One milliliter of C3Gel was placed in the test cup of a cone-plate viscometer (Anton Paar, ViscoQC 300). Using rotor CP52, the measurement was conducted under the following conditions: rotational speed of 30 rpm, temperature of 5.0°C, density set to 1 g/cm³, and a step width of 10 s. The dynamic viscosity was continuously recorded over a period of 200 s. The resulting data were plotted as a dynamic viscosity–time curve using Origin software.

C3Gel was diluted with physiological saline to concentrations of 4, 2, 1, and 0.5 mg/mL. For each concentration, 200 μL of the solution was immediately transferred to a cuvette with a 1 mm path length and subjected to spectral scanning using a circular dichroism spectrometer. The scanning protocol was configured with the following parameters: wavelength range 190–260 nm, bandwidth 1.0 nm, step size 1.0 nm, time-per-point 1 s, and scan rate 50 nm/min. A 0.5 mg/mL solution of bCol-I served as the reference standard.

The spectrum of physiological saline was recorded as the baseline. All acquired spectra were processed using the software Pro-Data Viewer for baseline subtraction and smoothing. The processed data was then exported to Origin for further graphical refinement.

A 10 μL aliquot of C3Gel was pipetted onto a sample carrier and rapidly plunged into liquid nitrogen slush for approximately 30 s. The cryo-fixed sample was then transferred under vacuum using a Quorum cryo-transfer device to a pre-cooled preparation chamber maintained at –140 °C. Under cryogenic and vacuum conditions, the sample was randomly fractured. Subsequently, it was subjected to freeze sublimation at –70 °C for 10 min, followed by sputter coating with platinum to enhance conductivity.

The prepared sample was transferred via a cryo-transfer shuttle onto the cold stage of a Helios G4 UC cryo-scanning electron microscope, where it was observed at –140 °C. Imaging was performed under an accelerating voltage of 10 kV and a beam current of 13 pA. Micrographs were acquired at appropriate magnifications.

Under conditions of 2–8°C, C3Gel was diluted with physiological saline to a concentration of 0.4 mg/mL, ensuring that the gel state was maintained throughout the dilution process. The mixture was gently inverted for homogenization. A 5 μL aliquot was aspirated and applied onto a grid using a Thermo Fisher Vitrobot. The blot force was set to 0 and the blot time was 3 s. A Quantifoil 1.2/1.3 300 mesh Au grid was used for sample support.

The prepared sample was subsequently plunge-frozen in liquid ethane. Imaging was performed using a Glacios cryo-electron microscope operated at 200 kV, with a magnification of 120,000 × and a total electron dose of 50 e^–/Ų.

A 0.1 mL volume of C3Gel was diluted with 1.5 mL of physiological saline to achieve a protein concentration of 1 mg/mL for the determination of its thermal denaturation temperature (Tm). After acquiring a baseline scan using physiological saline, which showed minimal response and good reproducibility, 250 μL of the diluted C3Gel was loaded for measurement. The temperature was scanned from 15 to 100°C at a heating rate of 90°C/h. All measurements were performed under a nitrogen atmosphere.

C3Gel was subjected to dilution with physiological saline and subsequently combined in a 1:1 ratio with DMEM complete medium supplemented with 10% FBS and 1% penicillin-streptomycin, yielding final C3Gel concentrations of 0, 0.25, 0.5, 1, 2, 4, and 8 mg/mL. The group containing 0 mg/mL C3Gel was designated as the control.

A cellular suspension at a density of 1 × 10⁵ cells/mL was introduced into a 96-well plate at a volume of 100 μL per well and maintained under culture conditions (37 °C, 5% CO₂) for 24 h. The supernatant was carefully aspirated, followed by two washing steps with PBS. Thereafter, 100 μL of the previously prepared C3Gel solutions, diluted in DMEM complete medium, were administered to each well. After an additional 24-h incubation period, the solutions were discarded, and the wells underwent two further washes with PBS. Next, 100 μL of CCK-8 solution, diluted to 5% in basal DMEM medium, was introduced into each well. Following a 3-h incubation, the absorbance was measured at 450 nm using a microplate reader.

Cell viability in the control group was established as 100%. The viability of cells exposed to varying concentrations of C3Gel was calculated as a percentage relative to the control group. Statistical significance was assessed through one-way analysis of variance (ANOVA).

C3Gel was diluted with D-PBS buffer to concentrations of 0.25, 0.5, 1, and 2 mg/mL for experimental use. A 1 mg/mL solution of bovine type I collagen served as the positive control (PC), while D-PBS buffer was used as the negative control (NC). A volume of 100 μL of each experimental group (diluted in D-PBS), PC, and NC was added into individual wells of a 96-well plate. Following covering, the plate was incubated overnight at 4°C.

After incubation, the supernatant was discarded, and each well was blocked with 100 μL of 1% BSA in D-PBS at 37 °C for 60 min. The solution was then removed, and the wells were gently washed twice with D-PBS. HaCaT cells were plated at a density of 2 × 10⁶ cells/mL in 100 μL aliquots per well. The cells were then cultured for 120 min under standard conditions (37°C, 5% CO₂). Following incubation, the supernatant was carefully aspirated using a multichannel pipette, and the wells were washed twice with 100 μL D-PBS. Subsequently, 100 μL of DMEM basal medium containing 5% CCK-8 solution was added to each well, and the plate was incubated for 2 h. A microplate reader was utilized to measure the absorbance at 450 nm.

Cell viability in the NC group was defined as 100%. The viability of cells in all other groups was calculated as a percentage relative to the NC group. Statistical significance was evaluated using one-way analysis of variance (ANOVA).

Cells were resuspended in medium supplemented with 10% FBS and plated into a 12-well plate with a final density of 3 × 10⁵ cells/mL (1 mL per well), using three replicate wells per group. To minimize the impact of cell proliferation on scratch healing and ensure HaCaT cells remain in optimal condition to achieve significant healing effects at the 24-h mark, we performed starvation treatment and subsequent experimental procedures using DMEM medium supplemented with 1% FBS after 24 h of cell attachment. Upon reaching over 85% confluence, a linear scratch was introduced employing a standard 200 μL pipette tip. Culture medium solutions for different experimental groups were prepared in advance. The negative control group (NC) was prepared using DMEM starvation medium containing 1% FBS. The experimental groups were established using C3Gel solutions with final concentrations of 0.125, 0.5, and 1 mg/mL prepared in the same medium. The formulated solutions for every experimental group, along with the NC group, were subsequently administered. Scratch images were acquired at intervals of 0, 12, 24, and 36 h employing an inverted fluorescence microscope under 10 × magnification. The scratch width was quantified utilizing Photoshop software, enabling computation of the cellular migration rate. For comparative analysis, statistical significance was determined by two-way ANOVA.

All data from cellular assays were processed and graphically represented with GraphPad Prism software, version 8. Quantitative findings are reported as mean ± standard deviation (SD). Statistical significance was assessed based on the following criteria: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

C3Gel exhibited a white, semi-transparent gel-like appearance (Figure 1D). The formation of this appearance arises from the primary sequence of C3Gel being rich in specific repetitive functional segments Gly-X-Y. This enables multiple single strands to twist into a triple helix structure through hydrogen bonds, salt bridges, and hydrophobic interactions. Furthermore, based on the presence of Cys residues in the primary sequence, it forms interchain covalent disulfide bonds, aggregating the triple helix structures into oligomers. The numerous charged side chains on the surface of these oligomers then undergo lateral alignment into fibrous bundles through electrostatic attraction and hydrogen bonding, ultimately cross-linking into a networked gel. Under the specified parameters, the measured shear rate was 59.94/s. The dynamic viscosity over a period of 200 s is shown in Figure 1G, with an average dynamic viscosity of 1202.4 mPa⋅s. This value, being greater than 1,000 mPa⋅s, indicates that the protein possesses favorable mechanical strength and can maintain a relatively stable gel state (3).

C3Gel is composed of 489 amino acids with a theoretical repeating sequence of (GERGAPGFRGPAGPNG IPGEKGPAGERGAP)16 + GAPGPCCGG and a theoretical molecular weight of 45,454.2 Da. Analysis of the LC-MS spectrum (Figure 1A) showed that the measured molecular weight was 45,455.3 Da, which is consistent with the theoretical value.

Lys-C is an enzymatic protein belonging to the serine protease family, which selectively hydrolyzes peptide bonds at the C-terminal end of lysine residues and digests large proteins into smaller peptide fragments. After enzymatic digestion of C3Gel with Lys-C, followed by LC-MS separation and scanning, the UV chromatogram (Figure 1B) and total ion current (TIC) mass spectrum (Figure 1C) of the resulting digest are shown. Peptide matching and identification were performed using UNIFI software (Waters), which revealed 100% coverage of the theoretical peptide sequence, confirming that the detected sequence is consistent with the theoretical one.

The CD results of proteins reflect their tertiary structure, and this technique is commonly used to analyze the secondary structure of biological protein molecules. To further assess the structural integrity of C3Gel, we subjected it to analysis using a CD spectrometer. As shown in Figure 1E, a positive absorption peak appeared above zero within the 200–230 nm range, accompanied by a distinct negative peak below zero at 190–200 nm. And this result applies to all tested concentrations. These features are consistent with those of the reference bCol-I, which are typical of a triple-helical tertiary structure (9). As the concentration went up, we observed a corresponding increase in peak intensity. It indicates a higher content of the triple-helical structure. Collectively, these data verify the preserved structural integrity of C3Gel.

SEM imaging (Figure 2) reveals that C3Gel successfully formed a uniform, three-dimensional network through self-crosslinking. This structure contains tightly arranged micropores measuring 10–30 μm in size. Notably, this range is comparable to the scale of skin cells like keratinocytes and fibroblasts (10, 11). This pore morphology mimics the physical characteristics of the natural ECM. It can create a biomimetic structure that is highly conducive to cell adhesion and function. The high porosity not only provides critical support for cell attachment and migration (12) but also enables efficient permeation of body fluids and nutrients (13), which is essential for tissue engineering applications.

The Cryo-EM image (Figure 2) reveals the morphology of C3Gel at a more detailed nanoscale, demonstrating that C3Gel self-assembles into a fibrous structure under cryogenic conditions. The measured fiber length ranges between 30 and 45 nm. This dimensional scale is closely linked to the mechanical properties of collagen fibers (14), cellular mechanotransduction, and tissue repair (15). Moreover, it suggests a potential key role in promoting interfacial integration within bone tissue (16).

Thermal analysis is commonly used to study physical changes—such as polymorphic transitions, melting, and evaporation—or chemical changes—including thermal decomposition and oxidation—that occur in substances upon heating, along with accompanying alterations in temperature, energy, or weight. DSC is a widely used method for assessing the thermal stability of proteins (17). It records the heat flow difference between a sample and a reference during a controlled temperature increase, enabling the determination of the material’s Tm.

The Tm refers to the melting temperature of a crystal or the denaturation temperature of a biomacromolecule. In the context of protein unfolding thermodynamics, it represents the thermal denaturation temperature at which half of the ordered triple-helical structure unwinds into a disordered random coil. For collagen-based materials, this transition reflects the thermal stability of the gel network and is observed as an endothermic peak in the DSC scan (18). A higher Tm indicates better thermal stability of the collagen protein.

Analysis of the DSC curve of C3Gel (Figure 1F) showed that as the ambient temperature increased, C3Gel began to absorb heat around 20 °C, with a distinct endothermic peak appearing at 27.25 °C. This temperature corresponds to the Tm of C3Gel, representing the midpoint of the triple helix-to-coil transition.

The cytotoxicity assay was conducted to evaluate whether C3Gel at various concentrations exerts any toxic effects on HaCaT cells, thereby assessing its in vitro biosafety. After 24 h of treatment, the group without C3Gel was used as the control, with its cell viability set as 100%. The viability of other groups was compared to that of this control.

The results of the cytotoxicity assay (Figure 3A) demonstrated that C3Gel exhibits excellent cytocompatibility. Furthermore, all concentration groups showed statistically significant differences compared to the control, with higher cell viability, indicating a pro-proliferative effect of C3Gel. This effect exhibited concentration dependence: within the range of 0.25–1 mg/mL, cell viability showed an increasing trend, with highly significant differences (P ≤ 0.001) compared to the control. In contrast, at concentrations ranging from 1 to 8 mg/mL, the promotive effect on cell proliferation gradually decreased. Based on these findings, subsequent experiments were conducted using a concentration of 1 mg/mL.

Cell adhesion, as the initial stage of cell-material interaction, serves as a critical prerequisite for anchoring, spreading, and subsequent cell proliferation on material surfaces. This process significantly influences cellular physiological activities and determines whether cells can stably survive and perform their biological functions in a new environment (19).

As shown in Figure 3B, the viability of adhered HaCaT cells was evaluated after 120 min of incubation in 96-well plates coated with different concentrations of C3Gel. The results demonstrated that within the concentration range of 0.25–2 mg/mL, the cell adhesion rates showed significant differences compared to the D-PBS negative control group, indicating that C3Gel enhances cell adhesion.

To examine the potential impact of recombinant collagen C3Gel on keratinocyte migration within a composite wound healing context, HaCaT cells were incubated in a culture medium containing C3Gel, followed by observation of cellular movement within the scratched region. As illustrated in Figures 3C,D, exposure to medium incorporating 0.125, 0.5, and 1 mg/mL C3Gel over periods of 12, 24, and 36 h resulted in varying degrees of increase in HaCaT cell migration into the wound area across all groups relative to the NC group. By 24 h, the 0.5 mg/mL group demonstrated the most effective promotion of scratch healing (P ≤ 0.001). At 36 h, the 0.5 and 1 mg/ml groups exhibited near-complete wound closure, with both concentrations demonstrating significantly higher rates of healing compared to the bCol-I group (P ≤ 0.05). These findings reveal that C3Gel especially in the concentration 0.5 mg/mL potently stimulates cellular migration and enhances the wound closure process in the scratch assay model.