.7 mm thick. With regard to future scaffold applications in regenerative medicine it will be very interesting to study how the thickness of self-assembled fibrinogen scaffolds can be increased even further by adjusting the protein concentration and other parameters in the self assembly process.

[0125] In contrast to previous approaches the new self assembly routine of this invention facilitates fibrillogenesis of fibrinogen in an enzyme-free environment. Another advantage of salt-induced self assembly is the fact that it does not require any high electric fields or denaturing organic solvents like 1,1,1,3,3,3-hexafluoro-2-propanol, which might impede the biological functionality of the resulting fibrinogen fiber scaffolds. The versatile choice of substrate materials to prepare fibrinogen nanofibers by salt-induced self assembly is another important advantage, which will make the new biofabrication strategy highly attractive for the modification of implant coatings or novel wound dressings.

[0126] Importantly, the use of different substrate materials also introduced the unique possibility to prepare either free-standing or immobilized fibrinogen scaffolds on demand. By adding a fixation in formaldehyde vapor to preserve the nanofiber morphology upon washing the inventors were able to induce controlled detachment of the 3D-scaffolds from glass slides. On the other hand, using silanized glass, polystyrene or gold surfaces as underlying substrate enabled the inventors to specifically immobilize self-assembled fibrinogen scaffolds on these surfaces upon fixation. This versatile and well-controllable routine is easy to use and can be implemented with standard lab equipment and low-cost consumables. Using only physiological buffer systems the novel self assembly method offers a powerful biofabrication platform to prepare immobilized or free-standing fibrinogen scaffolds for various biomedical applications.

[0127] Nano- or microfibrous scaffolds are prepared from aqueous fibrinogen solutions (i.e., at physiological pH) on glass substrates using different preparation methods (for instance salt-induced self-assembly for nanofibers or re-hydration of adhered fibrinogen films for microfibrous assemblies). If the fibrous scaffolds are prepared on piranha-cleaned glass, a subsequent fixation step in paraformaldehyde vapor followed by washing in aqueous solution facilitates controlled detachment of the fibrous scaffolds. If a silanization of the glass surface with (3-Aminopropyl)triethoxysilane (APTES) is introduced into the procedure prior to the respective fiber preparation method, the final fixation and subsequent washing will result in fibrous fibrinogen scaffolds, which are immobilized on the substrate. Likewise, the APTES modification can be used to immobilize planar fibrinogen on the underlying substrate.

[0128] Importantly, nano- or microfibrous fibrinogen scaffolds are prepared from fibrinogen solutions using different methods. Salt-induced self-assembly is, for instance, used to prepare fibrinogen nanofibers, or re-hydration of adsorbed fibrinogen films in aqueous buffer is used to produce microfibrous fibrinogen scaffolds. After a drying step the fibrous scaffolds are fixated in a chamber with formaldehyde vapor, followed by repeated washing steps in aqueous buffer. When fibrous scaffolds are prepared on bare glass the fixation and repeated washing steps induce controlled detachment of the fibrous scaffolds from the underlying glass. This procedure yields three-dimensional, free-floating fiber scaffolds in solution, which can be transferred, for instance, to cell cultures or injured tissue to facilitate wound healing. On the APTES-modified glasses the fibrous scaffolds stay immobilized upon washing and can be used as cell or tissue scaffold.

[0129] Accordingly, the inventors succeeded in the first-time preparation of either free-standing or immobilized fibrinogen scaffolds. This unique feature could be realized by tailoring the underlying substrate material and by introducing a customized fixation and washing procedure after the fiber assembly. Fibrillogenesis could be induced with a fibrinogen concentration of at least 2 mg/ml in a pH regime of 7 to 9. Fiber diameters ranged from 100 to 300 nm, thus resembling native fibrin and ECM protein fibers. By adjusting the salt concentration the inventors could prepare fibrinogen scaffolds with overall dimensions in the centimeter range and a thickness of 3 to 5 m. Using FTIR analysis the inventors observed peak shifts of the amide bands for fibrinogen nanofibers in comparison to planar fibrinogen, which indicates changes in the secondary structure. Since fibrillogenesis was only induced upon drying when salt ions were present the inventors assume that protein molecules were locally oriented in the respective buffers, whichin combination with the observed conformational changesled to the assembly of individual molecules into fibers. In summary, the inventors' novel self assembly process offers a simple and well controllable method to prepare large-scale 3D-scaffolds of fibrinogen nanofibers under physiological conditions. The unique possibility to chose between free-standing and immobilized scaffolds makes the inventors' novel biofabrication process highly attractive for the preparation of versatile tissue engineering scaffolds.

[0130] Using scanning electron microscopy the inventors observed that different buffers including phosphate buffered saline and sodium phosphate reproducibly yielded dense fiber networks on bare and silanized glass surfaces, gold as well as polystyrene upon drying. Finally, the three-dimensional, free-floating fiber scaffolds can be tested in vivo and in vitro, such as in cell culture experiments using fibroblasts or endothelial cells.