High-strength and high-toughness ECM films with the potential for peripheral nerve repair

Extracellular matrix (ECM) scaffolds are widely applied in the field of regeneration as the result of their irreplaceable biological advantages, and the preparation of ECM scaffolds into ECM hydrogels expands the applications to some extent. However, weak mechanical properties of current ECM materials limit the complete exploitation of ECM’s biological advantages. To enable ECM materials to be utilized in applications requiring high strength, herein, we created a kind of new ECM material, ECM film, and evaluated its mechanical properties. ECM films exhibited outstanding toughness with no cracks after arbitrarily folding and crumpling, and dramatically high strength levels of 86 ± 17.25 MPa, the maximum of which was 115 MPa. Such spectacular high-strength and high-toughness films, containing only pure ECM without any crosslinking agents and other materials, far exceed current pure natural polymer gel films and even many composite gel films and synthetic polymer gel films. In addition, both PC12 cells and Schwann cells cultured on the surface of ECM films, especially Schwann cells, showed good proliferation, and the neurite outgrowth of the PC12 cells was promoted, indicating the application potential of ECM film in peripheral nerve repair.


Introduction
Decellularized biological scaffold obtained from tissues or organs, as a kind of regenerative biological material, is widely used in tissue repair, reconstruction, and regeneration [1][2][3][4].Decellularization removes the immunogenicity of tissues or organs and preserves the natural biochemical complexity, nanostructure, and bio-inducible properties, so decellularized scaffolds have significant biological functions like receptor signaling, morphogenesis, cell proliferation and differentiation [5][6][7].Based on the platform of 3D cell culture and 3D printing and other new technologies, utilizing the irreplaceable biological advantages, researchers developed pure extracellular matrix (ECM) hydrogels to expand their applications in biomedical and tissue engineering [8][9][10].
Although the form of hydrogels has expanded the applications of ECM scaffolds in vivo and in vitro to some extent, limitation still exists.From the current reports, the advantages of ECM scaffolds have not been completely exploited.ECM hydrogels in the state of expansion exhibit weak mechanical strength [11,12], which makes them unsuitable for utilization under load pressure, also brings great difficulties in the preparation of 3D printing bio-inks, drug delivery carriers and other materials required mechanical strength [13,14].
To strengthen the mechanical properties of ECM hydrogels, ECM hydrogels are modified with various methods.Normally, high polymer networks and chemical crosslinking agents are added into ECM hydrogels [14,15], or ECM biomimetic hydrogels composed of natural high polymers and bioactive factors are fabricated [16], all of which are hoped to fully develop the application potential of ECM scaffolds based on their excellent biological advantages.However, poor biocompatibility cannot be avoided, and biomimetic ECM hydrogels cannot completely replace the natural ECM with complete biological function [17].Therefore, creating another form of pure ECM materials with high toughness, high strength, and no additives is becoming meaningful and necessary to achieve the high utilization of ECM scaffolds' advantages.
Gel films have been found in many applications, such as wound dressing, material coating, drug delivery, mucosal repair, artificial blood vessels, nerve tubes, etc [18][19][20], but this form of materials has not yet been developed in ECM scaffolds.Is it possible to prepare a high-strength and high-toughness ECM film without crosslinkers and thickeners?And, if it could be prepared, we still need to understand its physical and biological properties.If ECM films with great mechanical properties could be created, it means that ECM scaffolds could display their advantages in broader sceneries mentioned above.For example, current studies on nerve tubes are mainly focused on inert polymer materials, while ECM film fabricated tubes or ECM film coated nerve tubes might be another chose to facilitate nerve repair and reconstruction.
In this study, we prepared an ECM film with great toughness and high tensile strength with the maximum of 115 MPa, and the strain reached 24 ± 0.06%.The film had a plastic film like appearance, and its toughness allowed random folding and crumpling without breaking.These outstanding mechanical properties of pure ECM films which contain no crosslinking agents and other materials far exceed that of current pure natural polymer gel films and even many composite gel films and synthetic polymer gel films.At the same time, ECM films showed the ability to promote the neurite outgrowth of PC12 cells and the proliferation of PC12 cells and Schwann cells, indicating good application potential in promoting peripheral nerve repair.

Decellularized scaffolds preparation
The scaffolds were prepared as previously described [21].Briefly, the fresh porcine skin used was purchased from Central Fresh Market (Ningbo, China).The subcutaneous fat and hair were removed.Then the skin sheets were cut into 5 mm × 5 mm small pieces, and washed with phosphate buffer solution (PBS) for 5 min on magnetic stirrer, repeating 3 times to remove impurities.The washed porcine skin was stirred in 2% (v/v) Triton-X-100 (Sigma, USA) buffer on magnetic stirrer for 24 h, changing the buffer every 12 h.After 24 h, the buffer was changed to 0.1% (w/v) SDS (Sigma, USA)/0.1 M NaCl solution, stirring on magnetic stirrer for 48 h, changing the buffer every 12 h.Finally, the SDS/NaCl buffer was changed to deionized water and stirred for 24 h, changing the buffer every 8 h.Samples were stored at −80 • C.

Evaluation of decellularized efficiency 2.3.1. Histological and fluorescent staining
Native and decellularized samples were fixed in 4% paraformaldehyde for 16 h and dehydrated in alcohol series.Then, the samples were soaked in xylene series and embedded in paraffin to prepare 6 mm paraffin sections.Both H&E staining and DAPI (Silarbio, China) staining were performed according to manufacturer's protocol.

DNA quantification
Dried 100 mg ECM samples mixed with 1 ml of lysis buffer containing 0.5 M ethylene diamine tetraacetie acid, 5 M NaCl, 1 M Tris-HCl at pH 8.0, 10% SDS, and added with 2 µl Proteinase K (Solarbio, China), then incubated at 55 • C for 24 h.After DNA extraction, tubes containing the sample were centrifuged at 10 000 rpm for 15 min.The supernatant was divided into two tubes.Equal amounts of isopropanol were then added to each tube.After centrifugation at 1300 rpm for 30 min, the supernatant was discarded and 70% ethanol was added to the pellet for washing and centrifuged again at 10 000 rpm for 5 min.After the supernatant was discarded, the pellet was dried for 15 min.Then 30 µl of water was added to dissolve the pellet and 1 µl was analyzed using a nanophotometer.

Preparation of ECM films
Decellularized samples were freeze-dried and then ground into powder.The obtained powder was solubilized in 0.01 M HCl solution with 10 mg ml −1 pepsin (1:3000, Amresco, USA) overnight at 4 • C. The concentration of ECM powder was 10 mg ml −1 .The digested ECM solution was then neutralized with 1 M NaOH solution and 10 × PBS to a pH of about 7.4 and a 1 × PBS concentration.Subsequently, the pre-gel solution was incubated at 37 • C for 30 min to form ECM hydrogels.To prepare ECM films, the ECM hydrogel then dried for 48 h in a constant temperature oven at 37 • C. The dried films were soaked in ddH 2 O for 15 min and then dried again, repeating twice.Finally, a transparent high-tough and highstrength film is obtained.

Rheological test
Rheological investigations were conducted on HAAKE MARS iQ AIR Rotary rheometer (Thermo Scientific, USA) using 25 mm diameter plate geometry.The samples were loaded at an initial temperature of 4 •C, which was then raised to 37 •C and maintained for 40 min to induce gelation; during which the storage modulus and loss modulus of samples were monitored at a fixed oscillation frequency of 1 Hz and a strain of 5%.

Microstructure and chemical characterization of ECM samples
Native ECM, ECM hydrogels were dehydrated with alcohol series, and dried by critical point drying using Autosamdri® 931(Tousimis, USA).Those samples and ECM films were then coated with platinum using sputter coater prior to scanning electron microscopy (SEM) observation.Scanning SEM (ThermoFisher Phenom ProX, USA) with a working voltage of 2.0 kV was used to observe surface morphology and microarchitecture.
Fourier transform infrared (FTIR) analysis was carried out to determine changes in chemical compositions of ECM samples before and after films formation.FTIR spectra was collected using Transmittance Mode by obtaining 16 scans with 4 cm −1 resolution from 4000 to 400 cm −1 range ((ATR-FTIR Nicolet IS 10, Thermo, USA) equipped with OMNIC software.Major bands associated with chemical groups of FTIR spectra were evaluated.Original spectra were analyzed applying Fourierself deconvolution and second derivative techniques.The original protein spectra of amide I bands were used for Gaussian/Lorentzian curve-fitting by PeakFit v4.12 software (SeaSolve Software Inc., San Jose, CA).

Mechanical properties of ECM films
ECM films were cut into 0.5 cm wide stripes for the following tests.They were fixed on the fixture of the Universal testing machine (ElectroForce Testbench, TA Instruments, USA).The stretching speed was set to 2 mm min −1 , and the distance between two fixtures was set to 6 mm.Some cut films were soaked in water for 15 min to perform the same test.

Cell culture and cytotoxicity studies
Cytotoxicity was determined using CCK-8.Decellularize samples were sterilized by ultraviolet radiation for one hour and then soaked in high glucose DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37 • C for 24 h (6 cm 2 samples in 1 ml growth medium).The extract was then collected for subsequent use.L929 cells were seeded into 96-well plates at a density of 3000 cells per well and cultured at 37 • C in a humidified atmosphere of 5% CO2.After 24 h, the medium was removed from each well and replaced with 100 µl graded concentrations of extract (0%, 25%, 50%, 75%,100%).All medium and extracts were changed every day.After 24 h and 72 h, 10 µl CCK-8 solution was added to each well and incubated for 2 h at 37 • C to quantify cell metabolism.The absorbance of each well was measured by a micro-plate reader (SpectraMaxiD3, USA) at the 450 nm wavelength.Then the percentage survival was calculated compared with the control by subtracting the background reference.
PC12 cells were cultured in RPMI 1640 (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) solution.Schwann cells were cultured in high glucose DMEM (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) solution.All cells were cultured at 37 • C in a humidified atmosphere of 5% CO2 and passaged every 3 d, with the growth medium being changed every 2 d.PC 12 cells and Schwann cells of the third to sixth passages were used for subsequent experiments.Prepared ECM films were Sterilized by ultraviolet radiation for 30 min with prepared collagen (Corning, USA) films as control.The films of the two groups were cut into appropriate size and put into 24-well plate and incubated in medium for 24 h.PC-12 cells and Schwann cells were then seeded on the surface of these two different films at a density of 2 × 10 4 cells per well.After 24 h, the growth medium of the two groups was separately refreshed.The purchased PC12 cells were highly differentiated cells, so differentiation medium containing NGF was not required.The growth medium was refreshed every 2 d.The cells were cultured on film for 1, 3 and 5 d.

Analysis of cell counts and neurite outgrowth on ECM films
After 1, 3 and 5 d of cell culture on film, Schwann cells were stained with SF488-Phalloidin for detecting cell proliferation, and PC12 were immunofluorescence stained for detecting endogenous levels of β-III tubulin and evaluating neurite outgrowth.Briefly, the samples were rinsed with 1 × PBS and fixed in 4% paraformaldehyde for 15 min at room temperature followed by permeation with 0.5% Triton-X-100 for 10 min.After rinsed in 1 × PBS, samples containing Schwann cells were then stained with SF488-Phalloidin and Hoechst.For samples containing PC12 cells, non-specific labeling was blocked with incubating in permeabilization/blocking solution (0.1% v/v Triton X-100 and 5% w/v BSA in PBS) for 2 h at room temperature.Next, samples were immersed in anti β-III tubulin antibody overnight at 4 • C.After rinsed in 1× PBS, samples were incubated in secondary antibody solution for 2 h at room temperature.Subsequently, the samples were rinsed by 1× PBS and stained with Hoechst for 5 min.The stained samples were observed under laser confocal scanning microscopy (NCF950, Novel Optics, China).Ten random, separate fields per sample were recorded at 10× lens and 20× lens objective.The total cell counts, percentage of neurite-bearing cells, and neurite outgrowth were determined from acquired images with Image J software.Neurite-bearing cells were those have at least one neurite with a length equal to or greater than the cell body diameter.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8 and SPSS 18. Differences between two groups were determined by two-tailed unpaired t-test, and differences between multiple groups were calculated using one-way ANOVA.* P < 0.05, * * P < 0.01, * * * P < 0.001 and * * * * P < 0.0001 were considered statistically significant.

Decellularization efficiency
As the process shown in figure 1(A), we prepared decellularized skin scaffolds and grinded it into powder for following usage.Decellularization was confirmed in H&E and DAPI stained sections (figure 1(B)).The results showed that the cellular components of skin tissue were successfully removed during the decellularization process, while the most extracellular matrix components and architecture of native skin remained undisturbed.DNA content was quantified to determine the efficiency of the decellularization process using native skin as the control (figure 1(C)).Obviously, the DNA content of decellularized skin (410.31 ± 18.19 ng mg −1 ) showed significant decrease compared with native porcine skin (8.30 ± 1.73 ng mg −1 ).More than 95% DNA content was removed from native skin, which guaranteed the safe ECM biomaterials without immunogenicity.

Preparation and physical characterization of ECM films
Figure 2(A) showed the overview of the progression of ECM films fabrication.To obtain ECM films, ECM hydrogels must be prepared at first.After repeated dry and soaking, the final ECM films displayed a transparent and ultrathin appearance, like a sheet of plastic.The gelation of ECM was tested through rheological test.The storage and loss modulus of ECM pre-gels were measured with a temperature range from 4 • C-37 • C. As the temperature rose to 37 • C, the modulus curves of ECM pre-gel showed a rapid S-shaped increase, with higher storage modulus (G ′ ) than loss modulus (G ′′ ) (figure 2(B)).This observation indicates that ECM pre-gel exhibited gel-like properties at 37 • C.
The microstructure before and after film formation was observed using SEM with the native skin as the control.The images (figure 2(C)) displayed that although there was some difference between them, the microstructure of native skin, ECM hydrogels, and ECM films still exhibited relatively similar fibrous networks.The fibers of native skin, mainly collagen fibers, were thicker and more compact than that of ECM hydrogels and films.Before ECM films was formed, the microstructure of ECM hydrogels was a three-dimensional network structure.Drying caused the furthest compression of the three-dimensional space, finally resulting in an extremely dense and compact ECM film structure similar to that of native skin.These microstructures might, to some extent, explain the high strength of ECM films.

Chemical structure change of ECM films
FTIR spectroscopy (FTIR, Nicolet iS10, Thermo Fisher Scientific, USA) was carried out to functional chemical components before and after films formation (figure 3(A)).The FTIR plots of native skin, pepsin-solubilized ECM, ECM hydrogel and ECM film were basically similar.The peak of native skin spectra at amide A, amide B, amide II and amide III slightly shifted to low frequency, usually associated to more hydrogen bonds [22].The spectra of four groups (native skin, pepsin-solubilized ECM, ECM Hydrogel and ECM Film) showed a broad peak at 3289 −1 , 3301 −1 , 3289 −1 and 3300 −1 associated with N-H stretching vibration of amid A, showing the existence of hydrogen bonds.The peaks at 3080 −1 , 3078 −1 , 3075 −1 , 2926 −1 , 2929 −1 and 2925 −1 were both due to asymmetrical stretch of CH 2 stretching vibration of amide B. The peaks in three groups at 1630 −1 ,1631 −1 and 1634 −1 were due to amide I (C = O stretching) of protein and peptides, and peaks at 1547 −1 , 1544 −1 , 1543 −1 and 1516 −1 were due to  amide II (N-H bending) of protein and peptides.The peaks at 1236 −1 ,1237 −1 , 1235 −1 and 1231 −1 were associated to amide III (C-N stretching and N-H deformation) [23][24][25].
The shape of Amid I bands (1600-1700 cm −1 ) in the FTIR spectra of ECM materials provides information about the protein secondary structure [26][27][28].Single-subcomponent bands of the secondary protein structure were obtained by analyzing original amide I bands through deconvolution, secondaryderivative analysis, and curve fitting.Figure 3(C)-(F) showed single protein secondary structure bands of native skin, pepsin-solubilised ECM, ECM hydrogel and ECM film.Meanwhile, figure 3(B) summarized the quantity of each secondary structure in four groups.It could be seen that native skin contained the most β-sheet (48 ± 0.3%) and the least β-turn (11 ± 0.1%) than the other three groups.Compared to native skin, pepsin-solubilised ECM contained less β-sheet (41 ± 1.3%) and more β-turn (17 ± 0.8%).Compared to pepsin-solubilised ECM, the β-sheet structure of ECM hydrogel continued to decrease to 34 ± 1.1%, while the α-helix increased to 33 ± 0.7%.Compared to ECM hydrogel, a significant increase of  β-sheet (41 ± 0.2%) and β-turn (19 ± 0.6%) could be seen in ECM Film group, and the α-helix decreased to 19 ± 0.4%.

Mechanical properties of ECM films
Already prepared ECM films displayed a transparent and ultra-thin appearance, looking like a sheet of plastic (figure 4(A)).ECM films were highly tough with no cracks observed after arbitrarily folding and crumpling.We further tested the tensile strength of ECM films (figures 4(B) and (C)).Results showed a dramatically high strength of 86 ± 17.25 MPa, and the strain were 24 ± 0.06%.Spectacularly, the maximum stress of this film could reach 115 MPa.Even though the films were wet condition, their stress and strain could separately reach 2.8 ± 0.48 MPa and 44 ± 13.2%, and the maximum stress and strain could separately reach 3.43 MPa and 64.9%.

Cytotoxicity, cell proliferation, and neurite outgrowth on ECM film
The CCK-8 assay was utilized to evaluate the cytocompatibility of ECM film.L929 cultured in extracts of graded concentration (0%, 25%, 50%, 75%, 100%) for 24 h and 72 h to measure the cell viability.The results (figure 5(E)) showed that cell viability in different concentration of extracts has no significant change at 24 h and 72 h, which indicated that ECM films were safe.We cultured PC12 cells and Schwann cells on ECM films for evaluating the potential for nerve repair application.Phalloidin fluorescent staining of Schwann cells and β-III tubulin staining of PC12 cells (figure 5(A)) exhibited better cell proliferation than the control.Schwann cells proliferated more rapidly than control groups, with numerous proliferating cells aggregating into cell clusters on day 5.The counts of Schwann cell (figure 5(B)) similarly showed more cells proliferated on ECM film on day 3 and day 5. Figure 5(A) exhibited that the density of PC12 cells in both groups increased and the axon lengthened over time.The counts of PC12 cell (figure 5(C)) of ECM film group also showed better proliferation than control groups.To further evaluate the neurite outgrowth on the ECM film, we analyzed the neurite-bearing cell rate and neurite length of PC12 cells at different time point.The results (figure 5(D)) showed that compared to ECM film group, the control group had higher neurite-bearing cell rate on day 1 and day 3, while the rate in ECM film groups quickly caught up with that in control groups on day 5. Besides, cells on ECM film groups outgrowth axons over 60 µm significantly more quickly on day 1 (figure 5(F)).The control group had more cells at a neurite length between 40-60 µm on day 3, while exhibited no difference with ECM film group at a length over 60 µm on day3 (figure 5(G)).By the fifth day of culture, the number of cells with neurite elongation over 60 µm in ECM film groups far exceeded that in control groups, and the number of cells with neurite length below 60 µm were basically the same (figure 5(H)).

Discussion
In the last few decades, hydrogel have emerged as the most popular scaffolds to reconstitute artificial 3D environments that impart biochemical and biophysical cues to regulate cell fate and functions [29,30].While the weak mechanical properties of extracellular matrix hydrogels limit the utilization of extracellular matrix scaffolds in many applications.We prepared a new kind of ECM material, ECM film, which is more suitable for scenarios requiring high strength.ECM films were highly tough, no cracks could be observed after arbitrarily folding and kneading.The microstructure of films clearly showed compact fibrous networks, liking the tight collagen fibers in the native skin, which may contribute to its high mechanical strength.
FTIR analysis could show the biochemical composition and the inner change of chemical bonds.In our study, the preparation of ECM films requires pepsin digestion of ECM, gelation and dry.The FTIR plots of native skin, pepsin-solubilised ECM, ECM hydrogel and ECM film were basically similar and no new chemical bonds formed during this whole process, which indicated that only physical crosslinking happened in materials, including van der Waals forces, hydrogen bonds, electrostatic attraction, and intermolecular assemblies [8,31,32].
Digestion, gelation and dry could both affect the change of protein secondary structures.Pepsin digested native skin into a solution composed of protein monomeric components (mainly collagen), polysaccharide chains and peptide molecules [33].The amide A, amide B, amide II and amide III bonds of pepsinsolubilised ECM slightly shifted to low frequency, correlated to the breakage of hydrogen bonds in native skin [22].Pure collagen with intact triple helix conformation can be characterized by its feature amide bands in FTIR spectra [22].Our related results showed that during this process, pepsin did not affect the triple helical structure of collagen [34,35].The content of β-sheet structure decreased and more αhelix structure formed, which may result of the cleavage of pepsin on telopeptide regions of tropocollagen [36].
Under the temperature-, salt solution-and PH-controlled neutralization, the intramolecular bonds of the monomeric components spontaneously assemble into homogeneous gels [32,37], along with the content change of β-sheet structure, αhelix structure, and β-turn structure.Drying ECM hydrogels into films allowed further self-assemble of protein and peptides, aggregating fibers more compact [38].The breakage of hydrogen bonds between amides and water and the reconstruction of hydrogen bonds between amides happened during films formation, thus more β-sheet structure formed [39], and the increase of β-sheet content usually associates to high strength and film toughness [40].ECM films prepared in our study exhibited outstanding toughness and tensile strength with the maximum 115 MPa.Such spectacular high strength and toughness of ECM films, using only pure ECM without any crosslinking agents and other materials, far exceed the mechanical properties of current pure natural polymer gel films and even many composites gel films and synthetic polymer gel films, such as pure collagen films (7.63 ± 0.55 MPa), pure methylcellulose films (15.78 ± 1.57 MPa) [41], gelatin films (4.4 ± 0.26 MPa) [42], composite collagen films (49.2 ± 7.1 MPa) [43], hemicellulose nanocomposite films (44.4 ± 6.8 MPa) [44], polylactic acid films (29.3 ± 2.4 MPa) [45], PVA-gelatin composite films (36 ± 1.5 MPa) [46], et al.The maximum mechanical strength of the wet ECM film decreased to 3.43 MPa, while its strain increased to 2 times that of the dry film, which also superior to many pure natural polymer films in wet conditions.In some reports, the tensile strength of wet collagen films was 2.71 MPa, and the mechanical properties of PCL nanofibrous layer, with the ultimate tensile strength 2.23 ± 0.35 MPa at the elongation of 35%, were similar with the rat sciatic nerve [47,48].
ECM films preparation is a process based on collagen self-assembles.But unlike the films composed of individual components or composite materials, ECM films retain the abundant bioactive components of the native tissue, including glycosaminoglycans, proteoglycans, ECM proteins and various growth factors [49].Our results showed that PC12 cells and Schwann cells cultured on ECM films significantly proliferated, and neurite outgrowth of PC12 cells was promoted with earlier neurite elongation and more long-neurites cells over 60 µm, which may benefit from the multiple ECM proteins and binding sites for neurotrophic factors [50].Previous studies have shown that fibronectin existed in ECM promotes maximum Schwann cell diffusion area, while laminin is most effective in promoting Schwann cell proliferation, cell elongation and c-Jun expression [51].Compared to commercial rat tail type I collagen, ECM contains exclusive and abundant laminin and type V collagen which could regulate SC gene expression.Type V collagen could improve cell proliferation and laminin could promote Schwann cell spreading and neurite outgrowth [52].Laminins are axonal growth-promoting proteins and play a key role in guiding commissural axons to the midline.In addition, glycosaminoglycans and other minor growth factors are also preserved in the ECM and can absorb cytokines to promote cell adhesion and migration [53].On account of the irreplaceable biological advantages accompanied with great toughness and excellent mechanical strength, the application of ECM films on nerve repair could be explored, like being nerve tubes or coating films on synthetic polymer materials for nerve regeneration.

Conclusion
In summary, our study has created a new kind of pure ECM material with high toughness and high strength, which not only makes full use of the biological advantages of ECM, but also meaningfully expands its application sceneries.ECM films could be utilized in many sceneries required higher mechanical strength than ECM hydrogels, like biologic patch, coating film, peripheral nerve repair and small blood vessel repair and so on.Certainly, more in-depth exploration on application should to be carried out and developed later.

Figure 1 .
Figure 1.The process and efficiency of decellularization.(A) Preparation of decellularized skin.(B) H&E staining and fluorescent staining of native and decellularized skin.Scar bar, 200 µm.(C) DNA quantification of native and decellularized skin.The statistical method was independent sample t-test.* * * * P < 0.0001.Error bars are SD.

Figure 2 .
Figure 2. Fabrication and characterization of ECM hydrogels and films.(A) Overview of the preparation of ECM films.(B) Rheological characterization of ECM hydrogels at the temperature ramping from 4 • C to 37 • C and then holding constant.(C) SEM images of native skin, ECM hydrogel and ECM film.Scar bar, 8 µm.

Figure 4 .
Figure 4. Mechanical properties of ECM films.(A) An intuitive demonstration of the appearance and toughness of ECM films.(B) Tensile stress-strain curves of ECM films (N = 5).(D) Tensile stress-strain curves of ECM films after being completely soaked (N = 5).

Figure 5 .
Figure 5.In vitro cell tests of ECM films.(A) Phalloidin fluorescent staining of Schwann cells and β-III tubulin staining of PC12 cells.Scar car, 300 µm and 100 µm.(B) Total cell counts of Schwann cell (N = 10).(C) Total cell counts of PC12 cells (N = 10).(D) Neurite-bearing cells of PC12 cell counted under microscopy in ten random fields.(E) Cytotoxicity test of ECM films at 24 h and 72 h.(N = 5).(F)-(H) neurite length distribution as detected on materials on day 1, day 3, and day 5.All neurite-bearing cells were analyzed by Image J software.At least 500 cells in day 1, 1000 cells in day 3, and 1500 cells in day 5 per group were selected for calculating the neurite length.The statistical method was one-way analysis of variance.* P < 0.05, * * P < 0.01.Error bars are SD.

the
Natural Science Foundation of Zhejiang Province (Grant No. BY23H180015), the Natural Science Foundation of Ningbo city (Grant No. 2022J212), and the Public Welfare Science and Technology Project of Ningbo city (Grant No. 202002N3182), and the Medical Science and Technology Project of Zhejiang province (Grant No. 2022KY311)