A mussel protein self-assembled adhesive coating and a preparation method and application thereof

Abstract

The application belongs to the field of protein materials, and particularly relates to a mussel protein self-assembled adhesive coating and a preparation method and application thereof. The mussel protein self-assembled adhesive coating is obtained by standing a mixed solution of mussel protein and metal ions, and the specific preparation steps are as follows: dissolving mussel protein in an acetic acid solution to obtain a mussel protein solution, adding a solution containing metal ions to the mussel protein solution to obtain a mixed solution, and finally standing the mixed solution to obtain the mussel protein self-assembled adhesive coating. The mussel protein self-assembled adhesive coating is directly prepared by ion-induced protein self-assembly, and has the advantages of simple preparation process and easy operation. The prepared mussel protein coating not only has excellent moisture resistance, but also has good biocompatibility and biodegradability, and has the potential to be an ideal protein-based adhesive material.

Description

Technical Field

[0001] This invention belongs to the field of protein materials, specifically relating to a self-assembled adhesive coating of mussel protein, its preparation method, and its application. Background Technology

[0002] Marine mussels, such as the Mediterranean mussel (Mytilus galloprovincalis) and the thick-shelled mussel (Mytilus coruscus), anchor themselves to underwater surfaces like rocks and ship hulls using mussel foot proteins (Mfps) secreted by their byssal threads. Mfps not only possess excellent adhesive properties but also have various functions in the biomedical field, such as antibacterial, anti-inflammatory, and antioxidant effects. However, the small molecular weight of Mfps makes them susceptible to thermal, chemical, or biological factors under certain conditions, reducing their stability and durability. Furthermore, the small molecular weight of mussel foot proteins may lead to their rapid metabolism and clearance in the body, thus affecting their bioavailability and duration within the organism.

[0003] Self-assembly mechanisms utilize non-covalent bonds between proteins, such as hydrogen bonds, ionic bonds, and hydrophobic interactions, to form aggregates and other structures. Protein self-assembly may enhance protein stability and biological activity, expanding its applications in tissue engineering, cell culture, and other fields. Natural mussel proteins are synthesized by byssal gland cells and secreted into the foot groove for assembly; this process is complex, and dopa is often the inducing factor. However, reports on in vitro induced self-assembly of mussel proteins are currently unknown, making the development of novel self-assembling protein materials a new focus. Currently, most research on protein self-assembly processes concentrates on factors such as protein concentration, self-assembly time, solution pH, and electrolytes; therefore, discovering a simple and effective self-assembly method remains essential.

[0004] Therefore, developing a novel self-assembling adhesive coating for mussel proteins is of great significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a mussel protein self-assembly adhesive coating that addresses the shortcomings of the prior art. This coating has excellent moisture resistance, biocompatibility and biodegradability, and can be used as a potential bioadhesive material and cell culture scaffold material.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned mussel protein self-assembly adhesive coating.

[0007] The final technical problem to be solved by this invention is to provide the application of the above-mentioned mussel protein self-assembly adhesive coating.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A self-assembly adhesive coating for mussel protein is obtained by adding a solution containing metal ions to a mussel protein solution to obtain a mixed solution, and then allowing the mixed solution to stand.

[0010] The concentration of the mussel protein solution is 0.2–1 mg / mL, and the solvent is 5–25% (v / v) acetic acid; the concentration of the metal ions in the solution containing the metal ions is 5–50 mM.

[0011] Preferably, the concentration of the mussel protein solution is 1 mg / mL, and the solvent is 5% (v / v) acetic acid; the concentration of the metal ions in the solution containing the metal ions is 50 mM.

[0012] The mussel protein is a synthetic mussel foot protein obtained by fermentation and purification of engineered Escherichia coli.

[0013] The mussel protein in question is either wild-type Mediterranean mussel protein 3B (Mgfp-3B) or a mutant of wild-type thick-shelled mussel protein 3B. m 3B mutant of the 3B protein in wild-type Mediterranean mussels m Any one or more of the following combinations.

[0014] Specifically, the mutant 3 of the wild-type thick-shelled mussel protein type 3 m The mutant 3B of the 3B protein in wild-type Mediterranean mussels m All include corresponding single mutants and combined mutants.

[0015] Specifically, the single mutants of the wild-type thick-shelled mussel type 3 protein are N33Y, N46Y, or N62Y, and the combined mutants are N33Y / N46Y, N33Y / N62Y, N46Y / N62Y, or N33Y / N46Y / N62Y.

[0016] Specifically, the single mutants of the wild-type Mediterranean mussel 3B protein are G33Y, N43Y, or N76Y, and the combined mutants are G33Y / N43Y, N43Y / N76Y, G33Y / N76Y, and G33Y / N43Y / N76Y.

[0017] Furthermore, detailed information on the wild-type Mediterranean mussel 3B protein 3B (Mgfp-3B), the single mutant of wild-type thick-shelled mussel 3 protein, and the single mutant of wild-type Mediterranean mussel 3B protein has been disclosed in patent CN115181169A.

[0018] Furthermore, the construction process of the combined mutant of wild-type thick-shelled mussel type 3 protein and the combined mutant of wild-type Mediterranean mussel type 3B protein can refer to the construction process of the single mutant in patent CN115181169A.

[0019] The adhesion of mussel protein obtained from the above combined mutants through expression and fermentation purification was measured using a glass overlap experiment. It was found that the three mutants had the best adhesion among the combined mutants.

[0020] Preferably, the mutant 3 of the wild-type thick-shelled mussel type 3 protein... m The mutant 3B of the wild-type Mediterranean mussel protein is described as N33Y / N46Y / N62Y. m It is G33Y / N43Y / N76Y.

[0021] Wherein, the metal ion is Mg 2+ or Ca 2+ One of them; preferably Ca 2+ .

[0022] Specifically, the metal ion Mg 2+ It is provided by magnesium acetate solution, the metal ions Ca 2+ It is provided by calcium acetate solution.

[0023] Specifically, the metal ion Mg 2+ The induced mussel protein forms irregular aggregates in the central region of the protein mica, where the metal ions Ca... 2+ The induced mussel proteins self-assemble into a uniform and dense coating.

[0024] The volume ratio of the mussel protein solution to the solution containing metal ions is 20 to 200:1; preferably 20:1.

[0025] The mussel protein self-assembled adhesive coating has excellent moisture resistance, biocompatibility, and biodegradability. It is non-toxic to cells and blood and can withstand certain mechanical damage.

[0026] This invention also provides a method for preparing a self-assembled adhesive coating of mussel protein, specifically including the following steps:

[0027] S1. Dissolve mussel protein in acetic acid solution to obtain mussel protein solution;

[0028] S2. Add a solution containing metal ions to the mussel protein solution obtained in S1 to obtain a mixed solution;

[0029] S3. Let the mixed solution obtained in S2 stand to obtain the mussel protein self-assembled adhesive coating.

[0030] In step S3, the standing time is 1 to 6 hours at room temperature.

[0031] Preferably, in step S3, the protein in the mixed solution rapidly aggregates within 1 hour, and the protein is basically assembled after 3 hours.

[0032] The application of the above-mentioned mussel protein self-assembly adhesive coating in the preparation of bioadhesive materials is also within the scope of protection of this invention.

[0033] The application of the above-mentioned mussel protein self-assembly adhesive coating in the preparation of cell culture scaffold materials is also within the scope of protection of this invention.

[0034] Beneficial effects:

[0035] This invention utilizes mussel protein and its mutants, obtained through microbial fermentation, as raw materials to form a uniform and dense adhesive coating under the induction of metal ions. The process is simple and easy to operate, and ion induction is effective for various mussel proteins. This invention develops a universal method for preparing self-assembled adhesive coatings from mussel proteins. The adhesive coating prepared using this method exhibits stable moisture resistance and tolerance to certain mechanical damage; it also possesses excellent biocompatibility, is non-toxic to cells and blood, and is biodegradable. This adhesive coating can serve as an ideal bioadhesive material and cell culture scaffold material, showing broad application prospects. Attached Figure Description

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0037] Figure 1 This is a macroscopic experimental diagram of the self-assembled adhesive coating of mussel protein in Example 2 of the present invention.

[0038] Figure 2 This is an AFM image of mussel protein and the formed adhesive coating in Example 2 of the present invention.

[0039] Figure 3 Mg in Example 3 of the present invention 2+ and Ca 2+ The effect on changes in mussel protein turbidity.

[0040] Figure 4 Mg in Example 4 of this invention 2+ and Ca 2+ The effect on the particle size variation of mussel protein.

[0041] Figure 5 This image shows the coatings on a glass plate, titanium plate, and PS plate being rinsed with a water tap in Embodiment 5 of the present invention. From left to right, the plates are labeled 3B. m and 3m via Ca 2+ Induced spots.

[0042] Figure 6 The surface morphology and residual area of ​​the coating after ultrasonic cleaning for 3 hours in Example 5 of this invention are calculated.

[0043] Figure 7 The surface morphology and residual area of ​​the coating after immersion in water for 10 days in Example 5 of the present invention are calculated.

[0044] Figure 8 The surface morphology of the coating after 2 days of protease digestion in Example 6 of this invention.

[0045] Figure 9 These are fluorescence microscopy images of L929 cells cultured for 1 day and 3 days with and without mussel protein coatings in Example 7 of this invention.

[0046] Figure 10 This is for calculating the viability of L929 cells in Example 7 of the present invention.

[0047] Figure 11 The images and statistical analysis of hemolysis rates for each group in Example 8 of this invention are shown. Detailed Implementation

[0048] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0049] In the following examples, the trypsin was purchased from Sinopharm Chemical Reagent Co., Ltd. (catalog number 64008860); the L929 cells were purchased from Nanjing Yifeixue Biotechnology Co., Ltd.; the mice were C57 mice, 7-8 week old male mice, weighing 24-26g, purchased from Spiefol (Beijing) Biotechnology Co., Ltd.; and the live / dead cell staining kit was purchased from Thermo Fisher Scientific Co., Ltd. (catalog number L32250).

[0050] Example 1: Construction of mussel protein combinatorial mutants

[0051] In the early stages, the laboratory conducted single mutations on the type 3 protein of wild-type thick-shelled mussels and the type 3B protein of wild-type Mediterranean mussels, respectively, and obtained the corresponding single mutants N33Y, N46Y and N62Y; G33Y, N43Y and N76Y.

[0052] In this embodiment, the mutation sites of the obtained wild-type thick-shelled mussel type 3 protein and wild-type Mediterranean mussel type 3B protein were further combined in pairs and in triplicates to obtain N33Y / N46Y, N33Y / N62Y, N46Y / N62Y and N33Y / N46Y / N62Y combined mutants; and G33Y / N43Y, N43Y / N76Y, G33Y / N76Y and G33Y / N43Y / N76Y combined mutants. The specific construction process of the combined mutants can be referred to the construction process of the single mutant in patent CN115181169A.

[0053] Single mutants and combined mutants of type 3 protein in wild-type thick-shelled mussels are collectively referred to as mutant 3. m Single mutants and combined mutants of the 3B protein in wild-type Mediterranean mussels are collectively referred to as mutant 3B. m .

[0054] The adhesive force of mussel protein obtained after expression, fermentation and purification of the above-mentioned combined mutants was measured using a glass overlap experiment. The results are shown in Table 1. The glass overlap experiment, expression of the combined mutants, and fermentation and purification process can be referred to the experimental process in patent CN115181169A.

[0055] Table 1. Adhesion strength of the combined mutant of wild-type thick-shelled mussel type 3 protein and wild-type Mediterranean mussel type 3B protein.

[0056]

[0057] The results showed that the wild-type thick-shelled mussel type 3 protein and the wild-type Mediterranean mussel type 3B protein, which underwent the 33 mutation, exhibited the best adhesion. Subsequent experiments were conducted using mutants of wild-type thick-shelled mussel type 3 protein, N33Y / N46Y / N62Y, and mutants of wild-type Mediterranean mussel type 3B protein, G33Y / N43Y / N76Y.

[0058] Example 2: Preparation and morphological observation of mussel protein self-assembled coating

[0059] The purified 3B(Mgfp-3B), 3B m (G33Y / N43Y / N76Y) and 3 m (N33Y / N46Y / N62Y) proteins were dissolved in 5% (v / v) acetic acid at a concentration of 1 mg / mL. 50 μL of metal ions (to a final concentration of 50 mM) were added to each 1 mL protein solution. To simulate a marine environment, potassium acetate, sodium acetate, magnesium acetate, and calcium acetate solids were weighed and dissolved in water to prepare corresponding solutions, providing K+ ions for each. + Na + Mg 2+ and Ca2+ After mixing the solutions, quickly transfer 30 μL of the mixture onto a glass slide, let it stand at room temperature for 3 hours, and then rinse thoroughly with ultrapure water. Stain the surface coating with Coomassie Brilliant Blue R-250 to make the coating visible. Place the remaining solution in a sample vial, let it stand for 3 hours, and then invert the vial.

[0060] When observing the microstructure of the coating using atomic force microscopy (AFM), a protein solution was first prepared to a concentration of 0.6 mg / mL. Then, 5 μL of metal ions with a final concentration of 5 mM were added to 100 μL of the protein solution. The mixture was transferred to mica and allowed to deposit completely on the surface for 12 hours. The morphology of the mica plate covered by the coating was then scanned using AFM.

[0061] Figure 1 Macroscopic experimental diagram of mussel protein self-assembly adhesive coating, showing 3B, 3B m and 3 m The aggregation states of proteins induced by different metal ions. The results showed that Mg... 2+ and Ca 2+ It can induce the formation of a protein coating, forming blue spots on the glass slide, while a distinct white coating appears on the bottom of the bottle, which is sticky and adheres to the bottom of the bottle.

[0062] Figure 2 AFM images of mussel proteins and the resulting adhesive coating confirm the relationship between them. Figure 1 The same experimental results showed that the protein was evenly distributed on the mica plate, K + Na + Without induction, there was no significant difference in protein morphology, Mg 2+ After induction, irregular aggregates form in the central region of the protein mica, Ca 2+ The induced mussel proteins self-assemble into a uniform and dense coating.

[0063] Example 3: Turbidity Assembly Dynamics Detection of Mussel Protein Self-Assembled Coating

[0064] The purified 3B(Mgfp-3B), 3B m (G33Y / N43Y / N76Y) and 3 m (N33Y / N46Y / N62Y) proteins were dissolved in 5% v / v acetic acid at a concentration of 1 mg / mL. 200 μL of the protein solution was then added to each solution with 10 μL of Mg(II) metal ions to a final concentration of 50 mM. 2+ and Ca 2+The mixed solution was placed in a microplate reader at room temperature, and the absorbance at 340 nm was measured. The absorbance of the aggregate at 340 nm was read at different time points. Turbidity (Δ340) was defined as T-To, where T and To are the absorbances of the protein solutions containing and without metal ions at 340 nm, respectively. Each sample was measured three times.

[0065] Figure 3 Mg 2+ and Ca 2+ The effect of Mg on the turbidity changes of three mussel proteins showed that 2+ and Ca 2+ It can induce protein aggregation and Mg 2+ The induction speed is faster, and each protein assembles rapidly within 1 hour. Self-assembly is basically completed by 3 hours, and the turbidity remains stable.

[0066] Example 4: Particle size detection of mussel protein self-assembled coating

[0067] The purified 3B(Mgfp-3B), 3B m (G33Y / N43Y / N76Y) and 3 m (N33Y / N46Y / N62Y) proteins were dissolved in 5% v / v acetic acid at a concentration of 1 mg / mL. 1 mL of each protein solution was then added to 50 μL of Mg(II) metal ions to a final concentration of 50 mM. 2+ and Ca 2+ The protein was transferred to a cuvette. Data was automatically read every two minutes at 25°C using a nanoparticle size analyzer, while the particle size of the protein solution without metal ions was measured.

[0068] Figure 4 Mg 2+ and Ca 2+ The effect of particle size changes on three types of mussel proteins is shown in the figure, confirming the influence of Mg. 2+ and Ca 2+ Induction of 3B, 3Bm, and 3m proteins, Mg 2+ The induced particle size is larger, approximately 255 nm, while Ca... 2+ The induced final particle size was 190 nm. This change in particle size demonstrates that Mg... 2+ Ca 2+ It interacted with mussel proteins, affecting the original structure of the mussel proteins.

[0069] Example 5: Moisture Resistance Test of Mussel Protein Self-Assembled Coating

[0070] Mussel protein mutant 3B m 3 mCompared to the main protein 3B, it has a similar secondary structure but higher viscosity, so this embodiment selects 3B. m 3 m The self-assembled coating was used to determine its moisture resistance. Furthermore, a uniform coating ensures that proteins maintain the same concentration and activity throughout the surface, preserving biological activity. Therefore, Ca was selected in subsequent studies. 2+ This triggers the formation of the coating.

[0071] Purified 3B m and 3 m Proteins were dissolved in 5% v / v acetic acid at a concentration of 1 mg / mL. 1 mL of the protein solution was then added to 50 μL of a 50 mM CaO metal ion solution. 2+ A mixed solution was obtained. 30 μL of the mixed solution was rapidly transferred to glass, titanium (Ti), and Ps plates and deposited at room temperature until completely dry. The slides were placed under running water or immersed in beakers for sonication and soaking. After staining, the coating morphology on the slides was observed using an optical microscope, and the coating area was calculated using ImageJ software.

[0072] Figure 5 Images showing the coatings on a glass plate, titanium plate, and PS plate rinsed with a tap, with the plates labeled 3B from left to right. m and 3 m via Ca 2+ The induced spots, shown in the figure, are treated with Ca. 2+ 3B after induction m 3 m It exhibits strong adsorption on various substrates, and the blue spots remain stable under water flow. Figure 6 The surface morphology and residual area of ​​the coating were calculated after 3 hours of ultrasonic cleaning. The figures show that the coating remained essentially stable on the glass plate after 3 hours of ultrasonic treatment. Furthermore, Figure 7 The surface morphology and residual area of ​​the coating after immersion in water for 10 days were calculated by... Figure 7 It can be seen that the coating showed only slight damage after 10 days of immersion, with an area retention rate exceeding 80%, demonstrating excellent wet stability. Overall, Ca... 2+ The induced coating exhibits strong adhesion under humid conditions.

[0073] Example 6: Biodegradability Test of Mussel Protein Self-Assembled Coating

[0074] The 3B prepared on the glass plate of Example 4 m 3 m The coating was placed in a petri dish containing 10 mL of trypsin solution (50 U / mL). After incubation at 37°C for 2 days, the substrate was washed with distilled water, and the surface coating was analyzed.

[0075] Figure 8 The image shows the surface morphology of the coating two days after trypsin digestion. It indicates that after trypsin digestion, the pores of the coating increased and the network structure became looser, suggesting that Ca... 2+ The induced coating can be degraded.

[0076] Example 7: Cell compatibility experiment of mussel protein self-assembled coating

[0077] mussel protein 3B m 3 m via Ca 2+ The induced mixed solution was frozen and then lyophilized. A solution with a density of 5 × 10⁻⁶ was prepared. 4 L929 cells / mL were seeded in 96-well plates containing DMEM medium and cultured at 37°C for 24 h in a 5% CO2 incubator. Freeze-dried mussel protein was dissolved in 5% v / v acetic acid and added to the DMEM cell culture medium at a final concentration of 1 μg / mL. A protein-free 5% v / v acetic acid solution was used as a negative control. Three replicates were provided for each group. After 1 and 3 days of culture, 20 μL of MTT (5 mg / mL) was added to the cell culture medium, and the cells were co-cultured for 4 h to form water-soluble formaldehyde. The absorbance was measured at 570 nm. Cell viability was calculated using the following formula:

[0078] Cell viability (%) = (AS / AC) × 100

[0079] AS and AC represent the absorbance of the sample and control cultured cells, respectively, with cell viability of the control defined as 100%. Live / dead cell staining of L929 cells was performed using the experimental procedures of a live / dead cell staining kit.

[0080] Figure 9 Fluorescence microscopy images of L929 cells cultured for 1 and 3 days with and without mussel protein coating, by [Author Name] Figure 9 It can be seen that as time goes on, the cells begin to proliferate. The cells in all protein-coated groups and the control group adhere to the wall and fully extend, with the cell morphology showing a spindle shape. Furthermore, the fluorescence images show that there are almost no dead cells in all groups. Figure 10 For L929 cell viability calculation, by Figure 10 It can be seen that, compared with the control group, the viability of L929 cells after co-incubation with the coating for 1, 2 and 3 days was greater than 99%, which confirms the good biocompatibility of the coating.

[0081] Example 8: Blood compatibility test of mussel protein self-assembled coating

[0082] Blood was collected from the tail vein of mice, and approximately 10 times the volume of 0.9% sodium chloride (v:v = 1:9) was added and gently mixed. The mixture was centrifuged at 1000–1500 rpm for 15 min, and the supernatant was removed. The precipitated red blood cells were washed 2–3 times with 0.9% sodium chloride solution until the supernatant no longer appeared red. The obtained red blood cells were then suspended in a 4% sodium chloride solution. The lyophilized protein solution was dissolved in 5% acetic acid and incubated with the cell suspension at 37°C for 1 h at a final concentration of 1 μg / mL. Ultrapure water and 5% acetic acid were used as positive and negative controls, respectively. The samples were centrifuged at 1000 rpm for 10 min, and hemolysis was observed visually. The absorbance of the supernatant was measured at 540 nm (n = 3). The formula for calculating the hemolysis rate is as follows:

[0083] Hemolysis rate = (AS–AN) / (AP–AN) × 100

[0084] Where AS, AN, and AP represent the absorbance of the sample, negative control, and positive control at 540 nm, respectively.

[0085] Figure 11 For each group of hemolysis test photos and statistical analysis of hemolysis rate, such as Figure 11 As shown, no obvious hemolysis was observed in the hemolysis experiment, indicating that the sample did not have any adverse interactions with blood components. Furthermore, the hemolysis rate of each protein coating was less than 5%, indicating that it is non-toxic to blood and meets the requirements for clinical application. Therefore, it can be used as an ideal bioadhesive material and cell culture scaffold material in clinical practice.

[0086] This invention provides a concept and method for the self-assembly adhesive coating of mussel protein, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A self-assembling adhesive coating for mussel proteins, characterized in that, The mussel protein self-assembly adhesive coating is obtained by adding a solution containing metal ions to a mussel protein solution to obtain a mixed solution, and then allowing the mixed solution to stand. The concentration of the mussel protein solution is 0.2-1 mg / mL, and the solvent is 5-25% v / v acetic acid; the concentration of the metal ions in the solution containing the metal ions is 5-50 mM. The mussel protein is a mutant of type 3 protein from wild-type thick-shelled mussels. m 3B mutant of the 3B protein in wild-type Mediterranean mussels m Any one or a combination of two of them; The metal ion is Mg. 2+ or Ca 2+ .

2. The mussel protein self-assembly adhesive coating according to claim 1, characterized in that, The volume ratio of the mussel protein solution to the solution containing metal ions is 20~200:

1.

3. The mussel protein self-assembly adhesive coating according to claim 1, characterized in that, The concentration of the mussel protein solution was 1 mg / mL, and the solvent was 5% v / v acetic acid.

4. The mussel protein self-assembly adhesive coating according to claim 1, characterized in that, The concentration of the metal ions in the solution containing the metal ions is 50 mM.

5. The method for preparing the mussel protein self-assembly adhesive coating according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Dissolve mussel protein in acetic acid solution to obtain mussel protein solution; S2. Add a solution containing metal ions to the mussel protein solution obtained in S1 to obtain a mixed solution; S3. Let the mixed solution obtained in S2 stand to obtain the mussel protein self-assembled adhesive coating.

6. The preparation method according to claim 5, characterized in that, In step S3, the standing time is 1 to 6 hours at room temperature.

7. The application of the mussel protein self-assembly adhesive coating according to any one of claims 1 to 4 in the preparation of bioadhesive materials.

8. The application of the mussel protein self-assembly adhesive coating according to any one of claims 1 to 4 in the preparation of cell culture scaffold materials.

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