Preparation of novel double cross-linked hydrogels of dietary fibers and proteins from soybeans as scaffolds for cultured meat

Preparation of novel double cross-linked hydrogels of dietary fibers and proteins from soybeans as scaffolds for cultured meat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preparation of novel double cross-linked hydrogels of dietary fibers and proteins from soybeans as scaffolds for cultured meat Huicheng Fang, Wei Yu, Boyan Gao, Yuge Niu, Liangli Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4459544/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The composited hydrogels derived from natural materials are getting attention in the field of cultured meat due to their advantages of biocompatibility and degradability as cell scaffolds. In this work, two edible cross-linking agents, transglutaminase (TGase) and/or calcium ions, were successfully used to cross-link soy protein isolated (SPI) and soy dietary fiber (SDF) to fabricate different scaffolds. The prepared scaffolds were characterized by structural, hydration, rheological and mechanical analysis. The double cross-linked scaffolds exhibited highest compressive moduli compared to the single cross-linked scaffolds and had an excellent liquid absorbing ability up to 309.45%, while its porosity was as high as 72.66%. In addition, NIH 3T3 cells were used to evaluate the biocompatibility of the scaffolds in vitro . The double cross-linked scaffolds could promote the expression of differentiation-related genes and were beneficial for cell adhesion and proliferation. In conclusion, present research provides a new approach to prepare cell scaffolds using soybean resources, which could be used in cultured meat applications. Cross-linking technology Soybean Dietary fibers Soy proteins Cultured meat scaffolds Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION One focus area of the production of cultured meat is fabricating constructs as extracellular matrix (ECM) for culture cells (Xiang et al., 2022 ). ECM can provide cells with extremely complex three-dimensional microenvironment and different biological cues (Berger et al., 2020 ). The complexity supports muscle cells in growth, migration, and assembly into muscle tissue. The artificial scaffolds can mimic ECM in terms of structure, chemistry, and mechanical properties. For porous scaffolds, porosity and interconnected pores are essential to facilitate cell migration, cell growth and nutrient flow (Afjoul et al., 2020 ). An ideal scaffold should have good mechanical properties and high biocompatibility. Furthermore, the scaffold must be intrinsically edible (Holmes et al., 2022 ). Conventional scaffolds usually are synthetic materials. However, some of them may lack biological cues, or have the unqualified mechanical properties, nor be biodegradable (Lin et al., 2013 ). Taking these limitations into consideration, safe renewable sources, such as food-derived proteins and polysaccharides, are increasingly being mentioned as scaffold materials for cultured meat. Textured soy protein was used as a novel scaffold because it could support cell attachment and proliferation to create a 3D-engineered bovine muscle tissue (Ben-Arye et al., 2020 ). Soy protein isolated (SPI), a purified protein from soybean, has recently been used as an attractive raw material for synthetic materials with other reagents as scaffolds (Chien et al., 2013 ; Tansaz & Boccaccini, 2016 ; Wu et al., 2020 ). SPI exhibits good processing characteristics, especially its outstanding biodegradability and biocompatibility (Santin & Ambrosio, 2008). Chien and Shah fabricated porous 3D SPI scaffolds using the freeze-drying method to culture cells (Chien & Shah, 2012 ). Different strategies have been developed to modify the processing characteristics of SPI. Polysaccharides are often added in protein solutions to obtain a protein-polysaccharides mixed system (Nishinari et al., 2000 ). The two components play synergistic role in the hydrogel system (Hu et al., 2017 ). Soybean soluble dietary fiber (SDF) as a polysaccharide is also derived from soybean. TGase are often used to cross-link proteins in food industry (Kieliszek & Misiewicz, 2014 ). As an indispensable cross-linking agent in the process of tofu, calcium ions can also cross-link both SPI and SDF (Fang et al., 2021 ). The previous research confirmed that the scaffold based on gellan gum-gelatin and further cross-linked with high concentrations of Ca 2+ had higher biocompatibility than non-cross linked scaffolds. It was more conducive to cell spreading (Chen et al., 2023 ). The use of “green” cross-linking agents avoids the use of toxic agents, and can match the desirable mechanical property of scaffolds. These methods take the advantages of both protein and polysaccharide, which make high utilization of natural renewable materials. This study provides a method to combine soybean protein and soybean dietary fiber with two cross-linking agents to prepare a novel scaffold for the production of cultured meat. The mechanical properties, surface morphology, infrared spectroscopy and water absorption of the scaffolds were determined. Mouse fibroblasts were subjected to cell proliferation experiments, cell morphology experiments and cell death staining analysis. SPI-SDF hydrogels are expected to be used as an effective scaffold for the production of cultured meat due to its ideal mechanical strength and cytocompatibility. 2. MATERIALS AND METHODS 2.1 Materials Soybean protein isolate (SPI) (protein content ≥ 90%) was purchased from Macklin Inc. (Shanghai, China). Soybean dietary fiber (SDF) was a gift of Henan Wan Bang Industrial Co. Ltd (Henan, China). Transglutaminase (TGase, 100 U/g, Food Grade) was purchased from Jiangsu Yiming Biological Co., Ltd. (Jiangsu, China). Glycerol was purchased from Sigma–Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 assay kit (CCK-8) was purchased from Dojindo China Co., Ltd (Shanghai, China). Live/Dead assay kit assay and the cDNA kit (iScriptTM cDNA Synthesis Kit) were purchased from Thermo Scientific (Waltham, MA, USA). Rhodamine phalloidin and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.2 SPI-SDF scaffolds fabrication The SPI-SDF scaffolds were prepared according to a laboratory protocol (Fang et al., 2021 ). SPI solution of 5% (w/w) in deionized water were homogenized at 8000 rpm for 5 min. After using 6 M NaOH to adjust the pH of the SPI solution to pH 7.5, the solution was preheated in water bath at 100 ℃ for 1 h to fully denature soybean protein. The solution was then cooled to ambient temperature. The glycerol of 5% (w/w) was added as plasticizer. According to Table 1 , the SDF were added correspondingly to obtain mixed samples. The mixtures were then homogenized at 8000 rpm for 5 min. TGase and/or calcium chloride were added to each sample at different amounts recorded in Table 1 . For TG-induced scaffolds, the mixtures were incubated in incubator at 55 ℃ for 1 h to complete the reaction of enzyme cross-linking. For Ca-induced scaffolds, the 2 M calcium chloride was added directly into the mixtures to adjust the final calcium ion concentration to 100 mM followed by vortex for 5 min. The slurries were frozen at -25 ℃ overnight and then freeze-dried. Table 1 Formulation of SPI-SDF scaffolds Sample SPI (w/v) SDF (w/v) TGase (w/v) CaCl 2 (w/v) a M-TG b 5% 0.5% 5.5% 0 M-Ca c 5% 0.5% 0 5.8% M-TG-Ca d 5% 0.5% 5.5% 5.8% a Calcium chloride concentration was 2 M. b M-TG: Mixture of SPI and SDF with single cross-linked by TGase. c M-Ca: Mixture of SPI and SDF with single cross-linked by Ca 2+ . d M-TG-Ca: Mixture of SPI and SDF with double cross-linked by TGase and Ca 2+ . 2.3 Water uptake capacity The water uptake capacity of the scaffolds was measured according to the previous report (Perumal et al., 2014 ). Each scaffold was immersed in the phosphate buffered saline (PBS) solution for 30 min. Then, the scaffold was removed from the liquid. The excess liquid was dried with filter paper. The weight of dry or wet scaffold was measured. Measurements for each scaffold were repeated five times. The absorption ratio was then calculated by the following equation: $$\text{Water uptake capacity=}\frac{\text{(}{\text{M}}_{\text{1}}\text{-}{\text{M}}_{\text{0}}\text{)}}{{\text{M}}_{\text{0}}}\text{×100%}$$ 1 where M 0 is the weight of the dry scaffold and M 1 is the weight of wet scaffold. 2.4 Rheological test of scaffolds The rheological properties of SPI-SDF solutions were studied employing an AR G2 rheometer (TA Instrument, USA). All samples of M-Ca and M-TG-Ca group were determined using the 40 mm titanium flat plates with a gap of 500 µm. Since the modulus of M-TG was relatively low, the plates were applied with a gap of 28 µm. The linear viscoelasticity region of scaffolds was obtained by strain-sweep measurements. The frequency-sweep measurements were performed at a fixed strain of 1% from 0.1 to 10 rad/s. All the experiments were conducted at the temperature of 25 ℃. To prevent water evaporation, the shear viscosity was measured at shear rates from 0.01 to 100 s − 1 . 2.5 Mechanical characterization In order to investigate the mechanical properties of the SPI -SDF scaffolds, both dry and wet samples (n = 5, respectively) were measured through compression test. The compression testing was conducted on TA-XT plus texture analyzer (Stable Micro Systems, UK). Each Scaffold was prepared as a cylinder (diameter: 25 mm; height: 10 mm). Samples were compressed at 0.5 mm/s up to 10% strain. The compressive modulus of scaffold was determined by TA texture analysis software. The wet scaffolds were prepared by completely immersed in PBS for 1 h until measurement. Both dry and water-hydrated scaffolds were tested. Measurements for each scaffold were repeated five times. 2.6 Porosity analysis The porosity analysis was conducted using liquid replacement method according to the previous report (Li et al., 2021 ). The cylindrical samples of scaffold formulations (diameter: 20mm, height: 8mm, n = 5) were fabricated. The volume of the scaffold was measured and calculated by a digital Vernier caliper. The dry weight (W i ) of the scaffold was measured by electronic analytical balance. The samples were placed in a graduated cylinder containing 30 mL of isopropanol (ρ i = 0.785 g/mL) under vacuum for 15 min to allow the liquid to fully infiltrate. The weight (W f ) of scaffolds filled with isopropanol was measured. Measurements for each scaffold were repeated three times. The porosity was calculated by the following formula: \(\text{Porosity=}\frac{\text{(}{\text{W}}_{\text{f}}\text{-}{\text{W}}_{\text{i}}\text{)/}{\text{ρ}}_{\text{i}}}{{\text{V}}_{\text{i}}}\text{×100%}\) (2). 2.7 Scanning electron microscopy (SEM) The SPI -SDF scaffolds were sputter coated with Au and were scanned at a voltage of 5 kV on Sirion200 SEM (FEI Company, USA). 2.8 Fourier Transform infrared spectroscopy (FT-IR) FT-IR analysis for the determination of functional groups of materials was conducted in an IR/Nicolet 6700 Fourier-transform infrared spectrometer (FT-IR) (Thermo Fisher Scientific, American). The device was equipped with an attenuated total reflectance (ATR). The 1 mg of each sample and 100 mg of KBr were mixed and then tableted. The spectra frequency range was from 500 to 4000 cm − 1 . The resolution was 4 cm − 1 . No. of scans was 32. 2.9 Cell adhesion Cell adhesion was examined using a commercial bioluminescent stain (Sigma-Aldrich, St. Louis, MO, USA). In brief, the NIH 3T3 cells were cultured in the Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% antibiotic mix of penicillin-streptomycin and 10% fetal bovine serum (FBS). The NIH 3T3 cells were then incubated in an incubator at 37 ℃ with a humidified atmosphere of 5% CO 2 . The scaffolds were sheared into regular shape with 10 mm*10 mm*1 mm. Then the scaffolds were sterilized with 75% ethanol overnight and washed 3 times by PBS for total 3 h. The fresh DMEM were added to immerse the scaffold overnight to pre-wet the scaffolds. The cells at the logarithmic growth phase were collected with tryp-sin-EDTA. For each scaffold, the NIH 3T3 cells were seeding at a cell density of 1 × 10 4 cells/mm 2 . In order to examine the cell adhesive behavior of the scaffolds, after incubation for 24 h, the cells were fixed with 4% formaldehyde at ambient temperature for 30 min. After being washed three times by PBS, rhodamine phalloidin and DAPI were used to stain the F-actin and nucleus of cells, respectively. The scaffolds were imaged using a Leica SP5 confocal microscope (Leica, Germany). 2.10 Cell proliferation and viability assays The culture and seeding procedures for the evaluation of cell proliferation and viability assay on the scaffolds were performed using a commercial kit. To examine the cell viability of the scaffolds, the proliferation of NIH 3T3 on the scaffolds was determined by a Cell Counting Kit-8 assay kit ((CCK-8), Dojindo, Japan) using well plates as a control. After 1 and 3 days of culture, the original medium was replaced by the new DMEM medium (10% CCK-8). The system was incubated at 37 ℃ with a humidified atmosphere of 5% CO 2 for 1 h. The absorbance of each well was measured at 450 nm. Measurements for each scaffold were repeated three times. For live/dead detection, the cells were stained by Live/Dead assay kit assay (Thermo Scientific, USA) according to the manufacturer's recommendations. 2.11 Reverse transcription-polymerase chain reaction (RT-PCR) NIH 3T3 cells were co-cultured with the scaffold material. The cells were seeded on a 6-well plate at a density of 1 × 10 6 cells/mm 2 . After co-cultured with scaffolds for 2 days, the harvested cells were collected and washed by PBS. Total RNA was extracted from the cells with TRIzol. Chloroform and isopropanol were added to isolate and precipitate the RNA, respectively. The RNA extract was purified with 75% DEPC-ethanol water by three times. The purified RNA was then resuspended in 20 µL of DEPC-treated water and quantified by Nano Drop spectrophotometer (Thermo Scientific, USA). The ratio of OD 260 and OD 280 usually exceeded 1.8 at this stage. The cDNA synthesis was followed by the manufacturer's instructions using the cDNA kit (iScriptTM cDNA Synthesis Kit) for reverse transcription of total RNA. 10 µL of total RNA, 4uL of 5×iScript Reaction Mix, 1uL of iScript Reverse Transcriptase, 5 µL of DEPC water were mixed to obtain 20 µL of solution. RT-PCR was done on the ABI 7900 HT (Applied Biosystems, Carlsbad, CA, USA). The amplification parameters used for PCR were as follows: 50 ℃ for 2 min, 95 ℃ for 10 min, and 46 cycles of amplification at 95 ℃ for 15 s and 60 ℃ for 1 min. Statistical analysis of the quantitative real time PCR was performed using the 2 −ΔΔCt method. GAPDH was used as a reference gene and amplified with the target gene in the same cDNA sample. Measurements for each scaffold were repeated three times. The forward and reverse primer sequences are shown in supplementary Table S1 . 2.12 Statistical analysis All data were analyzed using SPSS version 22.0 (IBM Co., USA) by one-way ANOVA. And P < 0.05 was regarded as being statistically significant. The differences among groups were determined with a Duncan means test. 3. RESULTS 3.1 Rheological behavior of SPI-SDF scaffolds Rheological test is an effective way to investigate the microstructure of materials. The storage modulus (G′) and loss modulus (G″) of scaffolds were shown in the Fig. 1 a. In the angular frequency sweeping model, all the scaffolds showed solid-like properties since the G′ of the samples was higher than the G″. The trends of G′ and G″ were almost stable in the angular frequency sweeping range. It indicated that all the samples were ideal gels because structural relaxation does not occur. This phenomenon suggested that the addition of TGase and calcium ions allowed the samples to form a gel network, which caused the samples to take longer time to relax. The G′ of M-TG-Ca sample was significantly higher than that of M-Ca and M-TG. For example, when angular frequency is 0.1 rad/s, G′ of the M-TG-Ca sample was 23 Pa, which was nearly 10 times stiffer than the M-Ca sample. This also indicated that the double cross-linking gels of M-TG-Ca had greater stiffness than single linked gels of M-TG or M-Ca. The result was consistent with our previous research (Fang et al., 2021 ). The frequency dependence of G′ and G″ was described by a power-law equation (Liao et al., 2015 ): $${\text{η}}^{\text{*}}\text{(}\text{ω}\text{)=}\frac{{\text{(}{\text{G'}}^{\text{2}}\text{+}{\text{G''}}^{\text{2}}\text{)}}^{\frac{1}{2}}}{\text{i}\text{ω}}$$ 3 $${\text{η}}^{\text{*}}\text{(}\text{ω}\text{)=}{\text{K}}_{\text{d}}{\text{ω}}^{\text{-}{\text{n}}_{\text{d}}}$$ 4 where ω is the angular frequency and η * ( ω ) is the complex viscosity. The constant K d stands for dynamic consistency index, whereas the exponent n d is the dynamic power-law factor. Figure 1 b showed the variation of shear viscosity of the scaffolds with shear rate in the form of double logarithmic coordinates. The relationship between viscosity and shear rate can also be described by a power-law equation: $$\text{η}\text{(}\text{γ}\text{)}\text{=}{\text{K}}_{\text{s}}{\gamma }^{{\text{n}}_{s}-1}$$ 5 where γ is the shear rate and η ( γ ) is the shear viscosity. The constant K s stands for shear consistency index, whereas the exponent n s is the shear power-law factor. In Table 2 , the n s of all samples are less than 1, which indicated that all of the scaffolds were pseudoplastic non-Newtonian fluid type and had shear thinning characteristics. M-TG scaffolds had the largest n d and the smallest n s , meaning that it was more elastic and non-Newtonian than the other two scaffolds. And M-TG-Ca had highest K d and K s , which further indicated that it had strongest mechanical property. In conclusion, SPI and SDF could form interpenetrating cross-linking networks by TGase and calcium ions. M-TG-Ca scaffolds formed by double cross-linking may have higher mechanical strength to support cell growth. Table 2 Regression parameters representing viscoelasticity of different SPI-SDF scaffolds M-TG M-Ca M-TG-Ca K d 0.067 3.685 331.538 n d 0.873 0.611 0.803 R 2 0.995 0.961 0.998 K s 0.078 0.279 0.868 n s 0.030 0.209 0.148 R 2 0.997 0.996 0.984 K d , n d , K s and n s were obtained by curve-fitting using Equations ( 3 ), ( 4 ) and (5) and based on the data presented in Fig. 1 . 3.2 Porosity analysis and morphology of scaffolds Porosity is particularly important for the cell culture during the production of cultured meat. The materials with high porosity can provide cells large surface areas and are more conducive to the transportation of nutrients to promote the growth of cells (Chong et al., 2007 ; Lanno et al., 2020 ). In order to determine the porosity of the scaffolds, isopropanol was used as displacement liquid because the scaffolds will not swell or shrink when immersed in the solvent. The porosity of different scaffolds was presented in Table 3 . The M-TG-Ca scaffold showed the porosity of 72.66%. The M-TG scaffold showed the porosity of 71.87% and the M-Ca showed the porosity of 69.44%. The M-Ca scaffold had the lowest porosity. There is no significant difference between the porosity of scaffolds induced by TGase. This may be due to the stronger density of covalent bond type cross-linking than ionic bond type. So, the additive of Ca 2+ did not significantly influence the porosity of the scaffolds bonded by TGase. The microstructure and porosity of scaffolds are usually related to each other. The microstructure of scaffolds was presented in Fig. 2 . The surface morphology of M-TG was similar with that of M-TG-Ca. The result was consistent with the porosity analysis. In Fig. 2 C, the struts of M-TG-Ca appeared thicker compared with M-TG scaffolds. Figure 2 B showed that the morphology of M-Ca was significantly different and exhibited looser and discontinuous network. Compared Fig. 2 A with Fig. 2 B, the pore surface of M-Ca was rough, while the surface of M-TG samples was smooth. The pore shapes of all samples were irregular. The small pores of scaffold could help cell adhesion and infiltration. Meanwhile, the large pores could facilitate cell growth since the oxygen can be transferred to the host easier (Ninan et al., 2013 ). The pore size within the field of view in Fig. 2 was measured using the image J software (NIH, USA). The pore diameter of M-Ca scaffold ranges from 26 to 60 µm with a median pore size of 38 µm. The pore diameter of M-TG scaffold ranges from 25 to 66 µm with a median pore size of 40 µm. The pore diameter of M-TG-Ca ranges from 23 to 50 µm with a median pore size of 34 µm. The pore size of M-TG-Ca was smallest which may be due to the highest extent of cross-linking, further explained the difference between double cross-linked scaffolds and single cross-linked scaffolds. To sum up, M-TG-Ca scaffolds had the highest porosity and the smallest pore size, which may allow M-TG-Ca to be attached by more cells. Table 3 Characterization of SPI-SDF scaffolds Porosity (%) Water uptake capacity (%) Compressive moduli (Kpa; wet) Compressive moduli (Kpa; dry) M-TG 71.87 ab ± 3.53 413.94 a ± 13.08 0.94 c ± 0.18 6.33 c ± 0.55 M-Ca 69.44 b ± 2.23 318.82 b ± 2.57 1.32 b ± 0.27 19.05 b ± 1.76 M-TG-Ca 72.66 a ± 0.90 309.45 b ± 2.51 3.04 a ± 0.04 80.29 a ± 9.35 Data are presented as mean ± SD (n = 5). Values with different letters were significantly different ( P < 0.05). 3.3 Water up taking capacity Excellent water up taking capacity are vital to hydrogel scaffolds. The high capacity on water up taking of scaffolds can keep the cells in the moist environment. Meanwhile, the strong ability of water absorption can provide nutrients for the colonized cells and elimination of waste metabolites (Han et al., 2014 ). These features can enhance cell proliferation. According to Table 3 , M-TG had the highest liquid absorption ability which may be due to the high hydrophilicity and large pore size of M-TG scaffolds. Polysaccharides are more discrete and have higher degrees of freedom than proteins. So, polysaccharides in M-TG scaffolds that were not cross-linked by calcium ion could absorb more water. The cross-linking of M-TG-Ca scaffolds is more intensive resulting in the low water up taking capacity. The water absorption rate of the M-TG sample was up to 413.94%, while the water absorption rates of the other two samples were both higher than 300%. These results showed that these scaffolds have strong ability of fluid absorption and retention. For cells, the scaffolds could be conducive to supply the essential nutrients and eliminate the excessive metabolites in the process of the production of cultured meat. Therefore, considering the good water up taking capacity, all scaffolds including M-TG-Ca scaffolds can be used as a potential material in the production of cultured meat. 3.4 Compressive modulus of scaffolds The compression modulus can reflect the mechanical property of the scaffolds. The dry M-TG-Ca scaffold had the highest compressive modulus, which was 12.68 times higher than that of the dry M-TG and 4.21 times higher than the dry M-Ca (Table 3 ). The compressive modulus of all SPI-SDF scaffolds in wet state decreased compared to them in dry state. The treatment of TGase combined with calcium ions significantly increased the compressive modulus of both dry and wet scaffolds. Good mechanical property is essential for cell culture since the high compressibility can provide a dynamic microenvironment for cell attaching and thus to regulate the cell behavior (Chung et al., 2002 ). In conclusion, these results indicated that double cross-linking methods could significantly increase the mechanical property of scaffolds. The result was consistent with rheological test. So, M-TG-Ca scaffolds, cross-linked by two kinds of agents, are suitable for cultured meat. 3.5 FT-IR analysis The FT-IR spectra of SPI, SDF, and the SPI-SDF scaffolds were showed in Fig. S1 as supplementary materials. The spectrum showed the characteristic peaks of the SPI including the peaks at 1658 cm − 1 (amide I, C = O) and 1533 cm − 1 (amide II, N–H). The pure SPI spectrum also showed an absorption band at 3302–3150 cm − 1 . This absorption represented the stretching vibration of the OH group, which may be attributed to the hydrogen bond and the moisture of the SPI (Qi et al., 2018 ). Characteristic peaks in the SDF included the peaks at 3420 cm − 1 (O–H stretching) and 1646 cm − 1 (C = O) (Qi et al., 2018 ). From the spectra of SPI-SDF scaffolds, it can be found that all the characteristic absorption bands of SPI and SDF were existed in the FTIR spectra. This indicated that the presence of SPI and SDF in the scaffold matrix. It also showed that there was no significant difference among SPI-SDF scaffolds formed with different cross-linking agents, suggesting that the chemical compositions had no detectable changes. 3.6 Cell proliferation of the SPI-SDF scaffolds The cell proliferation of the composite scaffolds was evaluated using CCK assay. The results of cell proliferation of the SPI-SDF scaffolds were presented in Fig. 3 . The proliferation of NIH 3T3 on the scaffolds was observed and quantified after incubation from 1 to 3 days. The results showed that the cell number of NIH 3T3 in all scaffolds continued to increase from the first day to the third day, indicating that the scaffold was conducive to cell proliferation. Moreover, the cell viability of M-TG-Ca was significantly higher than those of M-Ca and M-TG scaffolds ( P < 0.05). It may be related to the excellent mechanical strength of M-TG-Ca. The interconnected pores of scaffolds can provide cells with a 3D environment and offer the nutrient, as well as transportation channels of metabolic waste (Tufan et al., 2021 ). Therefore, the cell could grow well on the scaffolds. The cell proliferation on the scaffolds is extremely important since it provides evidences that muscle tissue can grow on SPI-SDF scaffolds, which is the first step of the production of cultured meat. Therefore, M-TG-Ca scaffolds have great potential to apply as cultured meat scaffolds. 3.7 Cell viability In order to further investigate the impact of scaffolds on the cell proliferation and the cytotoxicity, the NIH-3T3 cells after being cultured on scaffolds for day 1, 2 and 3, were harvested and then stained using Live/Dead Reagent. The live cells were stained with green color by calcein-AM. The dead cells were stained with red color by ethidium homodimer. Live/Dead fluorescence images showed similar results to CCK-8. Figure 4 showed that most of the cells seeding on scaffolds were stained with green, which indicated that NIH 3T3 cells were able to adhere on the SPI-SDF scaffolds and maintained excellent viability. Meanwhile, the number of live cells of all the scaffolds were increased at the third day, which presented the cell proliferation on the scaffolds. Therefore, these findings suggested that the SPI-SDF scaffolds presented excellent cytocompatibility in vitro and can support the adhesion and proliferation of NIH 3T3 cells. It was demonstrated that all three kinds of scaffolds, including M-TG-Ca scaffolds, could be used as cultured meat scaffolds for cell growth. 3.8 Cell morphological characterizations The scaffolds will dramatically affect the cell behaviors shown as cell morphology (Singhvi et al., 1994 ). Therefore, the cytoskeletal organization of cells attached on the scaffold was then investigated. The NIH 3T3 morphologies on M-TG, M-Ca and M-TG-Ca scaffolds were investigated after culturing for 24 h. To observe the morphology of NIH 3T3 on the scaffolds, rhodamine-phalloidin and DAPI were used to stain F-actin and nuclei, respectively. As shown in Fig. 5 , NIH 3T3 with a typical morphology adhered and spread on all the scaffolds. Meanwhile, there were no distinguishable differences among the cell morphologies adhered on the scaffolds. These results indicated that the cross-linking types of SPI-SDF scaffolds had little impact on the cell morphology of NIH 3T3. Furthermore, the F-actin filaments morphology of NIH-3T3 on the three scaffolds were all well-formed. The NIH 3T3 had contacted each other and formed a cell sheet on all scaffolds. The F-actin filament of NIH 3T3 can target cell proliferation to increase cell’s function and thus plays a key role in the early maturation of cells (Yun et al., 2010 ). Therefore, the well-grown F-actin indicated that the scaffolds could promote cell proliferation and cell differentiation at the same time. Surprisingly, M-Ca scaffolds showed the best ability to promote F-actin formation. At the same time, M-TG-Ca also could promote cell proliferation and differentiation greatly as scaffolds for cultured meat. 3.9 RT-PCR Integrin, which plays a vital role in cell migration and cell adhesion and proliferation, is the main receptor of ECM protein (Pan et al., 2013 ). Increased expression of integrin β1 (Fig. 6 a) was due to the increase of the migration of cells, which could promote cell proliferation and differentiation in progress of the production of cultured meat. The M-TG-Ca scaffolds group had the highest expression of integrin β1, which indicated that the M-TG-Ca scaffold showed great ability to promote cell migration. According to Fig. 6 b, the expression of integrin αV on the SPI-SDF scaffolds were all down-regulated, which suggested the scaffolds had less help on enhancing the adhesion of endothelial cells. Differentiation-related genes expression in cell were shown in the Fig. 6 c and Fig. 6 d. The expression of α-SMA indicated that the expression of collagen-related genes increased, which led to the deposition of collagen (Chogan et al., 2020 ). Compared with the M-TG-Ca and M-Ca groups, NIH 3T3 cell cultured in the M-TG scaffolds group showed the lowest expression of α-SMA. It indicated that M-TG group had lower ability to promote cell differentiation, which was consistent with the morphological characterizations result. In Fig. 6 c, the expression of α-SMA in the M-Ca group a was higher than that in the M-TG and M-TG-Ca groups. It also explained why M-Ca scaffolds had the strongest ability of F-actin formation in morphological characterization result. It could be seen from the Fig. 6 d that M-TG-Ca up-regulated the expression of β-catenin gene relative to M-Ca and M-TG. β-catenin can activate the transcription of MRFs, which is the signal pathway regulating the differentiation of muscle cells (Guan et al., 2022 ). According to the results of gene expression, M-TG-Ca scaffolds through double cross-linking methods had the great potential to promote cell differentiation, which meant it was suitable as cultured meat scaffolds. 4. CONCLUSION The novel SPI-SDF cross-linked hydrogels with three-dimensional porous structure by different crosslinking methods were prepared and be applied as scaffolds for cultured meat. Soy protein isolated and dietary fiber are both from soybeans, avoiding the use of synthetic polymers. Meanwhile, the cross-linking reagents, the enzyme and calcium ions, were in the list of food additives. Therefore, the scaffolds prepared in this study were safe, biocompatible and edible. The results showed that the physical and chemical properties of porous SPI-SDF scaffolds can be changed by using one or both of cross-linking agents. TGase cross-linked scaffolds exhibited excellent porosity and liquid absorption. TGase combined with calcium ions further enhanced the mechanical strength of the scaffolds. According to the biocompatible assays of the scaffolds, the SPI-SDF scaffolds could support cell proliferation and adhesion, which was the first step in large-scale production of cultured meat. The results of PCR represented that the scaffolds could promote the expression of differentiation-related genes. Among them, M-TG-Ca scaffolds formed by double cross-linking had the best physical properties and showed great cellular compatibility. Such double cross-linked scaffolds may have limits. For instance, the preparation of these scaffolds requires the freeze-drying process, which consumes high energy leading to the huge cost of cultured meat in industrial production. We envision that such double cross-linked hydrogels will provide a new candidate for the potential scaffolding materials from natural resources in the production of cultured meat. Declarations Conflict of Interest The authors declare no competing interests. FUNDING This research was funded by the National Key R&D Program of China, China (Grant No. 2022YFD2101304). Author Contribution Huicheng Fang: Investigation, Formal analysis, Data Curation, Writing - Original Draft, Visualization.Wei Yu: Investigation, Formal analysis, Data Curation, Writing - Original Draft, Visualization. Boyan Gao: Resources, Software, Validation.Yuge Niu: Conceptualization, Funding acquisition, Methodology, Project administration, Writing - Review & Editing.Liangli Yu: Supervision. References Afjoul, H., Shamloo, A., & Kamali, A. (2020). Freeze-gelled alginate/gelatin scaffolds for wound healing applications: An in vitro, in vivo study. Materials Science and Engineering C-Materials for Biological Applications 113 , 110957. https://doi.org/10.1016/j.msec.2020.110957. Ben-Arye, T., Shandalov, Y., Ben-Shaul, S., Landau, S., Zagury, Y., Ianovici, I., Lavon, N., & Levenberg, S. (2020). 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ACS Omega 5 , 30011-30022. https://doi.org/10.1021/acsomega.0c04402. Li, T. T., Zhang, Y., Ren, H. T., Peng, H. K., & Lin, J. H. J. C. P. (2021). Two-step strategy for constructing hierarchical pore structured chitosan–hydroxyapatite composite scaffolds for bone tissue engineering. Carbohydrate Polymers 260 , 117765. https://doi.org/10.1016/j.carbpol.2021.117765. Liao, H., Ai, W., Zhang, K., Nakauma, M., Funami, T., Fang, Y., Nishinari, K., Draget, K. I., & Phillips, G. O. (2015). Mechanisms of oligoguluronate modulating the calcium-induced gelation of alginate. Polymer 74 , 166-175. https://doi.org/10.1016/j.polymer.2015.08.007. Lin, L., Perets, A., Har-el, Y. E., Varma, D., Li, M., Lazarovici, P., Woerdeman, D. L., & Lelkes, P. I. (2013). Alimentary 'green' proteins as electrospun scaffolds for skin regenerative engineering. Journal of Tissue Engineering and Regenerative Medicine 7 , 994-1008. https://doi.org/10.1002/term.1493. Ninan, N., Muthiah, M., Park, I. K., Elain, A., Wong, T. W., Thomas, S., & Grohens, Y. (2013). Faujasites incorporated tissue engineering scaffolds for wound healing: in vitro and in vivo analysis. ACS Applied Materials & Interfaces 5 , 11194-11206. https://doi.org/10.1021/am403436y. Nishinari, K., Zhang, H., Ikeda, S. J. C. O. i. C., & Science, I. (2000). Hydrocolloid gels of polysaccharides and proteins. Current Opinion in Colloid & Interface Science 5 , 195-201. https://doi.org/10.1016/S1359-0294(00)00053-4. Pan, H.A., Liang, J.Y., Hung Y.C., Lee C.H., Chiou J.C., Huang G.S. (2013). The spatial and temporal control of cell migration by nanoporous surfaces through the regulation of ERK and integrins in fibroblasts. Biomaterials 34 , 841-853. https://doi.org/10.1016/j.biomaterials.2012.09.078. Perumal, S., Ramadass, S., & Madhan, B. (2014). Sol-gel processed mupirocin silica microspheres loaded collagen scaffold: a synergistic bio-composite for wound healing. European Journal of Pharmaceutical Sciences 52 , 26-33. https://doi.org/10.1016/j.ejps.2013.10.006. Qi, X., Yuan, Y., Zhang, J., Bulte, J. W. M., & Dong, W. (2018). Oral Administration of Salecan-Based Hydrogels for Controlled Insulin Delivery. Journal of Agricultural and Food Chemistry 66 , 10479-10489. https://doi.org/10.1021/acs.jafc.8b02879. Santin, M., & Ambrosio, L. J. E. R. o. M. D. (2008). Soybean-based biomaterials: Preparation, properties and tissue regeneration potential. Expert Review of Medical Devices 5 , 349-358. https://doi.org/10.1586/17434440.5.3.349. Singhvi, R., Stephanopoulos, G., & Wang, D. I. C. (1994). Effects of substratum morphology on cell physiology. Biotechnology and Bioengineering 43 , 764-771. https://doi.org/10.1002/bit.260430811. Tansaz, S., & Boccaccini, A. R. (2016). Biomedical applications of soy protein: A brief overview. Journal of Biomedical Materials Research Part A 104 , 553-569. https://doi.org/10.1002/jbm.a.35569. Tufan, Y., Öztatlı, H., Garipcan, B., & Ercan, B. (2021). Development of electrically conductive porous silk fibroin/carbon nanofiber scaffolds. Biomedical Materials 16 , 025027. https://doi.org/10.1088/1748-605X/abc3db. Wu, M., Wu, P., Xiao, L., Zhao, Y., Yan, F., Liu, X., Xie, Y., Zhang, C., Chen, Y., & Cai, L. (2020). Biomimetic mineralization of novel hydroxyethyl cellulose/soy protein isolate scaffolds promote bone regeneration in vitro and in vivo. International Journal of Biological Macromolecules 162 , 1627-1641. https://doi.org/10.1016/j.ijbiomac.2020.08.029. Xiang, N., Yuen, J. S. K., Jr., Stout, A. J., Rubio, N. R., Chen, Y., & Kaplan, D. L. (2022). 3D porous scaffolds from wheat glutenin for cultured meat applications. Biomaterials 285 , 121543. https://doi.org/10.1016/j.biomaterials.2022.121543. Yun, S. P., Ryu, J. M., Jang, M. W., & Han, H. J. (2010). Interaction of profilin‐1 and F‐actin via a β‐arrestin‐1/JNK signaling pathway involved in prostaglandin E2‐induced human mesenchymal stem cells migration and proliferation. Journal of Cellular Physiology 226 , 559-571. https://doi.org/10.1002/jcp.22366. Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Jul, 2024 Reviews received at journal 18 Jun, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers agreed at journal 12 Jun, 2024 Reviewers agreed at journal 12 Jun, 2024 Reviewers invited by journal 27 May, 2024 Editor assigned by journal 24 May, 2024 Submission checks completed at journal 22 May, 2024 First submitted to journal 22 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4459544","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309355141,"identity":"37c9c96e-6cde-4ef3-a3c3-484ee004511b","order_by":0,"name":"Huicheng Fang","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Huicheng","middleName":"","lastName":"Fang","suffix":""},{"id":309355142,"identity":"d019212d-d4b4-44c7-8b55-db3c633ba62b","order_by":1,"name":"Wei Yu","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Yu","suffix":""},{"id":309355143,"identity":"a361c807-2c23-4505-aa4f-717595ca1885","order_by":2,"name":"Boyan Gao","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Boyan","middleName":"","lastName":"Gao","suffix":""},{"id":309355144,"identity":"a88908f3-4649-46df-a77a-b459e1874251","order_by":3,"name":"Yuge Niu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYNACAwY5BoYDIBYz8VqMGRgOk6SFgSGxAaKaCC0GN9IvPi4oOJw+v/H8MQmGCuvEBvazBwhoySk2nmFwOHfDgcNsEgxn0hMbePISCGlJk+YBaWEAamFsO5zYIMFjQEhL+m+glnT5BpCWf0RpST/GDNSSwAByGGMDEVokz7xhBjos3RDoF2OLhGPpxm08Ofi18B1Pf/iZ54+1vPyMgw9vfKixlu1nP4Nfi8IBmDMkDjAwJABpNrzqgUC+gf0BhMXfQEjtKBgFo2AUjFQAANXqRyE6j3BPAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":true,"prefix":"","firstName":"Yuge","middleName":"","lastName":"Niu","suffix":""},{"id":309355145,"identity":"775d8246-24dd-4def-9267-a458e2c0b3ad","order_by":4,"name":"Liangli Yu","email":"","orcid":"","institution":"University of Maryland","correspondingAuthor":false,"prefix":"","firstName":"Liangli","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-05-22 08:55:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4459544/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4459544/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57616446,"identity":"0c3c4114-3c13-4bc9-a138-89a72d1898e8","added_by":"auto","created_at":"2024-06-03 11:43:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67283,"visible":true,"origin":"","legend":"\u003cp\u003eRheological properties of different SPI-SDF scaffolds. (a) G′ vs angular frequency: M-TG, M-Ca, M-TG-Ca and G″ vs angular frequency: M-TG, M-Ca, M-TG-Ca; (b) Shear viscosity vs shear rate: M-TG, M-Ca, M-TG-Ca\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/26f20ae7ce207ddb8dfb4fc5.jpg"},{"id":57616028,"identity":"5c11f8f4-c555-4605-97dc-48afcfe331c2","added_by":"auto","created_at":"2024-06-03 11:35:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91654,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM of different SPI-SDF scaffolds. (A)M-TG,500x; (B)M-Ca,500x; (C)M-TG-Ca,500x\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/b062985dcbbea76cd8688b0d.jpg"},{"id":57616031,"identity":"0dfaf7af-7a61-42df-bc44-a480ccdf13d2","added_by":"auto","created_at":"2024-06-03 11:35:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48756,"visible":true,"origin":"","legend":"\u003cp\u003eNIH 3T3 viabilities evaluated by the CCK-8 assay on SPI-SDF scaffolds after culturing for 1 and 3 days. Data are presented as mean ± SD (n = 3)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/2376fa842684fa66d3c523e1.jpg"},{"id":57616032,"identity":"eeda7ec6-0504-4997-a910-2470bff33a68","added_by":"auto","created_at":"2024-06-03 11:35:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":139583,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal microscopy images of live/dead stained NIH 3T3 in (A) M-TG, (B) M-Ca and (C) M-TG-Ca at the 1, 2 and 3 day time points. Live cells are stained fluorescent green, and dead cells appear red. Scale bars represent 100 μm\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/d36df3d9481f70a6880c88b5.jpg"},{"id":57616029,"identity":"1ae40f74-10a8-49d3-80b9-fa95229fb370","added_by":"auto","created_at":"2024-06-03 11:35:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86654,"visible":true,"origin":"","legend":"\u003cp\u003eNIH 3T3 morphologies on (A) M-TG, (B) M-Ca and (C) M-TG-Ca scaffolds after culturing for 24 h (blue: nucleus; red: F-actin). Scale bars represent 50(left), 7.5(medium) and 200(right) μm, respectively\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/958c4ff0224770c94e8e82a7.jpg"},{"id":57616033,"identity":"e03003fa-be0f-4cf5-b623-c7d1916cfd48","added_by":"auto","created_at":"2024-06-03 11:35:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78297,"visible":true,"origin":"","legend":"\u003cp\u003eNIH 3T3 cell related gene expression in different SPI-SDF scaffolds: (a) intergrin β1, (b) intergrin αV, (c) α-SMA and (d) β-catenin. Data are presented as mean ± SD (n = 3). Values with different letters were significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/a9618a7410d6733abe3d00b6.jpg"},{"id":57616734,"identity":"c007842f-a776-42c9-92b0-7c52b9dbb945","added_by":"auto","created_at":"2024-06-03 11:51:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1215075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/abc0e420-dffb-4eff-9063-4add7b3bbd5e.pdf"},{"id":57616447,"identity":"ad9e5a0a-a1f9-42a6-b22c-839fa92a4a7d","added_by":"auto","created_at":"2024-06-03 11:43:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":74918,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4459544/v1/cd5374d7bd120bec133b1fad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation of novel double cross-linked hydrogels of dietary fibers and proteins from soybeans as scaffolds for cultured meat","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eOne focus area of the production of cultured meat is fabricating constructs as extracellular matrix (ECM) for culture cells (Xiang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ECM can provide cells with extremely complex three-dimensional microenvironment and different biological cues (Berger et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The complexity supports muscle cells in growth, migration, and assembly into muscle tissue. The artificial scaffolds can mimic ECM in terms of structure, chemistry, and mechanical properties. For porous scaffolds, porosity and interconnected pores are essential to facilitate cell migration, cell growth and nutrient flow (Afjoul et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). An ideal scaffold should have good mechanical properties and high biocompatibility. Furthermore, the scaffold must be intrinsically edible (Holmes et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Conventional scaffolds usually are synthetic materials. However, some of them may lack biological cues, or have the unqualified mechanical properties, nor be biodegradable (Lin et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Taking these limitations into consideration, safe renewable sources, such as food-derived proteins and polysaccharides, are increasingly being mentioned as scaffold materials for cultured meat.\u003c/p\u003e \u003cp\u003eTextured soy protein was used as a novel scaffold because it could support cell attachment and proliferation to create a 3D-engineered bovine muscle tissue (Ben-Arye et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Soy protein isolated (SPI), a purified protein from soybean, has recently been used as an attractive raw material for synthetic materials with other reagents as scaffolds (Chien et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tansaz \u0026amp; Boccaccini, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). SPI exhibits good processing characteristics, especially its outstanding biodegradability and biocompatibility (Santin \u0026amp; Ambrosio, 2008). Chien and Shah fabricated porous 3D SPI scaffolds using the freeze-drying method to culture cells (Chien \u0026amp; Shah, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent strategies have been developed to modify the processing characteristics of SPI. Polysaccharides are often added in protein solutions to obtain a protein-polysaccharides mixed system (Nishinari et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The two components play synergistic role in the hydrogel system (Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Soybean soluble dietary fiber (SDF) as a polysaccharide is also derived from soybean. TGase are often used to cross-link proteins in food industry (Kieliszek \u0026amp; Misiewicz, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As an indispensable cross-linking agent in the process of tofu, calcium ions can also cross-link both SPI and SDF (Fang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The previous research confirmed that the scaffold based on gellan gum-gelatin and further cross-linked with high concentrations of Ca\u003csup\u003e2+\u003c/sup\u003e had higher biocompatibility than non-cross linked scaffolds. It was more conducive to cell spreading (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The use of \u0026ldquo;green\u0026rdquo; cross-linking agents avoids the use of toxic agents, and can match the desirable mechanical property of scaffolds. These methods take the advantages of both protein and polysaccharide, which make high utilization of natural renewable materials.\u003c/p\u003e \u003cp\u003eThis study provides a method to combine soybean protein and soybean dietary fiber with two cross-linking agents to prepare a novel scaffold for the production of cultured meat. The mechanical properties, surface morphology, infrared spectroscopy and water absorption of the scaffolds were determined. Mouse fibroblasts were subjected to cell proliferation experiments, cell morphology experiments and cell death staining analysis. SPI-SDF hydrogels are expected to be used as an effective scaffold for the production of cultured meat due to its ideal mechanical strength and cytocompatibility.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Materials\u003c/h2\u003e\n \u003cp\u003eSoybean protein isolate (SPI) (protein content\u0026thinsp;\u0026ge;\u0026thinsp;90%) was purchased from Macklin Inc. (Shanghai, China). Soybean dietary fiber (SDF) was a gift of Henan Wan Bang Industrial Co. Ltd (Henan, China). Transglutaminase (TGase, 100 U/g, Food Grade) was purchased from Jiangsu Yiming Biological Co., Ltd. (Jiangsu, China). Glycerol was purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 assay kit (CCK-8) was purchased from Dojindo China Co., Ltd (Shanghai, China). Live/Dead assay kit assay and the cDNA kit (iScriptTM cDNA Synthesis Kit) were purchased from Thermo Scientific (Waltham, MA, USA). Rhodamine phalloidin and 4\u0026apos;,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 SPI-SDF scaffolds fabrication\u003c/h2\u003e\n \u003cp\u003eThe SPI-SDF scaffolds were prepared according to a laboratory protocol (Fang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). SPI solution of 5% (w/w) in deionized water were homogenized at 8000 rpm for 5 min. After using 6 M NaOH to adjust the pH of the SPI solution to pH 7.5, the solution was preheated in water bath at 100 ℃ for 1 h to fully denature soybean protein. The solution was then cooled to ambient temperature. The glycerol of 5% (w/w) was added as plasticizer. According to Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the SDF were added correspondingly to obtain mixed samples. The mixtures were then homogenized at 8000 rpm for 5 min. TGase and/or calcium chloride were added to each sample at different amounts recorded in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. For TG-induced scaffolds, the mixtures were incubated in incubator at 55 ℃ for 1 h to complete the reaction of enzyme cross-linking. For Ca-induced scaffolds, the 2 M calcium chloride was added directly into the mixtures to adjust the final calcium ion concentration to 100 mM followed by vortex for 5 min. The slurries were frozen at -25 ℃ overnight and then freeze-dried.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFormulation of SPI-SDF scaffolds\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSPI (w/v)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSDF (w/v)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTGase (w/v)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e (w/v) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM-TG \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM-Ca \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM-TG-Ca \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Calcium chloride concentration was 2 M.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e M-TG: Mixture of SPI and SDF with single cross-linked by TGase.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e M-Ca: Mixture of SPI and SDF with single cross-linked by Ca\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003ed\u003c/sup\u003e M-TG-Ca: Mixture of SPI and SDF with double cross-linked by TGase and Ca\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Water uptake capacity\u003c/h2\u003e\n \u003cp\u003eThe water uptake capacity of the scaffolds was measured according to the previous report (Perumal et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Each scaffold was immersed in the phosphate buffered saline (PBS) solution for 30 min. Then, the scaffold was removed from the liquid. The excess liquid was dried with filter paper. The weight of dry or wet scaffold was measured. Measurements for each scaffold were repeated five times. The absorption ratio was then calculated by the following equation:\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\text{Water uptake capacity=}\\frac{\\text{(}{\\text{M}}_{\\text{1}}\\text{-}{\\text{M}}_{\\text{0}}\\text{)}}{{\\text{M}}_{\\text{0}}}\\text{\u0026times;100%}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere M\u003csub\u003e0\u003c/sub\u003e is the weight of the dry scaffold and M\u003csub\u003e1\u003c/sub\u003e is the weight of wet scaffold.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Rheological test of scaffolds\u003c/h2\u003e\n \u003cp\u003eThe rheological properties of SPI-SDF solutions were studied employing an AR G2 rheometer (TA Instrument, USA). All samples of M-Ca and M-TG-Ca group were determined using the 40 mm titanium flat plates with a gap of 500 \u0026micro;m. Since the modulus of M-TG was relatively low, the plates were applied with a gap of 28 \u0026micro;m. The linear viscoelasticity region of scaffolds was obtained by strain-sweep measurements. The frequency-sweep measurements were performed at a fixed strain of 1% from 0.1 to 10 rad/s. All the experiments were conducted at the temperature of 25 ℃. To prevent water evaporation, the shear viscosity was measured at shear rates from 0.01 to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Mechanical characterization\u003c/h2\u003e\n \u003cp\u003eIn order to investigate the mechanical properties of the SPI -SDF scaffolds, both dry and wet samples (n\u0026thinsp;=\u0026thinsp;5, respectively) were measured through compression test. The compression testing was conducted on TA-XT plus texture analyzer (Stable Micro Systems, UK). Each Scaffold was prepared as a cylinder (diameter: 25 mm; height: 10 mm). Samples were compressed at 0.5 mm/s up to 10% strain. The compressive modulus of scaffold was determined by TA texture analysis software. The wet scaffolds were prepared by completely immersed in PBS for 1 h until measurement. Both dry and water-hydrated scaffolds were tested. Measurements for each scaffold were repeated five times.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Porosity analysis\u003c/h2\u003e\n \u003cp\u003eThe porosity analysis was conducted using liquid replacement method according to the previous report (Li et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The cylindrical samples of scaffold formulations (diameter: 20mm, height: 8mm, n\u0026thinsp;=\u0026thinsp;5) were fabricated. The volume of the scaffold was measured and calculated by a digital Vernier caliper. The dry weight (W\u003csub\u003ei\u003c/sub\u003e) of the scaffold was measured by electronic analytical balance. The samples were placed in a graduated cylinder containing 30 mL of isopropanol (\u0026rho;\u003csub\u003ei\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.785 g/mL) under vacuum for 15 min to allow the liquid to fully infiltrate. The weight (W\u003csub\u003ef\u003c/sub\u003e) of scaffolds filled with isopropanol was measured. Measurements for each scaffold were repeated three times. The porosity was calculated by the following formula:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\text{Porosity=}\\frac{\\text{(}{\\text{W}}_{\\text{f}}\\text{-}{\\text{W}}_{\\text{i}}\\text{)/}{\\text{\u0026rho;}}_{\\text{i}}}{{\\text{V}}_{\\text{i}}}\\text{\u0026times;100%}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e (2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Scanning electron microscopy (SEM)\u003c/h2\u003e\n \u003cp\u003eThe SPI -SDF scaffolds were sputter coated with Au and were scanned at a voltage of 5 kV on Sirion200 SEM (FEI Company, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Fourier Transform infrared spectroscopy (FT-IR)\u003c/h2\u003e\n \u003cp\u003eFT-IR analysis for the determination of functional groups of materials was conducted in an IR/Nicolet 6700 Fourier-transform infrared spectrometer (FT-IR) (Thermo Fisher Scientific, American). The device was equipped with an attenuated total reflectance (ATR). The 1 mg of each sample and 100 mg of KBr were mixed and then tableted. The spectra frequency range was from 500 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The resolution was 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. No. of scans was 32.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Cell adhesion\u003c/h2\u003e\n \u003cp\u003eCell adhesion was examined using a commercial bioluminescent stain (Sigma-Aldrich, St. Louis, MO, USA). In brief, the NIH 3T3 cells were cultured in the Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 1% antibiotic mix of penicillin-streptomycin and 10% fetal bovine serum (FBS). The NIH 3T3 cells were then incubated in an incubator at 37 ℃ with a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. The scaffolds were sheared into regular shape with 10 mm*10 mm*1 mm. Then the scaffolds were sterilized with 75% ethanol overnight and washed 3 times by PBS for total 3 h. The fresh DMEM were added to immerse the scaffold overnight to pre-wet the scaffolds. The cells at the logarithmic growth phase were collected with tryp-sin-EDTA. For each scaffold, the NIH 3T3 cells were seeding at a cell density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mm\u003csup\u003e2\u003c/sup\u003e. In order to examine the cell adhesive behavior of the scaffolds, after incubation for 24 h, the cells were fixed with 4% formaldehyde at ambient temperature for 30 min. After being washed three times by PBS, rhodamine phalloidin and DAPI were used to stain the F-actin and nucleus of cells, respectively. The scaffolds were imaged using a Leica SP5 confocal microscope (Leica, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Cell proliferation and viability assays\u003c/h2\u003e\n \u003cp\u003eThe culture and seeding procedures for the evaluation of cell proliferation and viability assay on the scaffolds were performed using a commercial kit. To examine the cell viability of the scaffolds, the proliferation of NIH 3T3 on the scaffolds was determined by a Cell Counting Kit-8 assay kit ((CCK-8), Dojindo, Japan) using well plates as a control. After 1 and 3 days of culture, the original medium was replaced by the new DMEM medium (10% CCK-8). The system was incubated at 37 ℃ with a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e for 1 h. The absorbance of each well was measured at 450 nm. Measurements for each scaffold were repeated three times. For live/dead detection, the cells were stained by Live/Dead assay kit assay (Thermo Scientific, USA) according to the manufacturer\u0026apos;s recommendations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 Reverse transcription-polymerase chain reaction (RT-PCR)\u003c/h2\u003e\n \u003cp\u003eNIH 3T3 cells were co-cultured with the scaffold material. The cells were seeded on a 6-well plate at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mm\u003csup\u003e2\u003c/sup\u003e. After co-cultured with scaffolds for 2 days, the harvested cells were collected and washed by PBS. Total RNA was extracted from the cells with TRIzol. Chloroform and isopropanol were added to isolate and precipitate the RNA, respectively. The RNA extract was purified with 75% DEPC-ethanol water by three times. The purified RNA was then resuspended in 20 \u0026micro;L of DEPC-treated water and quantified by Nano Drop spectrophotometer (Thermo Scientific, USA). The ratio of OD\u003csub\u003e260\u003c/sub\u003e and OD\u003csub\u003e280\u003c/sub\u003e usually exceeded 1.8 at this stage. The cDNA synthesis was followed by the manufacturer\u0026apos;s instructions using the cDNA kit (iScriptTM cDNA Synthesis Kit) for reverse transcription of total RNA. 10 \u0026micro;L of total RNA, 4uL of 5\u0026times;iScript Reaction Mix, 1uL of iScript Reverse Transcriptase, 5 \u0026micro;L of DEPC water were mixed to obtain 20 \u0026micro;L of solution. RT-PCR was done on the ABI 7900 HT (Applied Biosystems, Carlsbad, CA, USA). The amplification parameters used for PCR were as follows: 50 ℃ for 2 min, 95 ℃ for 10 min, and 46 cycles of amplification at 95 ℃ for 15 s and 60 ℃ for 1 min. Statistical analysis of the quantitative real time PCR was performed using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. GAPDH was used as a reference gene and amplified with the target gene in the same cDNA sample. Measurements for each scaffold were repeated three times. The forward and reverse primer sequences are shown in supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll data were analyzed using SPSS version 22.0 (IBM Co., USA) by one-way ANOVA. And \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as being statistically significant. The differences among groups were determined with a Duncan means test.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Rheological behavior of SPI-SDF scaffolds\u003c/h2\u003e \u003cp\u003eRheological test is an effective way to investigate the microstructure of materials. The storage modulus (G\u0026prime;) and loss modulus (G\u0026Prime;) of scaffolds were shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. In the angular frequency sweeping model, all the scaffolds showed solid-like properties since the G\u0026prime; of the samples was higher than the G\u0026Prime;. The trends of G\u0026prime; and G\u0026Prime; were almost stable in the angular frequency sweeping range. It indicated that all the samples were ideal gels because structural relaxation does not occur. This phenomenon suggested that the addition of TGase and calcium ions allowed the samples to form a gel network, which caused the samples to take longer time to relax. The G\u0026prime; of M-TG-Ca sample was significantly higher than that of M-Ca and M-TG. For example, when angular frequency is 0.1 rad/s, G\u0026prime; of the M-TG-Ca sample was 23 Pa, which was nearly 10 times stiffer than the M-Ca sample. This also indicated that the double cross-linking gels of M-TG-Ca had greater stiffness than single linked gels of M-TG or M-Ca. The result was consistent with our previous research (Fang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The frequency dependence of G\u0026prime; and G\u0026Prime; was described by a power-law equation (Liao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e):\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$${\\text{\u0026eta;}}^{\\text{*}}\\text{(}\\text{\u0026omega;}\\text{)=}\\frac{{\\text{(}{\\text{G\u0026#039;}}^{\\text{2}}\\text{+}{\\text{G\u0026#039;\u0026#039;}}^{\\text{2}}\\text{)}}^{\\frac{1}{2}}}{\\text{i}\\text{\u0026omega;}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$${\\text{\u0026eta;}}^{\\text{*}}\\text{(}\\text{\u0026omega;}\\text{)=}{\\text{K}}_{\\text{d}}{\\text{\u0026omega;}}^{\\text{-}{\\text{n}}_{\\text{d}}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eω\u003c/em\u003e is the angular frequency and \u003cem\u003eη\u003c/em\u003e\u003csup\u003e*\u003c/sup\u003e(\u003cem\u003eω\u003c/em\u003e) is the complex viscosity. The constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e stands for dynamic consistency index, whereas the exponent \u003cem\u003en\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e is the dynamic power-law factor.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb showed the variation of shear viscosity of the scaffolds with shear rate in the form of double logarithmic coordinates. The relationship between viscosity and shear rate can also be described by a power-law equation:\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\text{\u0026eta;}\\text{(}\\text{\u0026gamma;}\\text{)}\\text{=}{\\text{K}}_{\\text{s}}{\\gamma }^{{\\text{n}}_{s}-1}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eγ\u003c/em\u003e is the shear rate and \u003cem\u003eη\u003c/em\u003e(\u003cem\u003eγ\u003c/em\u003e) is the shear viscosity. The constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e stands for shear consistency index, whereas the exponent \u003cem\u003en\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e is the shear power-law factor.\u003c/p\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the \u003cem\u003en\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e of all samples are less than 1, which indicated that all of the scaffolds were pseudoplastic non-Newtonian fluid type and had shear thinning characteristics. M-TG scaffolds had the largest \u003cem\u003en\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e and the smallest \u003cem\u003en\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e, meaning that it was more elastic and non-Newtonian than the other two scaffolds. And M-TG-Ca had highest \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e, which further indicated that it had strongest mechanical property. In conclusion, SPI and SDF could form interpenetrating cross-linking networks by TGase and calcium ions. M-TG-Ca scaffolds formed by double cross-linking may have higher mechanical strength to support cell growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRegression parameters representing viscoelasticity of different SPI-SDF scaffolds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM-TG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM-Ca\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM-TG-Ca\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e331.538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.998\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003csub\u003es\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.868\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en\u003csub\u003es\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.984\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e, n\u003csub\u003ed\u003c/sub\u003e, K\u003csub\u003es\u003c/sub\u003e and n\u003csub\u003es\u003c/sub\u003e were obtained by curve-fitting using Equations (\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), (\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and (5) and based on the data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Porosity analysis and morphology of scaffolds\u003c/h2\u003e \u003cp\u003ePorosity is particularly important for the cell culture during the production of cultured meat. The materials with high porosity can provide cells large surface areas and are more conducive to the transportation of nutrients to promote the growth of cells (Chong et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lanno et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In order to determine the porosity of the scaffolds, isopropanol was used as displacement liquid because the scaffolds will not swell or shrink when immersed in the solvent.\u003c/p\u003e \u003cp\u003eThe porosity of different scaffolds was presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The M-TG-Ca scaffold showed the porosity of 72.66%. The M-TG scaffold showed the porosity of 71.87% and the M-Ca showed the porosity of 69.44%. The M-Ca scaffold had the lowest porosity. There is no significant difference between the porosity of scaffolds induced by TGase. This may be due to the stronger density of covalent bond type cross-linking than ionic bond type. So, the additive of Ca\u003csup\u003e2+\u003c/sup\u003e did not significantly influence the porosity of the scaffolds bonded by TGase.\u003c/p\u003e \u003cp\u003eThe microstructure and porosity of scaffolds are usually related to each other. The microstructure of scaffolds was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The surface morphology of M-TG was similar with that of M-TG-Ca. The result was consistent with the porosity analysis. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, the struts of M-TG-Ca appeared thicker compared with M-TG scaffolds. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB showed that the morphology of M-Ca was significantly different and exhibited looser and discontinuous network. Compared Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA with Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the pore surface of M-Ca was rough, while the surface of M-TG samples was smooth. The pore shapes of all samples were irregular. The small pores of scaffold could help cell adhesion and infiltration. Meanwhile, the large pores could facilitate cell growth since the oxygen can be transferred to the host easier (Ninan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The pore size within the field of view in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e was measured using the image J software (NIH, USA). The pore diameter of M-Ca scaffold ranges from 26 to 60 \u0026micro;m with a median pore size of 38 \u0026micro;m. The pore diameter of M-TG scaffold ranges from 25 to 66 \u0026micro;m with a median pore size of 40 \u0026micro;m. The pore diameter of M-TG-Ca ranges from 23 to 50 \u0026micro;m with a median pore size of 34 \u0026micro;m. The pore size of M-TG-Ca was smallest which may be due to the highest extent of cross-linking, further explained the difference between double cross-linked scaffolds and single cross-linked scaffolds. To sum up, M-TG-Ca scaffolds had the highest porosity and the smallest pore size, which may allow M-TG-Ca to be attached by more cells.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of SPI-SDF scaffolds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePorosity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWater uptake capacity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompressive moduli (Kpa; wet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCompressive moduli (Kpa; dry)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-TG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.87\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e413.94\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;13.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.33\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.44\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e318.82\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.32\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.05\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM-TG-Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.66\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e309.45\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.04\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.29\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5). Values with different letters were significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Water up taking capacity\u003c/h2\u003e \u003cp\u003eExcellent water up taking capacity are vital to hydrogel scaffolds. The high capacity on water up taking of scaffolds can keep the cells in the moist environment. Meanwhile, the strong ability of water absorption can provide nutrients for the colonized cells and elimination of waste metabolites (Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These features can enhance cell proliferation. According to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, M-TG had the highest liquid absorption ability which may be due to the high hydrophilicity and large pore size of M-TG scaffolds. Polysaccharides are more discrete and have higher degrees of freedom than proteins. So, polysaccharides in M-TG scaffolds that were not cross-linked by calcium ion could absorb more water. The cross-linking of M-TG-Ca scaffolds is more intensive resulting in the low water up taking capacity. The water absorption rate of the M-TG sample was up to 413.94%, while the water absorption rates of the other two samples were both higher than 300%. These results showed that these scaffolds have strong ability of fluid absorption and retention. For cells, the scaffolds could be conducive to supply the essential nutrients and eliminate the excessive metabolites in the process of the production of cultured meat. Therefore, considering the good water up taking capacity, all scaffolds including M-TG-Ca scaffolds can be used as a potential material in the production of cultured meat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Compressive modulus of scaffolds\u003c/h2\u003e \u003cp\u003eThe compression modulus can reflect the mechanical property of the scaffolds. The dry M-TG-Ca scaffold had the highest compressive modulus, which was 12.68 times higher than that of the dry M-TG and 4.21 times higher than the dry M-Ca (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The compressive modulus of all SPI-SDF scaffolds in wet state decreased compared to them in dry state. The treatment of TGase combined with calcium ions significantly increased the compressive modulus of both dry and wet scaffolds. Good mechanical property is essential for cell culture since the high compressibility can provide a dynamic microenvironment for cell attaching and thus to regulate the cell behavior (Chung et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In conclusion, these results indicated that double cross-linking methods could significantly increase the mechanical property of scaffolds. The result was consistent with rheological test. So, M-TG-Ca scaffolds, cross-linked by two kinds of agents, are suitable for cultured meat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 FT-IR analysis\u003c/h2\u003e \u003cp\u003eThe FT-IR spectra of SPI, SDF, and the SPI-SDF scaffolds were showed in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e as supplementary materials. The spectrum showed the characteristic peaks of the SPI including the peaks at 1658 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide I, C\u0026thinsp;=\u0026thinsp;O) and 1533 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide II, N\u0026ndash;H). The pure SPI spectrum also showed an absorption band at 3302\u0026ndash;3150 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This absorption represented the stretching vibration of the OH group, which may be attributed to the hydrogen bond and the moisture of the SPI (Qi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Characteristic peaks in the SDF included the peaks at 3420 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (O\u0026ndash;H stretching) and 1646 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O) (Qi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). From the spectra of SPI-SDF scaffolds, it can be found that all the characteristic absorption bands of SPI and SDF were existed in the FTIR spectra. This indicated that the presence of SPI and SDF in the scaffold matrix. It also showed that there was no significant difference among SPI-SDF scaffolds formed with different cross-linking agents, suggesting that the chemical compositions had no detectable changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Cell proliferation of the SPI-SDF scaffolds\u003c/h2\u003e \u003cp\u003eThe cell proliferation of the composite scaffolds was evaluated using CCK assay. The results of cell proliferation of the SPI-SDF scaffolds were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The proliferation of NIH 3T3 on the scaffolds was observed and quantified after incubation from 1 to 3 days. The results showed that the cell number of NIH 3T3 in all scaffolds continued to increase from the first day to the third day, indicating that the scaffold was conducive to cell proliferation. Moreover, the cell viability of M-TG-Ca was significantly higher than those of M-Ca and M-TG scaffolds (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It may be related to the excellent mechanical strength of M-TG-Ca. The interconnected pores of scaffolds can provide cells with a 3D environment and offer the nutrient, as well as transportation channels of metabolic waste (Tufan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the cell could grow well on the scaffolds. The cell proliferation on the scaffolds is extremely important since it provides evidences that muscle tissue can grow on SPI-SDF scaffolds, which is the first step of the production of cultured meat. Therefore, M-TG-Ca scaffolds have great potential to apply as cultured meat scaffolds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Cell viability\u003c/h2\u003e \u003cp\u003eIn order to further investigate the impact of scaffolds on the cell proliferation and the cytotoxicity, the NIH-3T3 cells after being cultured on scaffolds for day 1, 2 and 3, were harvested and then stained using Live/Dead Reagent. The live cells were stained with green color by calcein-AM. The dead cells were stained with red color by ethidium homodimer. Live/Dead fluorescence images showed similar results to CCK-8. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed that most of the cells seeding on scaffolds were stained with green, which indicated that NIH 3T3 cells were able to adhere on the SPI-SDF scaffolds and maintained excellent viability. Meanwhile, the number of live cells of all the scaffolds were increased at the third day, which presented the cell proliferation on the scaffolds. Therefore, these findings suggested that the SPI-SDF scaffolds presented excellent cytocompatibility in vitro and can support the adhesion and proliferation of NIH 3T3 cells. It was demonstrated that all three kinds of scaffolds, including M-TG-Ca scaffolds, could be used as cultured meat scaffolds for cell growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Cell morphological characterizations\u003c/h2\u003e \u003cp\u003eThe scaffolds will dramatically affect the cell behaviors shown as cell morphology (Singhvi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Therefore, the cytoskeletal organization of cells attached on the scaffold was then investigated. The NIH 3T3 morphologies on M-TG, M-Ca and M-TG-Ca scaffolds were investigated after culturing for 24 h. To observe the morphology of NIH 3T3 on the scaffolds, rhodamine-phalloidin and DAPI were used to stain F-actin and nuclei, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, NIH 3T3 with a typical morphology adhered and spread on all the scaffolds. Meanwhile, there were no distinguishable differences among the cell morphologies adhered on the scaffolds. These results indicated that the cross-linking types of SPI-SDF scaffolds had little impact on the cell morphology of NIH 3T3. Furthermore, the F-actin filaments morphology of NIH-3T3 on the three scaffolds were all well-formed. The NIH 3T3 had contacted each other and formed a cell sheet on all scaffolds. The F-actin filament of NIH 3T3 can target cell proliferation to increase cell\u0026rsquo;s function and thus plays a key role in the early maturation of cells (Yun et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, the well-grown F-actin indicated that the scaffolds could promote cell proliferation and cell differentiation at the same time. Surprisingly, M-Ca scaffolds showed the best ability to promote F-actin formation. At the same time, M-TG-Ca also could promote cell proliferation and differentiation greatly as scaffolds for cultured meat.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.9 RT-PCR\u003c/h2\u003e \u003cp\u003eIntegrin, which plays a vital role in cell migration and cell adhesion and proliferation, is the main receptor of ECM protein (Pan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Increased expression of integrin β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) was due to the increase of the migration of cells, which could promote cell proliferation and differentiation in progress of the production of cultured meat. The M-TG-Ca scaffolds group had the highest expression of integrin β1, which indicated that the M-TG-Ca scaffold showed great ability to promote cell migration. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, the expression of integrin αV on the SPI-SDF scaffolds were all down-regulated, which suggested the scaffolds had less help on enhancing the adhesion of endothelial cells. Differentiation-related genes expression in cell were shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed. The expression of α-SMA indicated that the expression of collagen-related genes increased, which led to the deposition of collagen (Chogan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Compared with the M-TG-Ca and M-Ca groups, NIH 3T3 cell cultured in the M-TG scaffolds group showed the lowest expression of α-SMA. It indicated that M-TG group had lower ability to promote cell differentiation, which was consistent with the morphological characterizations result. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, the expression of α-SMA in the M-Ca group a was higher than that in the M-TG and M-TG-Ca groups. It also explained why M-Ca scaffolds had the strongest ability of F-actin formation in morphological characterization result. It could be seen from the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed that M-TG-Ca up-regulated the expression of β-catenin gene relative to M-Ca and M-TG. β-catenin can activate the transcription of MRFs, which is the signal pathway regulating the differentiation of muscle cells (Guan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to the results of gene expression, M-TG-Ca scaffolds through double cross-linking methods had the great potential to promote cell differentiation, which meant it was suitable as cultured meat scaffolds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eThe novel SPI-SDF cross-linked hydrogels with three-dimensional porous structure by different crosslinking methods were prepared and be applied as scaffolds for cultured meat. Soy protein isolated and dietary fiber are both from soybeans, avoiding the use of synthetic polymers. Meanwhile, the cross-linking reagents, the enzyme and calcium ions, were in the list of food additives. Therefore, the scaffolds prepared in this study were safe, biocompatible and edible. The results showed that the physical and chemical properties of porous SPI-SDF scaffolds can be changed by using one or both of cross-linking agents. TGase cross-linked scaffolds exhibited excellent porosity and liquid absorption. TGase combined with calcium ions further enhanced the mechanical strength of the scaffolds. According to the biocompatible assays of the scaffolds, the SPI-SDF scaffolds could support cell proliferation and adhesion, which was the first step in large-scale production of cultured meat. The results of PCR represented that the scaffolds could promote the expression of differentiation-related genes. Among them, M-TG-Ca scaffolds formed by double cross-linking had the best physical properties and showed great cellular compatibility. Such double cross-linked scaffolds may have limits. For instance, the preparation of these scaffolds requires the freeze-drying process, which consumes high energy leading to the huge cost of cultured meat in industrial production. We envision that such double cross-linked hydrogels will provide a new candidate for the potential scaffolding materials from natural resources in the production of cultured meat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis research was funded by the National Key R\u0026amp;D Program of China, China (Grant No. 2022YFD2101304).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHuicheng Fang: Investigation, Formal analysis, Data Curation, Writing - Original Draft, Visualization.Wei Yu: Investigation, Formal analysis, Data Curation, Writing - Original Draft, Visualization. Boyan Gao: Resources, Software, Validation.Yuge Niu: Conceptualization, Funding acquisition, Methodology, Project administration, Writing - Review \u0026amp; Editing.Liangli Yu: Supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAfjoul, H., Shamloo, A., \u0026amp; Kamali, A. (2020). Freeze-gelled alginate/gelatin scaffolds for wound healing applications: An in vitro, in vivo study. \u003cem\u003eMaterials Science and Engineering C-Materials for Biological Applications\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 110957. https://doi.org/10.1016/j.msec.2020.110957.\u003c/li\u003e\n\u003cli\u003eBen-Arye, T., Shandalov, Y., Ben-Shaul, S., Landau, S., Zagury, Y., Ianovici, I., Lavon, N., \u0026amp; Levenberg, S. (2020). 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Interaction of profilin‐1 and F‐actin via a \u0026beta;‐arrestin‐1/JNK signaling pathway involved in prostaglandin E2‐induced human mesenchymal stem cells migration and proliferation. \u003cem\u003eJournal of Cellular Physiology\u003c/em\u003e \u003cstrong\u003e226\u003c/strong\u003e, 559-571. https://doi.org/10.1002/jcp.22366.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cross-linking technology, Soybean Dietary fibers, Soy proteins, Cultured meat scaffolds","lastPublishedDoi":"10.21203/rs.3.rs-4459544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4459544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe composited hydrogels derived from natural materials are getting attention in the field of cultured meat due to their advantages of biocompatibility and degradability as cell scaffolds. In this work, two edible cross-linking agents, transglutaminase (TGase) and/or calcium ions, were successfully used to cross-link soy protein isolated (SPI) and soy dietary fiber (SDF) to fabricate different scaffolds. The prepared scaffolds were characterized by structural, hydration, rheological and mechanical analysis. The double cross-linked scaffolds exhibited highest compressive moduli compared to the single cross-linked scaffolds and had an excellent liquid absorbing ability up to 309.45%, while its porosity was as high as 72.66%. In addition, NIH 3T3 cells were used to evaluate the biocompatibility of the scaffolds\u003cem\u003e in vitro\u003c/em\u003e. The double cross-linked scaffolds could promote the expression of differentiation-related genes and were beneficial for cell adhesion and proliferation. In conclusion, present research provides a new approach to prepare cell scaffolds using soybean resources, which could be used in cultured meat applications.\u003c/p\u003e","manuscriptTitle":"Preparation of novel double cross-linked hydrogels of dietary fibers and proteins from soybeans as scaffolds for cultured meat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 11:35:52","doi":"10.21203/rs.3.rs-4459544/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-05T23:25:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-18T06:42:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155635559846248570703387953888366398514","date":"2024-06-13T05:45:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115828818841497375134245395688666281753","date":"2024-06-12T08:04:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219867320094229670914376574441621605929","date":"2024-06-12T07:58:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-27T08:52:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-24T10:44:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T10:06:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-05-22T08:54:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e7105a6c-fd8f-4046-a7a3-447188e90c93","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-06T04:10:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-03 11:35:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4459544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4459544","identity":"rs-4459544","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}