A recombinant collagen / calcium alginate composite wound dressing and a preparation method thereof

Abstract

The application discloses a recombinant collagen / calcium alginate composite wound dressing and a preparation method thereof. The dressing is prepared by synthesizing recombinant collagen containing a GFPGER and / or GPP platelet recognition sequence through synthetic biology, and forming the recombinant collagen and sodium alginate into a shape through wet spinning and calcium ion cross-linking. The spinning parameters adopted in the application effectively maintain the biological activity of the recombinant collagen. The dressing can quickly absorb liquid to form glue, has good adhesion and interfacial adhesion. Meanwhile, the recombinant collagen actively recognizes platelet receptors, and cooperates with Ca²⁺ released by calcium alginate to cascade amplify the blood coagulation reaction, thereby significantly improving the hemostatic efficiency. In addition, the wet healing environment constructed by the dressing and the biological activity of the recombinant collagen synergistically accelerate the migration and proliferation of fibroblasts and endothelial cells, and promote angiogenesis and tissue remodeling. The dressing is suitable for the treatment of acute trauma, intraoperative bleeding and refractory wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and wound repair technology, specifically relating to a recombinant collagen / calcium alginate composite wound dressing and its preparation method. Background Technology

[0002] The repair of refractory wounds has become an increasingly serious clinical challenge worldwide. The healing process is often hindered by factors such as local ischemia, persistent infection, and inflammatory dysregulation. Acute bleeding and persistent oozing from the wound are core issues throughout the entire repair process. Blood outflow not only leads to insufficient oxygen supply to local tissues, but its accumulation on the wound surface can also form hematomas that compress microvessels and provide a breeding ground for bacteria, thereby exacerbating the local inflammatory response.

[0003] Currently, commonly used wound repair materials in clinical practice mainly include gauze, gelatin sponge, oxidized regenerated cellulose, animal-derived collagen dressings, and alginate dressings. However, existing hemostatic materials generally have the following limitations: long hemostasis time, easy allergic reactions, and limited contribution to wound regeneration.

[0004] Alginate dressings are widely used in the treatment of chronic wounds due to their excellent absorbency, biocompatibility, and biodegradability. However, after absorbing water, a smooth gel layer easily forms on the surface of alginate dressings, resulting in insufficient interfacial adhesion between the dressing and wound tissue. This makes it difficult to achieve a firm fit, and dressing displacement or detachment can further weaken the healing effect. Furthermore, in cases of severe bleeding, blood clots may cause the dressing to adhere to the wound surface, causing secondary damage.

[0005] Collagen is an important component of the human extracellular matrix and is one of the first natural biomolecules to participate in the hemostasis process after damaged tissue is exposed. However, currently reported collagen-based dressings mostly focus on enhancing its tissue repair function. Due to its large molecular weight and complex structure, natural collagen has insufficient effective coagulation site density per unit mass and significant steric hindrance, greatly limiting its hemostatic efficiency. In recent years, the rapid development of synthetic biology has provided new avenues; however, current research and applications of recombinant collagen mainly focus on the construction of conventional biomimetic repair sequences and the improvement of material physicochemical properties, or only on the design of the hemostatic functional sequence itself. For example, existing collagen hemostatic materials mostly use sponges. However, collagen sponges are generally brittle or have limited mechanical properties, with significantly insufficient absorbency and lack of adhesiveness, making them unsuitable for demanding wound application scenarios.

[0006] Existing technologies have reported the preparation of composite fibers from natural collagen and sodium alginate via wet spinning, which are then laid into dressings. However, these composite fibers suffer from drawbacks such as poor mechanical properties, low collagen content, insufficient structural stability, low density of active sites, and limited hemostatic effect, making it difficult to meet the clinical demand for high-performance wound dressings. Summary of the Invention

[0007] This invention discloses a platelet recognition and synergistic cascade coagulation-enhancing recombinant collagen / calcium alginate composite wound dressing and its preparation method. It provides a new approach to achieving rapid fluid absorption, efficient hemostasis, and promotion of tissue repair, and broadens the application scope of recombinant collagen in the field of wound healing.

[0008] This invention first constructs a recombinant collagen protein containing a high-density platelet-specific recognition fragment (GFPGER / GPP) using synthetic biology techniques, overcoming the shortcomings of traditional animal-derived collagen extraction. Furthermore, it utilizes microbial fermentation technology to achieve low-cost, high-batch-stability, large-scale production. Subsequently, the recombinant collagen protein is introduced into a sodium alginate wet spinning system. Through precise optimization of spinning parameters and in-situ cross-linking in an inorganic salt coagulation bath, a recombinant collagen / calcium alginate composite wound dressing is constructed.

[0009] Upon contact with the wound, the calcium alginate fibers form a hydrogel through physical liquid absorption, giving the dressing excellent adhesion. Simultaneously, the hydrophilic groups of the recombinant collagen molecules and their uniform distribution within the calcium alginate fiber network can form hydrogen bonds with the moist tissue surface, significantly enhancing the dressing's adhesion and interfacial stability, ensuring a tight fit to the wound during the repair process and preventing slippage. Furthermore, the dressing actively binds to GPVI and integrin α2β1 receptors on the platelet surface through recombinant collagen, and releases Ca from calcium alginate. 2+ This invention utilizes a dual synergistic cascade of concentrated coagulation factors to significantly improve hemostasis efficiency under dynamic blood flow conditions. Furthermore, after absorbing water and gelling, the dressing creates a moist healing environment, while recombinant collagen directly participates in regulating the biological behavior of fibroblasts and endothelial cells, accelerating angiogenesis and tissue remodeling, achieving comprehensive intervention from rapid hemostasis to moist healing. In summary, this invention creatively prepares a novel composite wound dressing that achieves a multi-effect synergy of high-speed fluid absorption, efficient hemostasis, and rapid healing through active platelet recognition and synergistic cascade coagulation functions, making it suitable for the treatment of acute trauma, intraoperative bleeding, and refractory wounds.

[0010] On one hand, the present invention provides a method for preparing a recombinant collagen / calcium alginate composite wound dressing, comprising the following steps: S1, Preparation of recombinant collagen with platelet recognition function: Construct and express the nucleic acid sequence encoding a recombinant collagen with platelet recognition function motif, and obtain recombinant collagen with platelet recognition function by gene recombination expression; the platelet recognition function motif is a GFPGER sequence and / or a GPP sequence; S2, Preparation of mixed spinning solution: The recombinant collagen with platelet recognition function is dissolved in phosphate buffer to obtain a recombinant collagen solution; sodium alginate is dissolved in purified water to obtain a sodium alginate solution; the recombinant collagen solution and the sodium alginate solution are mixed to obtain a mixed spinning solution; S3, Wet spinning: The mixed spinning solution described in step S2 is extruded into a coagulation bath containing calcium ions for ion cross-linking and molding to obtain recombinant collagen / calcium alginate composite fiber.

[0011] Furthermore, the method also includes preparing a recombinant collagen / calcium alginate composite wound dressing by washing, stretching, drying and sterilizing the composite fibers.

[0012] Further, step S1 includes: designing and synthesizing a nucleic acid sequence encoding a recombinant collagen protein with a platelet recognition motif; inserting the nucleic acid sequence into an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into host cells and screening for positive transformants; constructing a recombinant co-expression plasmid using a molecular chaperone gene and transforming it into a positive transformant to screen for molecular chaperone-enhanced genetically engineered bacteria; fermenting the molecular chaperone-enhanced genetically engineered bacteria and inducing the expression of recombinant collagen protein; collecting the fermentation supernatant, purifying and drying it to obtain recombinant collagen protein.

[0013] Furthermore, the nucleic acid sequence encoding the recombinant collagen with platelet recognition functional motif is shown in any one of SEQ ID NO. 1-3. Furthermore, the amino acid sequence of the recombinant collagen is shown in any one of SEQ ID NO. 4-6; Preferably, the expression vector in step S1 is a pPIC9K or pPICZαA vector; Preferably, the expression vector in step S1 is the pPIC9K vector; Preferably, the host cell is Pichia pastoris GS115; Preferably, it also includes: a high-copy screening step, using G418 antibiotic to screen genetically engineered bacteria; Preferably, the concentration of the G418 antibiotic is 1~2.5 mg / mL; Preferably, the molecular chaperone gene is selected from at least one of HAC1, BIP, and SEC1; Preferably, the molecular chaperone gene is a combination of three genes: HAC1, BIP, and SEC1. Preferably, in the mixed spinning solution, the mass ratio of recombinant collagen to sodium alginate is 1-15:100; Preferably, in the mixed spinning solution, the mass ratio of recombinant collagen to sodium alginate is 2.5-11.25:100; Preferably, in the mixed spinning solution, the mass ratio of recombinant collagen to sodium alginate is 7.5:100; Preferably, the concentration of recombinant collagen in the recombinant collagen solution is 0.1~20.0 mg / mL; Preferably, the sodium alginate solution contains 1.0~8.0 wt% sodium alginate. Preferably, the coagulation bath in step S3 contains CaCl2 and ethanol; Preferably, the calcium ion concentration in the coagulation bath is 2-8 wt%; Preferably, the calcium ion concentration in the coagulation bath is 6 wt%; Preferably, the ethanol concentration in the coagulation bath is 20%~60%; Preferably, the ethanol concentration in the coagulation bath is 30%; Preferably, the pH of the coagulation bath is 2-5; Preferably, the pH of the coagulation bath is 4; Preferably, the coagulation bath temperature is 25~55 ℃; Preferably, the coagulation bath temperature is 37 ℃; Preferably, the ionic crosslinking time is 0.5~1.5 h; Preferably, the ionic crosslinking time is 1.0 h; Preferably, in the wet spinning process, the spinneret diameter is 50~500 μm, the spinning speed is 10~100 m / min, the extrusion gas is nitrogen, helium or argon, and the winding speed is 40~100 m / min. Preferably, sterilization is performed using gamma rays or ethylene oxide for 0.5 to 6.0 hours.

[0014] The innovation of this invention compared to existing technologies lies in: This invention constructs a recombinant collagen containing a platelet-specific recognition fragment using synthetic biology techniques. Precise gene design and modification are achieved using the amino acid sequence of human collagen as a template, resulting in a product with a single component, high safety, and high batch-to-batch consistency. High-density tandem cascading of active sites is achieved through multiple repetitions of functional motifs, overcoming the structural bottleneck of low active site proportion and large steric hindrance in natural collagen. Simultaneously, combined with a high-density fermentation process, the yield reaches the g / L level, significantly reducing raw material costs and fully meeting the needs of industrial production.

[0015] Based on this, the present invention screened vectors suitable for the expression of recombinant collagen with specific sequences. At the same time, the present invention further introduced a co-expression strategy of molecular chaperones HAC1, BIP and SEC1, which achieved high yield and high stability expression of this functional recombinant collagen.

[0016] The recombinant collagen / calcium alginate composite wound dressing prepared in this invention synergistically combines the triple hemostatic mechanisms of "functional recombinant collagen," "sodium alginate," and "calcium ions," resulting in significantly superior hemostatic performance compared to fiber dressings made solely with sodium alginate and fiber dressings made from a combination of natural collagen and sodium alginate. Specifically, the GFPGER sequence specifically binds to the platelet surface integrin α2β1 receptor, and the GPP sequence targets the GPVI receptor, jointly achieving active capture, anchoring, and activation of platelets in dynamic blood flow or osmosis. Simultaneously, the calcium alginate fibers efficiently absorb water, concentrate peripheral blood, and release calcium ions. 2+ Cofactors amplify the coagulation reaction in a cascade manner, synergistically accelerating the formation of stable fibrin thrombi.

[0017] The recombinant collagen / calcium alginate composite wound dressing prepared in this invention possesses both excellent ability to construct a moist healing environment and tissue repair performance. Upon contact with wound exudate, the dressing rapidly absorbs the liquid and gels. The recombinant collagen molecules are rich in hydrophilic groups such as carboxyl, amino, and hydroxyl groups, which can form hydrogen bonds with the moist tissue surface, thereby enhancing the adhesion and interfacial stability of the dressing on the moist wound surface and preventing dressing displacement or detachment. Stable adhesion helps to construct a continuous, moist healing microenvironment on the wound surface, providing favorable space for cell migration and proliferation. Simultaneously, the recombinant collagen can directly regulate and induce the adhesion and migration of fibroblasts and endothelial cells, accelerating angiogenesis and tissue remodeling. The moist healing microenvironment constructed by the aforementioned physical gelation and the bioactivity of the recombinant collagen synergistically create a three-dimensional repair interface conducive to regeneration, significantly improving the quality of wound healing.

[0018] The triple helix structure of recombinant collagen is less stable than that of natural collagen, and the GFPGER / GPP active sites are highly sensitive to conformational perturbations. pH, ionic conditions, and dehydration during wet spinning can all lead to its inactivation. This invention achieves unexpected technical results by systematically controlling key process parameters of wet spinning, such as the ratio of recombinant protein to sodium alginate, the CaCl2 concentration in the coagulation bath, the ionic crosslinking time, and the pH of the coagulation bath, thereby protecting the molecular structure and bioactivity of recombinant collagen.

[0019] Collagen from different sources and with different sequences exhibits variations in physicochemical properties, including molecular weight, isoelectric point, charge distribution, and side chain group content. Recombinant collagen containing the GFPGER / GPP short peptide sequence differs fundamentally from natural collagen or conventional recombinant collagen in terms of molecular conformation, carboxyl group distribution, and steric hindrance. This invention improves the overall performance of composite fibers by controlling the ratio of recombinant collagen to sodium alginate. The ratio specified in this invention is not something that those skilled in the art could foresee through conventional formulation optimization, resulting in unexpected technical effects. Attached Figure Description

[0020] Figure 1 Transmission electron microscope images of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6; Figure 2 The fiber diameter distribution diagrams are shown for the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6. Figure 3 The tensile strength of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6; Figure 4 Quantitative graphs of elongation at break of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6; Figure 5 Transmission electron microscope images of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 after gelation; Figure 6 Pore ​​size distribution diagrams of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 after gelation; Figure 7 The absorbency ratio of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6; Figure 8 The swelling rate is the composite wound dressing prepared in Examples 1 and 7 and Comparative Examples 4 and 6. Figure 9 This is a schematic diagram of the peeling experiment between the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 and the pigskin. Figure 10The peel strength between the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 and the pigskin; Figure 11 Coagulation dynamics evaluation of the composite dressings prepared in Examples 1-10 and Comparative Examples 1-6; Figure 12 Images showing the live / dead staining results of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 after co-culturing with mouse embryonic fibroblasts (NIH / 3T3) for 1 and 3 days; Figure 13 The image shows the cytoskeleton spreading results of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 after co-culturing with mouse embryonic fibroblasts (NIH / 3T3) for 2 days. Figure 14 The composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 were co-cultured with mouse embryonic fibroblasts (NIH / 3T3) for 3 days, and the cell migration detection results were shown in the figure. Figure 15 Characterization of the hemostatic properties of the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 in mice. Figure 16 HE staining images of skin repair in mice using the composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6. Detailed Implementation

[0021] To better understand the present invention, preferred embodiments will be described below in conjunction with examples. It should be noted that these descriptions are intended to further illustrate the features and advantages of the present invention and do not constitute a limitation on the scope of the claims. Furthermore, it should be understood that after reading the disclosure of this invention, those skilled in the art can make various alterations or modifications to the invention, and equivalent forms of these alterations or modifications also fall within the scope defined by the appended claims. Example 1

[0022] 1. Design and synthesize a recombinant collagen nucleic acid sequence (e.g., SEQ ID NO. 1) encoding a platelet recognition motif (GFPGER), insert the nucleic acid sequence downstream of the α signal peptide of the pPIC9K plasmid vector without introducing any additional nucleotides, and obtain the recombinant expression vector (pPIC9K-GFPGER), labeled as S1; 2. S1 was transformed into E. coli Top10 competent cells for amplification. After plasmid extraction, it was linearized with restriction endonuclease SalI and then electroporated into Pichia pastoris GS115 competent cells. The transformed cells were plated on MD solid medium and cultured at 28 °C for 3 days to screen for positive transformants S2. 3. S2 cells were inoculated into glycerol enrichment medium and cultured at 30 °C and 220 rpm for 24 h with shaking. The cells were collected by centrifugation and transferred to methanol induction medium. The cells were induced and cultured at 28 °C and 220 rpm for 72 h. The fermentation supernatant was collected to determine the expression of the target protein. 4. Transfer S2 to YPD solid medium containing 2 mg / mL G418 antibiotic using a sterile inoculation needle and continue culturing to screen out high-copy integration positive engineered bacteria S3; 5. Molecular chaperone genes HAC1, BIP and SEC1 were amplified from the Pichia pastoris GS115 genome, and then cloned into the pPICZA plasmid to construct the recombinant co-expression plasmid S4. 6. After mixing S3, linearize it with the restriction endonuclease SacI, electroporate it into competent S3 cells, and then plate the transformation product on YPD solid medium containing Zeocin (100 μg / mL) and G418 (2 mg / mL) to obtain molecular chaperone-enhanced high-yield genetically engineered bacteria S5. 7. Inoculate S5 (inoculation dose is 10%) V / V The glycerol was transferred to a fermenter containing a basal salt medium. The pH of the fermentation broth was maintained at 5 by automatic addition of acid and alkali solutions. A staged feeding strategy and a gradient increase in methanol were employed to control nutrient and methanol content during fermentation. Specifically, when dissolved oxygen (DO) reached 70-80%, glycerol was added at a rate of 80 mL / h for 4 h; when DO reached 100%, feeding was stopped and the cells were starved for 1 h. Methanol was then added for induction, with the following rates: 13 mL / h for 0-6 h, 25 mL / h for 6-12 h, 40 mL / h for 12-24 h, and 43 mL / h for 24-40 h. The OD value of the fermentation broth was measured every 4 h during induction to monitor cell growth. After fermentation, the supernatant was collected and purified, eluted, desalted, and dried using a hollow fiber column and a multimode cation exchange chromatography column to obtain recombinant collagen lyophilized powder S6 with platelet recognition function. The amino acid sequence of the recombinant collagen is shown in SEQ ID NO. 4.

[0023] 8. Dissolve 150 mg of self-made recombinant collagen containing the GFPGER sequence in 10 mL of phosphate buffer (pH=6.0), stir at 4 ℃ for 30 min to disperse it evenly, and obtain a recombinant collagen solution with a concentration of 15 mg / mL, labeled as S7. 9. Take 10 mL of purified water, add 0.3 g of sodium alginate, stir at 80 ℃ for 20 min to completely dissolve it, and obtain a 3.0 wt% sodium alginate solution, labeled as S8; 10. Slowly add 1.5 mL of S7 solution to S8 solution, stir at room temperature for 30 min at 500 rpm to obtain a mixed solution of self-made recombinant collagen and sodium alginate in a mass ratio of 7.5:100, labeled as S9; 11. Solution S9 was extruded into a coagulation bath containing 6 wt% CaCl2 using a wet spinning process and subjected to ion cross-linking for 1 h to obtain recombinant collagen / calcium alginate composite fibers. The coagulation bath temperature was 37 ℃, the ethanol content was 30%, the pH was 4, the spinneret orifice diameter was 100 μm, the spinneret speed was 10 m / min, the extrusion gas was nitrogen, and the winding speed was 40 m / min. 12. The obtained composite fibers were washed three times with deionized water and then stretched to improve their strength and uniformity. After freeze-drying for 1 hour, the composite wound dressing was prepared by sterilization with ethylene oxide for 1 hour. Example 2

[0024] 1. Design and synthesize a recombinant collagen nucleic acid sequence GFPGER containing a platelet recognition functional motif. Insert the nucleic acid sequence downstream of the α signal peptide of the pPICZα A plasmid vector without introducing any additional nucleotides to obtain the recombinant expression vector (pPICZα A-GFPGER), labeled as S1. 2. S1 was transformed into E. coli Top10 competent cells for amplification. After plasmid extraction, it was linearized with restriction endonuclease SalI and then electroporated into Pichia pastoris GS115 competent cells. The transformed cells were plated on MD solid medium and cultured at 28 °C for 3 days to screen for positive transformants S2. 3. S2 cells were inoculated into glycerol enrichment medium and cultured at 30 °C and 220 rpm for 24 h with shaking. The cells were collected by centrifugation and transferred to methanol induction medium. The cells were induced and cultured at 28 °C and 220 rpm for 72 h. The fermentation supernatant was collected to determine the expression of the target protein. 4. Transfer S2 to YPD solid medium containing 2 mg / mL G418 antibiotic using a sterile inoculation needle and continue culturing to screen out high-copy integration positive engineered bacteria S3; 5. Molecular chaperone genes HAC1, BIP and SEC1 were amplified from the Pichia pastoris GS115 genome, and then cloned into the pPICZA plasmid to construct the recombinant co-expression plasmid S4. 6. After mixing S3, linearize it with the restriction endonuclease SacI, electroporate it into competent S3 cells, and then plate the transformation product on YPD solid medium containing Zeocin (100 μg / mL) and G418 (2 mg / mL) to obtain molecular chaperone-enhanced high-yield genetically engineered bacteria S5. 7. Inoculate S5 (inoculation dose is 10%) V / V The glycerol was transferred to a fermenter containing a basal salt medium. The pH of the fermentation broth was maintained at 5 by automatic addition of acid and alkali solutions. A staged feeding strategy and a gradient increase in methanol were employed to control nutrient and methanol content during fermentation. Specifically, when dissolved oxygen (DO) reached 70-80%, glycerol was added at a rate of 80 mL / h for 4 h; when DO reached 100%, feeding was stopped and the cells were starved for 1 h. Methanol was then added for induction, with the following rates: 13 mL / h for 0-6 h, 25 mL / h for 6-12 h, 40 mL / h for 12-24 h, and 43 mL / h for 24-40 h. The OD value of the fermentation broth was measured every 4 h during induction to monitor cell growth. After fermentation, the supernatant was collected and purified, eluted, desalted, and dried using a hollow fiber column and a multimode cation exchange chromatography column to obtain recombinant collagen lyophilized powder S6 with platelet recognition function. 8. Dissolve 150 mg of the self-made recombinant collagen containing the GFPGER sequence in 10 mL of buffer solution, stir at 4 °C for 30 min to disperse it evenly, and obtain a recombinant collagen solution with a concentration of 15 mg / mL, labeled as S7. 9. Take 10 mL of purified water, add 0.3 g of sodium alginate, stir at 80 ℃ for 20 min to completely dissolve it, and obtain a 3.0 wt% sodium alginate solution, labeled as S8; 10. Slowly add 0.5 mL of S7 solution to S8 solution, stir at room temperature for 30 min at 500 rpm to obtain a mixed solution of self-made recombinant collagen and sodium alginate in a mass ratio of 2.5:100, labeled as S9. 11. Solution S9 was extruded into a coagulation bath containing 6 wt% CaCl2 using a wet spinning process and subjected to ion cross-linking for 1 h to obtain recombinant collagen / calcium alginate composite fibers. The coagulation bath temperature was 37 ℃, the ethanol content was 30%, the pH was 4, the spinneret orifice diameter was 100 μm, the spinneret speed was 10 m / min, the extrusion gas was nitrogen, and the winding speed was 40 m / min. 12. The obtained composite fibers were washed three times with deionized water and then stretched to improve their strength and uniformity. After freeze-drying for 1 hour, the composite wound dressing was prepared by sterilization with ethylene oxide for 1 hour. Example 3

[0025] The procedure is basically the same as in Example 1, except that in step 4, the concentration of G418 antibiotic is changed from 2 mg / mL to 1 mg / mL. All other operations are the same as in Example 1 and will not be repeated here. Example 4

[0026] The procedure is basically the same as in Example 1, except that in step 5, the molecular chaperone gene is changed from three types (HAC1, BIP, and SEC1) to only one type (HAC1). All other operations are the same as in Example 1 and will not be repeated here. Example 5

[0027] The procedure is basically the same as in Example 1, except that in step 5, the molecular chaperone gene is changed from three types (HAC1, BIP, and SEC1) to only one type (BIP). All other operations are the same as in Example 1 and will not be repeated here. Example 6

[0028] The procedure is essentially the same as in Example 1, except that in step 8, the amount of recombinant collagen added is changed from 150 mg to 50 mg, while the volume of the buffer solution remains unchanged (10 mL), meaning the concentration of solution S7 is changed from 15 mg / mL to 5 mg / mL. Correspondingly, in step 10, the mass ratio of recombinant collagen to sodium alginate in the final mixed solution S9 is changed from 7.5:100 to 2.5:100. All other operations are the same as in Example 1 and will not be repeated here. Example 7

[0029] The procedure is essentially the same as in Example 1, except that in step 1, the recombinant collagen nucleic acid sequence is changed from containing only GFPGER to containing both GFPGER and GPP sequences. Specifically, the recombinant expression vector is pPIC9K-GFPGER / GPP, and the recombinant collagen nucleic acid sequence encoding a platelet recognition motif is shown in SEQ ID NO. 2. Correspondingly, the recombinant collagen in step 8 is a recombinant collagen containing the GFPGER / GPP sequence, and the amino acid sequence of the recombinant collagen is shown in SEQ ID NO. 5. All other operations are the same as in Example 1 and will not be repeated here. Example 8

[0030] The procedure is essentially the same as in Example 7, with the only difference being that in step 9, the amount of sodium alginate added is changed from 0.3 g to 0.2 g, while the volume of purified water remains unchanged (10 mL), meaning the concentration of the sodium alginate solution in step S8 is changed from 3.0 wt% to 2.0 wt%. In step 10, the amount of solution added in step S7 remains unchanged (1.5 mL), and the final mass ratio of recombinant collagen to sodium alginate in the mixed solution S9 is 11.25:100. All other operations are the same as in Example 7 and will not be repeated here. Example 9

[0031] The procedure is basically the same as in Example 7, except that in step 11, the ethanol content of the coagulation bath is changed from 30% to 50%. All other operations are the same as in Example 7 and will not be repeated here. Example 10

[0032] The procedure is essentially the same as in Example 7, except that in step 11, the pH of the coagulation bath is changed from 4 to 3. All other operations are the same as in Example 7 and will not be repeated here. Comparative Example 1

[0033] The procedure is basically the same as in Example 1, except that in step 1, the expression vector is changed from pPIC9K to pGAPZα A; the rest of the operations are the same as in Example 1, and will not be repeated here. Comparative Example 2

[0034] The procedure is essentially the same as in Example 1, except that in step 4, the concentration of the G418 antibiotic is changed from 2 mg / mL to 3 mg / mL. All other operations are the same as in Example 1 and will not be repeated here. Comparative Example 3

[0035] The process is basically the same as in Example 1, except that in step 8, the amount of recombinant collagen added is changed from 150 mg to 100 mg, while the volume of the buffer solution remains unchanged (10 mL), that is, the concentration of the S7 solution is changed from 15 mg / mL to 10 mg / mL. Accordingly, in step 10, the mass ratio of recombinant collagen to sodium alginate in the final mixed solution S9 is changed from 7.5:100 to 5:100. The remaining operations are the same as in Example 1, and will not be repeated here. Comparative Example 4

[0036] The procedure is essentially the same as in Example 1, except that in step 1, the recombinant collagen nucleic acid sequence is changed from containing only GFPGER to containing only GPP, i.e., the recombinant expression vector is pPIC9K-GPP, and the recombinant collagen nucleic acid sequence encoding the platelet recognition motif (GPP) is shown in SEQ ID NO. 3; correspondingly, the recombinant collagen in step 8 is a recombinant collagen containing the GPP sequence, and the amino acid sequence of the recombinant collagen is shown in SEQ ID NO. 6. All other operations are the same as in Example 1 and will not be repeated here. Comparative Example 5

[0037] The procedure is essentially the same as in Example 7, except that in step 9, the amount of sodium alginate added is changed from 0.3 g to 0.1 g, while the volume of purified water remains unchanged (10 mL), meaning the concentration of the sodium alginate solution in step S8 is changed from 3.0 wt% to 1.0 wt%. In step 10, the amount of solution added in step S7 remains unchanged (1.5 mL), and the final mass ratio of recombinant collagen to sodium alginate in the mixed solution S9 is still 22.5:100. All other operations are the same as in Example 7 and will not be repeated here. Comparative Example 6

[0038] (1) Take 10 mL of purified water, add 0.3 g of sodium alginate, stir at 80 °C for 20 min to dissolve it completely, and obtain a 3.0 wt% sodium alginate solution, labeled as S1; (2) Solution S1 was extruded into a coagulation bath containing 6 wt% CaCl2 using a wet spinning process and subjected to ion cross-linking for 1 h to obtain calcium alginate fiber. The coagulation bath temperature was 37 ℃, the ethanol content was 30%, the pH was 4, the spinneret orifice diameter was 100 μm, the spinneret speed was 10 m / min, the extrusion gas was nitrogen, and the winding speed was 40 m / min. (3) The obtained calcium alginate fibers were washed three times with deionized water and then stretched to improve their strength and uniformity. After freeze-drying for 1 h, the hemostatic dressing was prepared by sterilizing with ethylene oxide for 1 h. Comparative Example 7

[0039] 8. Dissolve 150 mg of natural collagen in 10 mL of buffer solution and stir at 4 °C for 30 min to disperse it evenly, to obtain a collagen solution with a concentration of 15 mg / mL, labeled as S1; 9. Take 10 mL of purified water, add 0.1 g of sodium alginate, stir at 80 ℃ for 20 min to completely dissolve it, and obtain a 1.0 wt% sodium alginate solution, labeled as S2; 10. Slowly add 1.5 mL of solution S1 to solution S2, stir at room temperature for 30 min at 500 rpm to obtain a mixed solution of collagen and sodium alginate in a mass ratio of 7.5:100, labeled as S3; 11. Solution S3 was extruded into a coagulation bath containing 6 wt% CaCl2 using a wet spinning process and subjected to ion cross-linking for 1 h to obtain collagen / calcium alginate composite fibers. The coagulation bath temperature was 37 ℃, the ethanol content was 30%, the pH was 4, the spinneret orifice diameter was 100 μm, the spinneret speed was 10 m / min, the extrusion gas was nitrogen, and the winding speed was 40 m / min. 12. The obtained composite fibers were washed three times with deionized water and then stretched to improve their strength and uniformity. After freeze-drying for 1 hour, the composite wound dressing was prepared by sterilization with ethylene oxide for 1 hour.

[0040] The composite wound dressing prepared in Example 1 was named Alg-GFPGER, the composite wound dressing prepared in Comparative Example 4 was named Alg-GPP, the composite wound dressing prepared in Example 7 was named Alg-GFPGER / GPP, the composite wound dressing prepared in Comparative Example 6 was named Alg, and the composite wound dressing prepared in Comparative Example 7 was named Alg-natural protein. Performance testing Fiber morphology and structural characterization

[0041] The wound dressings prepared in Examples 1, 7, 4, and 6 were observed using scanning electron microscopy. The results showed (e.g.) Figure 1-2 The addition of recombinant collagen increased the fiber diameter. The average fiber diameter of the recombinant collagen / calcium alginate composite wound dressing prepared in Example 1 was 1.55 ± 0.31 µm, the average fiber diameter of the recombinant collagen / calcium alginate composite wound dressing prepared in Comparative Example 4 was 1.80 ± 0.37 µm, and the average fiber diameter of the recombinant collagen / calcium alginate composite wound dressing prepared in Example 7 was 1.81 ± 0.35 µm. Simultaneously, with a suitable winding speed, most of the fiber filaments exhibited orientation, providing physical topological guidance for subsequent regenerative cell migration.

[0042] Mechanical property testing

[0043] The wound dressings prepared in Examples 1, 7, 4, and 6 were tested for tensile strength and elongation at break.

[0044] Cut the wound dressing into 1 cm × 2 cm rectangles. After calibrating the texture analyzer (UK SMS, model TA.XTplusC), place the cut dressing samples on the test platform, with the probe clamped firmly to the top and bottom ends of the samples. Start the tensile test program (1 mm / s before test; 2 mm / s during test) to perform the tensile property test, applying static tension along the longitudinal axis to the sample until it fractures. During the test, the texture analyzer records the tensile stress and strain data in real time, and saves the experimental values ​​of each sample after the test.

[0045] The results show (e.g.) Figure 3-4The wound dressing prepared with Comparative Example 6 (pure sodium alginate) exhibited the lowest tensile strength but the highest elongation at break, indicating that it was the most flexible and easily stretched, but lacked sufficient absolute strength to resist breakage. The addition of recombinant collagen (Examples 1 and 7, Comparative Example 4) significantly improved tensile strength, but decreased elongation at break. The introduction of recombinant collagen allows for the formation of more hydrogen bonds with calcium alginate, increasing the degree of cross-linking within the internal network, thereby improving the mechanical properties of the dressing and enabling it to resist external forces and prevent secondary damage to skin tissue. However, the increased rigidity due to hydrogen bond cross-linking sacrifices some extensibility, resulting in a lower elongation at break compared to pure sodium alginate.

[0046] The composite wound dressing prepared in Example 7 exhibited the best tensile strength (86.00 ± 13.03 MPa) and moderate elongation at break (10.85% ± 1.49%). The tensile strength and elongation at break of the composite wound dressings prepared in Example 1 and Comparative Example 4 were 85.33 ± 11.40 MPa and 87.00 ± 10.69 MPa, and 9.00% ± 1.18% and 9.22% ± 1.16%, respectively. Example 7, while maintaining high tensile strength, achieved a better elongation at break than Example 1 and Comparative Example 4, indicating that the synergistic effect of the GFPGER and GPP bifunctional peptides achieved a better balance between rigidity and toughness. Liquid absorption performance test

[0047] The microstructure and absorbance of the wound dressings prepared in Examples 1, 7, 4, and 6 after gelation were observed. Transmission electron microscopy results showed (e.g.) Figure 5-6 Each dressing has a three-dimensional porous structure.

[0048] The experimental procedures for measuring absorbance and swelling rate are as follows: A 5.0 cm × 5.0 cm sample of wound dressing of known mass is placed in a petri dish, and a test solution preheated to (37.0 ± 1.0) °C is added. The test solution is 40 times the mass of the sample, ± 0.5 g. The test solution consists of a sodium chloride and calcium chloride solution containing 142 mmol of sodium ions and 2.5 mmol of calcium ions. The sample is then transferred to a drying oven and kept at 37.0 ± 1.0 °C for 30 min. After holding the sample by one corner with tweezers for 30 s, it is weighed. The weight is calculated per 100 cm³ of sample. 2 The absorption ratio is expressed as the average mass of the absorbed solution / initial mass (dry state). Under the same experimental conditions for the absorption test, after the sample reaches swelling equilibrium, remove the sample with tweezers, gently blot away the free liquid on the surface with filter paper (avoid squeezing), and weigh it immediately. The equilibrium swelling rate is expressed as wet weight (wet weight - initial mass) / initial mass × 100%.

[0049] The experimental steps for microstructure observation are as follows: The prepared wound dressing was dried in a 50 °C oven for 1 hour to remove moisture, and then cut into 1 cm × 1 cm squares. Before scanning electron microscopy (SEM) observation, all samples were sputtered with a thin gold layer. The pore size of the dressing was observed using a Nova Nano SEM 230 (FEI Company, Hillsboro), and the pore diameter in the SEM images was measured using Nano Measure software (version 1.2).

[0050] The recombinant collagen / calcium alginate composite wound dressing prepared in Example 1 had an average pore size of 25.19 ± 4.63 µm after gelation, the recombinant collagen / calcium alginate composite wound dressing prepared in Comparative Example 4 had an average pore size of 26.77 ± 6.06 µm after gelation, and the recombinant collagen / calcium alginate composite wound dressing prepared in Example 7 had an average pore size of 28.20 ± 6.17 µm after gelation. Compared with Comparative Example 6, the addition of recombinant collagen significantly reduced the pore size. The recombinant collagen / calcium alginate composite wound dressing provided by this invention has the potential to act as a strong absorbent, enabling rapid hemostasis in practical applications.

[0051] The absorbency of the wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 was characterized as follows: Figure 7 As shown. The recombinant collagen / calcium alginate composite wound dressings prepared in Examples 1, 4, and 6 showed better absorption of buffer solutions compared to the wound dressing prepared in Comparative Example 6, with Example 7 being the best. The main reasons are as follows: Recombinant collagen molecules carry a large number of hydrophilic groups such as amino and carboxyl groups, increasing the number of water molecule binding sites. During freeze-drying, recombinant collagen induces the formation of a three-dimensional porous sponge-like structure within and between fibers, significantly increasing the physical liquid storage space. At the same time, the recombinant collagen and alginate network are interpenetrating, synergistically regulating the pore structure during freeze-drying, preventing the alginate from rapidly swelling on the surface after absorbing water to form a dense gel layer, making the dressing easier to achieve uniform and sufficient absorption of exudate.

[0052] Example 7 showed the optimal liquid absorption ratio, which may be attributed to the following combined factors: First, the two functional peptides, GFPGER and GPP, resulted in the largest average pore size among the recombinant collagen-containing groups, providing more ample physical liquid storage space; Second, the combined introduction of GFPGER and GPP functional peptides enriched the hydrophilic groups (amino, carboxyl, etc.) on the recombinant collagen molecules, enhancing their ability to chemically bind water.

[0053] The swelling ratio of the wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6 was characterized as follows: Figure 8As shown. There was no significant difference in swelling rate among the recombinant collagen / calcium alginate composite wound dressings prepared in Example 1, Comparative Examples 2 and 6. Adhesion test

[0054] The adhesion properties of the wound dressings prepared in Examples 1 and 7 and Comparative Examples 4, 6, and 7 to pigskin were tested. Wound dressings cut into 1 cm × 1 cm squares were pasted onto fresh pigskin after degreasing. After being rolled with a pressure roller and left to stand for 10 minutes, they were stretched at a constant speed of 300 mm / min using a tensile testing machine. The stress value that caused the sample to detach from the pigskin after stretching was recorded.

[0055] The results show (see Figure 10 The peel strengths of Example 1, Comparative Example 4, and Example 7 were 37.25 ± 0.94 kPa, 36.82 ± 2.13 kPa, and 38.83 ± 3.14 kPa, respectively, all significantly higher than the 25.42 ± 0.84 kPa of Comparative Example 6 (pure sodium alginate) and Comparative Example 7 (containing natural collagen). Compared with Comparative Example 6 and Comparative Example 7, the addition of recombinant collagen significantly improved the peel strength. This significant improvement is mainly attributed to the organic combination of recombinant collagen and the alginate matrix: on the one hand, the precise control of wet spinning parameters effectively maintained the three-dimensional helical conformation of recombinant collagen, allowing its hydrophilic groups (carboxyl, amino, hydroxyl, etc.) to be fully exposed, enabling it to form a dense hydrogen bond network with the surface of moist tissue, thereby enhancing the adhesion and interfacial stability of the dressing on the wet wound surface; on the other hand, the uniform cross-linking of recombinant collagen in the calcium alginate network further enhanced the overall structural integrity and interfacial stability of the dressing, jointly achieving a significant improvement in peel strength. coagulation dynamics evaluation

[0056] The coagulation dynamics of the recombinant collagen / calcium alginate composite wound dressings prepared in Examples 1-10 and Comparative Examples 1-6 are as follows: Figure 11 As shown. Weigh 70 mg of the sample to be tested and place it in a 50 mL culture dish. Prepare a mixed blood sample according to the volume ratio of heparinized whole blood: 0.2 M CaCl2 solution = 10:1. Take 500 μL of the mixed blood sample and slowly add it to the sample surface to completely cover the sample. Then, incubate the sample at 37 ℃ for 30 s, 60 s, 90 s, 120 s and 150 s respectively. After reaching the preset incubation time, add 10 mL of deionized water and place it on a biological shaker to shake at 50 rpm for 10 min. Finally, measure the absorbance at 540 nm wavelength using a spectrophotometer. At the same time, set up a blank control: take 500 μL of the same mixed blood sample and add 10 mL of deionized water directly for simultaneous treatment.

[0057] In Example 7, the recombinant collagen exhibited optimal blood coagulation properties due to the presence of both GFPGER and GPP sequences. Examples 1 and Comparative Example 4, containing only GFPGER or GPP sequences, showed slightly higher blood coagulation indices. Other examples and comparative examples, due to differences in sequence, content, and concentration, all had higher blood coagulation indices than Examples 1, 7, and Comparative Example 4. The recombinant collagen / calcium alginate composite wound dressing prepared in this invention is rich in GFPGER and GPP sequences and thus exhibits excellent hemostatic effects: the GFPGER sequence specifically binds to the platelet surface integrin α2β1 receptor, and the GPP sequence targets the GPVI receptor, jointly achieving active platelet capture, activation, and aggregation; simultaneously, calcium alginate fibers release Ca... 2+ It also absorbs concentrated blood clotting factors, amplifies the cascade of clotting reactions, and synergistically accelerates the formation of stable fibrin thrombi. Biocompatibility assessment

[0058] The biocompatibility characterization of the recombinant collagen / calcium alginate composite wound dressings prepared in Examples 1, 7 and Comparative Examples 4, 6, and the collagen / alginate composite dressing prepared in Comparative Example 7 is as follows: Figure 12 As shown, mouse embryonic fibroblasts (NIH / 3T3) were seeded onto five different composite hemostatic dressings and co-cultured for 1 and 3 days. Live and dead cells were simultaneously stained with fluorescence using a live / dead cell double staining kit, and the cells were monitored under a fluorescence microscope. Green fluorescence represents live cells, and red fluorescence represents dead cells. The fluorescence images show that the series of recombinant collagen / calcium alginate composite wound dressings and collagen / alginate composite dressings prepared in this invention have excellent biocompatibility.

[0059] The spread of mouse embryonic fibroblast NIH / 3T3 scaffolds was observed in the wound dressings prepared in Examples 1, 7, 4, 6, and 7 (e.g., ...). Figure 13 (As shown). NIH / 3T3 cells were seeded in confocal dishes covered with wound dressing samples at a density of approximately 2 × 10⁻⁶. 4 Cells were cultured in wells at 37 °C for 24 h to allow for full cell adhesion and spread. The culture medium was then discarded, and the cells were gently washed with PBS, fixed with 4% paraformaldehyde for 20 min, washed with PBS, and then permeabilized with 0.1% Triton X-100 for 10 min. Next, fluorescently labeled phalloidin was added and incubated at room temperature in the dark for 30 min to label the F-actin backbone. After washing with PBS, DAPI was added for nuclear counterstaining. Finally, the confocal dish was placed under a microscope for observation and image acquisition.

[0060] Compared with the recombinant collagen / calcium alginate composite wound dressings prepared in Example 1 and Comparative Example 4, the composite wound dressing prepared in Example 7 showed a significantly increased cytoskeleton spreading area and also promoted cell proliferation. Compared with the collagen / alginate composite dressing, the composite wound dressing prepared in Example 7 showed significantly improved cell spreading number, spreading area, and proliferation capacity. This is mainly attributed to the precise control of wet spinning parameters, a strategy that synergistically maintains the three-dimensional structure and bioactivity of recombinant collagen, as well as its Ca2+-based... 2+ The bridging is highly effective and anchored on the fiber substrate, thereby enhancing the dressing's positive regulation of cell behavior. Evaluation of cell migration promotion performance

[0061] NIH / 3T3 cell migration experiments were conducted on the wound dressings prepared in Examples 1, 7, 4, 6, and 7. Mouse embryonic fibroblasts (NIH / 3T3) were seeded onto four different composite hemostatic dressings and co-cultured for 3 days. The cytoskeleton was then stained with phalloidin dye, and migration was observed under a confocal microscope. The experimental results (e.g.) Figure 14 As shown in the figure, the series of recombinant collagen / calcium alginate composite wound dressings prepared by the present invention can effectively guide the directional migration of mouse embryonic fibroblasts. Among them, the migration-promoting effect of Example 7 is the most significant, indicating that the material is expected to promote skin wound healing and tissue regeneration by accelerating cell migration, and provide a bioactive dressing with active guiding function for skin repair. Evaluation of hemostatic effect in animals

[0062] The hemostatic effect of the wound dressings prepared in Examples 1, 7, 4, 6, and 7 was evaluated using a rat full-thickness skin wound defect model (e.g., ...). Figure 15 (As shown).

[0063] Adult healthy male SD rats, weighing 220–250 g, were selected. All rats were acclimatized for 7 days prior to the experiment, with free access to food and water, and maintained a circadian rhythm of 12 h light / 12 h dark. The environment was kept clean, with a room temperature of 20–25 ℃ and a relative humidity of 50%–60%. One day before modeling, the rats' backs were shaved after 7 days of acclimatization. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg). They were fixed in a prone position, and the hair on their backs was removed using an animal shaving razor. The wounds were disinfected routinely with povidone-iodine and then deiodized with 75% ethanol. A circular wound with a diameter of 2 cm and a depth of 2 mm was then made on the rat's back with a scalpel, exposing the wound surface. Immediately after modeling, the wound was covered with the series of composite wound dressings prepared according to this invention to achieve hemostasis.

[0064] As can be clearly seen from the photographs, Examples 1 and 7 exhibit significant hemostatic effects, with very little residual blood on the wound surface, far less than in Comparative Example 4. Comparative Example 7, a collagen / alginate composite dressing, also shows some hemostatic effect, but the amount of residual blood is greater than in Example 7. These results suggest that the excellent hemostatic performance of the recombinant collagen / calcium alginate composite dressing prepared in this invention may be attributed to its triple synergistic effect: physical absorption, Ca... 2+ Sustained release activates the coagulation cascade, and the specific binding of active sites on recombinant collagen molecules to platelets / coagulation factors accelerates the hemostasis process. Animal wound healing evaluation

[0065] The recombinant collagen / calcium alginate composite wound dressings prepared in Examples 1 and 7 and Comparative Examples 4 and 6, as well as the collagen / alginate composite dressing in Comparative Example 7, were evaluated in the treatment of full-thickness skin wound defects in rats. Figure 16 As shown. Adult healthy male SD rats, weighing 220–250 g, were selected. All rats were acclimatized for 7 days before the experiment, with free access to food and water, and a circadian rhythm of 12h light / 12h darkness. The housing environment was kept clean, with a room temperature of 20–25 ℃ and a relative humidity of 50%–60%. One day before modeling, the rats' backs were shaved after 7 days of acclimatization. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40–50 mg / kg). They were fixed in a prone position, and the hair on their backs was removed with an experimental animal shaving razor. The wound was disinfected with iodine and then deiodized with 75% ethanol. A circular wound with a diameter of 2 cm and a depth of 2 mm was then made on the back of the rat with a scalpel to expose the wound. The prepared sample was used to cover the wound, and the dressing was changed daily. Samples were collected after 14 days of treatment, and the treatment effect was observed.

[0066] After 14 days of treatment, the healing effects of each group differed significantly. Comparative Example 6 showed significant inflammatory infiltration, disordered collagen arrangement, and poor repair quality; Example 1 showed increased collagen deposition and improved arrangement; Comparative Example 4 showed reduced inflammation and enhanced angiogenesis; while Example 7 showed sufficient inflammation resolution, collagen arrangement approaching normal skin, and good regeneration of skin appendages, demonstrating the best repair effect; Comparative Example 7 had a rich collagen matrix, good tissue structure, but the number of skin appendages and tissue density were weaker than in Example 7. In summary, the moist healing hydrogel formed after the dressing prepared in this invention provides basic physical support for wound repair; and compared to the ordinary collagen used in Comparative Example 7, recombinant collagen retains a more complete three-dimensional structure and abundant cell recognition sites, enabling it to more efficiently mediate the adhesion, migration, and proliferation of fibroblasts and endothelial cells, thereby accelerating angiogenesis and tissue remodeling. The regenerative microenvironment synergistically constructed by both resulted in significantly better healing quality in Example 7 than in Comparative Example 7, confirming the clear advantage of recombinant collagen in promoting functional skin regeneration. Effect of expression vector on recombinant collagen production

[0067] Table 1 shows a comparison of recombinant collagen shake-flask yields obtained under the same expression conditions as described in Examples 1, 2, and Comparative Example 1, with only the expression vector type changed. As shown in Table 1, the recombinant collagen shake-flask yield of the pPIC9K group reached 450 mg / L, which was 3.85 times and 8.33 times that of the pPICZαA group (117 mg / L) and the pGAPZαA group (54 mg / L), respectively.

[0068] Table 1. Comparison of recombinant collagen yields from different expression vectors pPIC9K AOX1 (Methanol-induced) 1% methanol 450 mg / L pPICZαA AOX1 (Methanol-induced) 1% methanol 117 mg / L pGAPZαA GAP (combined type) No inducement required 54 mg / L Effect of G418 screening concentration on screening efficiency of high copy integration strains

[0069] Table 2 shows the effect of different G418 screening concentrations on the survival rate of positive transformants, where 1 mg / mL, 2 mg / mL, and 3 mg / mL correspond to the screening conditions of Example 3, Example 1, and Comparative Example 2, respectively. As shown in Table 2, 20 transformants picked from MD plates were sequentially spotted onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 using sterile toothpicks and incubated at 30 ℃ for 3 days. Table 2 shows that on the 2 mg / mL G418 plate, 18 out of 20 transformants grew; on the 1 mg / mL G418 plate, 11 grew; and on the 3 mg / mL G418 plate, only 5 grew.

[0070] Table 2 Comparison of growth data of transformants with different G418 concentrations. 1 20 11 55% 2 20 18 90% 3 20 5 25% Influence of wet spinning process parameters on the overall performance of dressings

[0071] Based on the wet spinning process parameters (6% CaCl2, crosslinking time 1.0 h, pH 4) described in Example 1, the effects of CaCl2 concentration, ionic crosslinking time, and coagulation bath pH on the cell proliferation rate, blood coagulation index, and tensile strength of the composite wound dressing were further investigated. The results are shown in Table 3. The data under pH 3 conditions correspond to Example 10.

[0072] Table 3 compares the effects of different wet spinning parameters on the cell proliferation rate, blood coagulation index, and tensile strength of recombinant collagen / calcium alginate composite wound dressings. As shown in Table 3, under fixed coagulation temperature and ethanol concentration, the overall performance of the recombinant collagen / calcium alginate composite wound dressings exhibits a clear trend change with variations in CaCl2 concentration, ionic cross-linking time, and pH. Specifically, with increasing CaCl2 concentration, the wound dressings show a clear trend of initial improvement followed by decline. The cell proliferation rate reaches a peak of 379% at a CaCl2 concentration of 6 wt%, while the blood coagulation index drops to a minimum of 0.75, indicating the optimal hemocoagulant effect. Appropriate CaCl2 concentration... 2+ On the one hand, it mediates the formation of alginate into a uniform ionic cross-linked framework; on the other hand, it can regulate the aggregation behavior of recombinant collagen molecules, maintain its triple helix structure, and retain binding sites with integrins and platelets. Extending the cross-linking time from 0.25 h to 1.0 h significantly increased cell proliferation rate, continuously decreased blood coagulation index, and steadily increased tensile strength. However, after 1.0 h, only tensile strength maintained an increasing trend. This is because when the cross-linking time is insufficient, the alginate cross-linked network is incompletely formed, the fiber structure is unstable, and recombinant collagen in wound dressings is easily dissolved, making it difficult to exert its cell proliferation and hemostatic effects. The bioactivity of recombinant collagen is highly dependent on pH changes; the cell proliferation rate is highest and the blood coagulation index is lowest at pH = 4. Deviating from this pH, whether decreasing or increasing, weakens both cell proliferation capacity and hemostatic effect. The triple helix structure of recombinant collagen has a stable range; under strongly acidic conditions, hydrogen bonds are broken, causing the triple helix structure to unwind, denature, and become inactive; under neutral conditions, aggregation and precipitation occur, reducing cell recognition ability.

[0073] Table 3. Effects of different wet spinning parameters on cell proliferation rate, blood coagulation index, and tensile strength of recombinant collagen / calcium alginate composite wound dressing. 37 2 1.0 4 30 156 0.93 52.7 37 4 1.0 4 30 210 0.82 60.4 37 6 1.0 4 30 379 0.75 87.6 37 8 1.0 4 30 147 1.04 94.2 37 6 0.25 4 30 121 0.99 41.6 37 6 0.5 4 30 135 0.91 66.2 37 6 1.25 4 30 132 0.85 89.8 37 6 1.5 4 30 127 0.83 101.4 37 6 1.0 2 30 111 0.94 52.3 37 6 1.0 3 30 238 0.88 62.4 37 6 1.0 5 30 219 0.84 119.3 37 6 1.0 7 30 152 1.08 127.0 Evaluation of hemostatic effect on mouse liver

[0074] Table 4 shows the total blood loss in the liver hemorrhage model. The composite wound dressings prepared in Examples 1-10 and Comparative Examples 1-7 were used in mouse liver hemostasis experiments. Male mice were anesthetized with trichloroacetaldehyde (0.2–0.4 mg / g). The liver was exposed through an abdominal incision, and pre-weighed filter paper was placed beneath the liver. After inducing bleeding at the liver incision, the prepared alginate dressing was applied to the wound to achieve hemostasis. The blood loss was calculated by weighing the filter paper before and after blood absorption, and the time from dressing application to complete cessation of bleeding was recorded as the hemostasis time.

[0075] Table 1 shows that the series of composite wound dressings provided in Examples 1-10 and Comparative Examples 1-7 of this invention resulted in lower blood loss in the liver hemorrhage model compared to Comparative Example 6, significantly shortening the hemostasis time. The hemostatic effect of Example 7 was significantly stronger than that of Comparative Example 7, indicating that when alginate and Ca... 2+ When the mediated coagulation activation background is the same, the difference in hemostatic effect depends entirely on the biological properties of the collagen itself. The ordinary collagen in Comparative Example 7 is derived from animal extraction and has inherent limitations such as uneven molecular weight and large batch-to-batch variations in helical structure. Furthermore, the wet spinning parameters (calcium ion concentration, crosslinking time, pH) are optimized for the characteristic sequence of recombinant collagen and do not provide specific conformational protection for this type of heterogeneous ordinary collagen. Therefore, after fiber formation, a large number of active sites are lost, resulting in limited platelet-binding capacity. In contrast, the recombinant collagen in Example 7 has a precise sequence and uniform conformation. In addition, the aforementioned process parameters provide targeted protection for its three-dimensional structure and active epitopes during the forming stage, fully preserving its activity. Therefore, under the same Ca2+ activation background, the hemostatic effect is significantly improved. 2+ The hemostatic efficacy under the background of assisted coagulation was significantly better than that of the control group 7.

[0076] Table 4. Data on hemostasis of mouse liver using the series of recombinant collagen / calcium alginate composite wound dressings prepared in this invention. Example 1 84.7581.2883.84 125.41112.68114.95 Example 2 86.2489.0385.72 120.98124.09118.63 Example 3 80.3186.5681.11 117.73121.29116.80 Example 4 69.6556.5474.92 98.67108.53116.84 Example 5 64.7561.2873.84 95.41112.68114.95 Example 6 68.5573.6461.49 103.78115.43100.88 Example 7 52.4367.1459.09 86.7988.2693.72 Example 8 64.3365.3361.67 90.33113.6770.33 Example 9 54.3255.1251.57 80.45112.1975.56 Example 10 60.8270.8353.41 94.57113.9087.62 Comparative Example 1 91.3388.4586.81 124.67124.17124.30 Comparative Example 2 98.4188.5188.24 109.31117.48107.64 Comparative Example 3 86.8785.9686.08 113.85120.52113.43 Comparative Example 4 87.5986.9387.63 113.56121.05125.42 Comparative Example 5 106.08109.11105.35 186.67193.17184.85 Comparative Example 6 110.49112.82108.97 197.89201.74196.12 Comparative Example 7 90.9588.4794.63 133.14127.18129.71 In summary, the recombinant collagen / calcium alginate composite wound dressings prepared in the various embodiments of this invention exhibit excellent performance in terms of fiber morphology, mechanical properties, absorbency, adhesion, coagulation dynamics, biocompatibility, cell migration promotion, and in vivo hemostasis and healing effects. Example 7, which simultaneously contains both GFPGER and GPP bifunctional sequences, is the optimal implementation scheme. The comparative results further verify that the expression vector type, G418 screening concentration, recombinant collagen sequence composition, protein content, sodium alginate concentration, and wet spinning parameters have a significant impact on the final product performance, and their preferred range should be within the conditions defined by this invention. Those skilled in the art can make adaptive adjustments to each parameter based on the disclosure of this invention and according to actual needs; such adjustments all fall within the protection scope of this invention.

Claims

1. A method for preparing a recombinant collagen / calcium alginate composite wound dressing, characterized in that, Includes the following steps: S1. Preparation of recombinant collagen with platelet recognition function: Design and synthesize a nucleic acid sequence encoding a recombinant collagen with a platelet recognition motif; insert the nucleic acid sequence into an expression vector to obtain a recombinant expression vector; transform the recombinant expression vector into host cells and screen for positive transformants; construct a recombinant co-expression plasmid using a molecular chaperone gene and transform it into positive transformants to obtain molecular chaperone-enhanced genetically engineered bacteria; ferment the molecular chaperone-enhanced genetically engineered bacteria and induce expression of recombinant collagen; collect the fermentation supernatant, purify and dry it to obtain recombinant collagen; the platelet recognition motif is a GFPGER sequence and / or a GPP sequence; S2, Preparation of mixed spinning solution: The recombinant collagen with platelet recognition function is dissolved in phosphate buffer to obtain a recombinant collagen solution; sodium alginate is dissolved in purified water to obtain a sodium alginate solution; the recombinant collagen solution and the sodium alginate solution are mixed to obtain a mixed spinning solution; S3, Wet spinning: The mixed spinning solution described in step S2 is extruded into a coagulation bath containing calcium ions for ion cross-linking and molding to obtain recombinant collagen / calcium alginate composite fiber.

2. The method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, characterized in that, Also includes: The high-copy screening step uses G418 antibiotic to screen genetically engineered bacteria, with the concentration of G418 antibiotic being 1~2.5 mg / mL.

3. The method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, wherein the molecular chaperone gene is selected from one or more of HAC1, BIP, or SEC1.

4. The method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, characterized in that, The expression vector is pPIC9K or pPICZα A.

5. The method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, characterized in that, In the mixed spinning solution, the mass ratio of recombinant collagen to sodium alginate is 1-15:

100.

6. A method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, characterized in that, The calcium ion concentration in the coagulation bath during step S3 is 2-8 wt%.

7. The method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, characterized in that, The pH of the coagulation bath is 2-5.

8. A method for preparing a recombinant collagen / calcium alginate composite wound dressing according to claim 1, characterized in that, In the wet spinning process, the spinneret orifice diameter is 50~500 μm, the spinneret speed is 10~100 m / min, the extrusion gas is nitrogen, helium or argon, and the winding speed is 40~100 m / min.

9. The method according to claim 1, characterized in that, It also includes a post-processing step, in which the recombinant collagen / calcium alginate composite fiber is washed, stretched, freeze-dried, and sterilized to prepare a composite wound dressing.

10. A recombinant collagen / calcium alginate composite wound dressing prepared by the method described in any one of claims 1-9.

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