A recombinant humanized type Ⅰ collagen, a DLP 3D printing biological ink, a preparation method and applications thereof

Abstract

The application belongs to the field of biomedical materials, and particularly relates to a recombinant humanized type I collagen, a DLP 3D printing biological ink, a preparation method and application, an amino acid sequence of the recombinant humanized type I collagen is shown as SEQ ID NO. 1; the biological ink comprises 50-150 mg / mL methacrylated recombinant humanized type I collagen, 2.0-5.0 mg / mL photoinitiator and 0.1-0.5 mg / mL light absorber; the biological ink has excellent printability and biological activity, a printed recombinant type I collagen scaffold has excellent biological activity, can significantly promote cell migration, proliferation and differentiation; a double-layer cell-loaded scaffold simulating a natural skin structure can be prepared, and the double-layer cell-loaded scaffold shows the effects of promoting epidermal regeneration and dermal remodeling, reducing a wound ROS level, improving a local immune microenvironment and enhancing angiogenesis, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a recombinant humanized type I collagen, a DLP 3D printing bio-ink, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by persistent hyperglycemia. Long-term hyperglycemia can lead to various complications, including diabetic retinopathy, diabetic nephropathy, peripheral neuropathy, autonomic neuropathy, and diabetic wounds. Among these, diabetic wounds are a particularly serious complication. Approximately 25% of diabetic patients develop chronic, non-healing wounds, and about one-third of these may eventually require amputation. Amputation not only severely impacts a patient's quality of life but also increases the risk of cardiovascular disease, depression, and other complications. Therefore, the treatment of diabetic wounds has become a crucial clinical problem that urgently needs to be addressed.

[0003] Difficulty in healing diabetic wounds is closely related to pathological abnormalities such as chronic inflammation, persistent oxidative stress, and impaired angiogenesis. In diabetic wounds, the abnormal microenvironment, including high glucose levels and infection, interferes with macrophage polarization, leading to the continuous accumulation of M1 macrophages and the release of large amounts of pro-inflammatory mediators, reactive oxygen species (ROS), and proteases. This exacerbates tissue damage and prolongs the inflammatory response. The high glucose environment also inhibits angiogenesis, causing insufficient blood supply and hypoxia at the wound site. Furthermore, the continuous accumulation of excessive ROS triggers oxidative damage, further inhibiting cell proliferation and angiogenesis, thus hindering wound repair. Traditional treatment strategies, such as debridement, infection control, and reduction of local pressure, often fail to effectively regulate the complex microenvironment of diabetic wounds, resulting in unsatisfactory treatment outcomes.

[0004] Skin wound repair involves multiple processes, including epidermal re-epithelialization, dermal extracellular matrix remodeling, and vascular network reconstruction. Therefore, there is an urgent need to construct biomaterials with spatially layered characteristics to simulate the epidermal and dermal microenvironment and combine their functions of scavenging reactive oxygen species, anti-inflammation, and promoting angiogenesis to achieve synergistic repair of diabetic wounds.

[0005] Collagen-based biomaterials possess excellent biodegradability and biocompatibility, showing promising application prospects in the field of diabetic wound repair. However, single-collagen systems still have limitations in addressing the complex pathological microenvironment of chronic diabetic wounds, typically requiring further integration with antioxidant, anti-inflammatory, and pro-angiogenic components to achieve comprehensive regulation of the wound microenvironment. Furthermore, natural collagen biomaterials often suffer from poor mechanical properties and swelling capacity, which may reduce their therapeutic efficacy. Compared to traditional animal-derived collagen, recombinant collagen prepared based on genetic engineering offers advantages such as clear origin, minimal batch-to-batch variation, and low immunogenicity. Simultaneously, its structure and function can be regulated through sequence design, giving it unique advantages in the development of functionalized wound repair materials.

[0006] To address the aforementioned technical challenges, this invention develops a methacrylamide-based recombinant type I collagen bio-ink (RCIMA) and constructs a bilayer cell-loaded recombinant type I collagen scaffold (RCIMA-Cell) using DLP 3D printing technology for regulating the microenvironment and repair of diabetic wounds. This scaffold mimics the layered structure of the epidermis and dermis of natural skin by encapsulating immortalized human keratinocytes (HaCaT) in the upper layer and loading human foreskin fibroblasts (HFF-1) in the lower layer. In vitro experiments show that the 3D-printed RCIMA scaffold possesses excellent bioactivity, significantly promoting the migration, proliferation, and differentiation of HaCaT and HFF-1 cells. In a diabetic rat chronic wound model, the cell-loaded scaffold RCIMA-Cell demonstrated effects in promoting epidermal regeneration and dermal remodeling, reducing wound ROS levels, improving the local immune microenvironment, and enhancing angiogenesis. This scaffold provides a novel tissue engineering strategy for the repair of chronic diabetic wounds and shows promising clinical application prospects. Summary of the Invention

[0007] The primary objective of this invention is to provide a recombinant humanized type I collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] A second objective of this invention is to provide a recombinant vector or recombinant genetically engineered bacteria carrying a gene sequence encoding the amino acid sequence of the recombinant humanized type I collagen described herein.

[0009] Preferably, the carrier includes pCold or pET.

[0010] Preferably, the genetically engineered bacterium is Escherichia coli.

[0011] A third objective of this invention is to provide a recombinant type I collagen bio-ink for DLP 3D printing, the bio-ink comprising the following components: 50-150 mg / mL methacrylamide recombinant humanized type I collagen, 2.0-5.0 mg / mL photoinitiator, and 0.1-0.5 mg / mL light absorber; wherein the methacrylamide recombinant humanized type I collagen is a photocrosslinkable collagen derivative obtained by chemically modifying the primary amine groups in the recombinant humanized type I collagen molecule with methacrylic anhydride.

[0012] Preferably, the photoinitiator is one or more of phenyl-2,4,6-trimethylbenzoylphosphine lithium and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone; the light absorber is selected from one or more of tartrazine and brilliant blue.

[0013] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid with a concentration of 2.0 mg / mL.

[0014] Preferably, the light absorber is lemon yellow, and its concentration is 0.5 mg / mL.

[0015] A fourth objective of this invention is to provide a method for preparing the bio-ink, comprising the following steps:

[0016] (1) Dissolve methacrylamide recombinant type I collagen in PBS buffer to obtain a collagen solution;

[0017] (2) Add a photoinitiator and a light absorber to the collagen solution described in step (1);

[0018] (3) After mixing evenly, a recombinant type I collagen bio-ink for DLP 3D printing is obtained;

[0019] The methacrylated recombinant type I collagen described in step (1) was prepared by the following method: recombinant humanized type I collagen was dissolved in PBS buffer; methacrylic anhydride was added to the recombinant humanized type I collagen solution at 4°C and reacted under alkaline conditions for 12-24 hours; after the reaction was completed, dialysis was performed for purification; and lyophilized recombinant humanized type I collagen was obtained.

[0020] A fifth objective of this invention is to provide a recombinant humanized type I collagen 3D scaffold prepared by DLP 3D printing technology using the bio-ink as described in claim 3.

[0021] Preferably, the scaffold is a double-layered cell-carrying scaffold, with both the upper and lower layers printed using RCIMA bio-ink. The upper and lower layers of the scaffold are loaded with different live cells, which are selected from one or more of keratinocytes, fibroblasts, vascular endothelial cells, and mesenchymal stem cells.

[0022] A sixth objective of this invention is to provide the application of the aforementioned bio-ink or the aforementioned 3D scaffold in the fabrication of medical devices.

[0023] A seventh objective of the present invention is to provide the use of the described bio-ink or the described 3D scaffold in the preparation of skin repair dressings, implants or biomaterials.

[0024] The eighth object of the present invention is to provide the application of the bio-ink or the 3D scaffold in the preparation of diabetic wound repair dressings.

[0025] The beneficial effects of the present invention are: (1) The present invention modifies recombinant type I collagen by methacrylylation, thereby introducing photocurable functional groups while retaining the biological characteristics of collagen, so that the resulting bio-ink can meet the requirements of DLP. (1) Requirements for rapid prototyping and structural stability in 3D printing; (2) By using RCIMA in combination with lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator and lemon yellow light absorber, the photocuring process can be effectively controlled, and the controllability of photocuring during printing can be improved, which is conducive to obtaining a three-dimensional printed structure with better forming effect and clearer boundary; (3) The recombinant type I collagen scaffold prepared by the present invention has excellent biological properties and can significantly promote the migration, proliferation and differentiation of HaCaT cells and HFF-1 cells; (4) By loading HaCaT cells and HFF-1 cells into bio-ink respectively, the present invention constructs a biomimetic double-layer cell-carrying scaffold, which can better simulate the layered structure of natural skin and shows good effects in promoting epidermal regeneration, dermal remodeling, reducing oxidative stress level, regulating local immune microenvironment and promoting angiogenesis; (5) The cell-carrying scaffold provided by the present invention can provide a new tissue engineering treatment approach for the repair of chronic diabetic wounds, which has high application value and good clinical prospects. Attached Figure Description

[0026] Figure 1 Synthesis, characterization and printability analysis of methacrylamide recombinant type I collagen (RCIMA);

[0027] Note: (A) Schematic diagram of RCIMA synthesis; (B) 1H NMR spectra, a is 5.6 ppm, b is 5.3 ppm, c is 1.8 ppm; (C) FTIR spectrum; (D) CD spectrum; (E) gelation properties of RCIMA bio-ink before and after 405 nm light irradiation; (F) in-situ photocuring rheological curve; (G) amplitude scan curve; (H) frequency scan curve; (IK) printability verification of RCIMA bio-ink.

[0028] Figure 2 Physicochemical characterization of RCIMA and ColMA scaffolds;

[0029] Note: ColMA's (A) SEM image and (B) pore size statistics; RCIMA's (C) SEM image and (D) pore size statistics; (E) swelling properties; (F) degradation properties; (G) stress-strain curves; (H) compressive strength; (I) compressive modulus.

[0030] Figure 3 HFF-1 cell viability of RCIMA and ColMA scaffolds;

[0031] Note: (A) Cell migration; (B) Cell migration rate; (C) Live / dead cell staining; (D) Cell proliferation; (E) Cell differentiation.

[0032] Figure 4 HaCaT cell activity of RCIMA and ColMA scaffolds;

[0033] Note: (A) Cell migration; (B) Cell migration rate; (C) Live / dead cell staining; (D) Cell proliferation; (E) Cell differentiation.

[0034] Figure 5 Cell-loaded printing and cell viability evaluation using RCIMA bio-ink;

[0035] Note: (A) Schematic diagram of HFF-1 cells and (B) HaCaT cells (a); (b) Staining of live / dead cells after 1, 4 and 7 days of culture; (c) Cell viability.

[0036] Figure 6 RCIMA-Cell is used for wound repair in diabetes;

[0037] Note: (A) Schematic diagram of diabetic wound treatment; (B) Wound appearance diagram; (C) Quantitative analysis of wound closure rate.

[0038] Figure 7 Histological analysis of diabetic wounds;

[0039] Note: (A) H&E staining; (B) Masson staining; (C) wound length statistics; (D) epidermal thickness statistics; (E) quantitative analysis of collagen volume fraction; (F) Sirius red staining; (G) ratio of type I to type III collagen in the wound.

[0040] Figure 8 Immunofluorescence staining and quantitative analysis of diabetes-related wound markers;

[0041] Note: (A) DCFH-DA staining; (B) Quantitative analysis of DCFH-DA positive area; (C) CD86 immunofluorescence staining; (D) CD206 immunofluorescence staining; (E) Quantitative analysis of M2 to M1 ratio; (F) CD31 immunofluorescence staining; (G) Quantitative analysis of CD31 positive area; (H) α-SMA immunofluorescence staining; (I) Quantitative analysis of α-SMA positive area. Detailed Implementation

[0042] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are further illustrations of the present invention, but are not intended to limit the scope of the invention. All other equivalent substitutions or obvious modifications that can be conceived by those skilled in the art without creative effort based on the technical solutions of the present invention should be considered to be included within the protection scope of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods in the art, and the reagents used are all commercially available.

[0044] Example 1: Preparation and Characterization of Recombinant Humanized Type I Collagen

[0045] 1. Preparation of recombinant humanized type I collagen

[0046] The amino acid sequence of the recombinant humanized type I collagen is shown in SEQ ID NO.1. This protein is obtained by treating precursor collagen with protease. The amino acid sequence of the precursor collagen is shown in SEQ ID NO.2, and the gene sequence is shown in SEQ ID NO.3.

[0047] 2. Synthesis of precursor collagen gene sequence and construction of expression vector

[0048] The gene sequence for synthesizing precursor collagen was constructed into the E. coli expression vector pCold, and the successful synthesis of the plasmid was confirmed by DNA sequencing. The plasmid was transformed into E. coli BL21-DE3 strain to obtain a precursor collagen expression strain. The successfully transformed strain was stored in glycerol at -80 °C.

[0049] 3. Expression of precursor collagen

[0050] Add 20 μL of the cryopreserved bacterial culture to 200 mL of LB broth containing antibiotics. Incubate overnight at 37 °C using a shaker. Then, transfer the culture to 1 L of LB broth containing antibiotics at a 2% inoculation rate and continue amplification in a shaker at 37 °C. Wait for OD... 600 When the value reaches the range of 1.2-2.0, adjust the temperature of the shaker to 25 ℃, add 1 mMIPTG to induce expression, and incubate overnight at a constant temperature; centrifuge the bacterial culture in a low temperature centrifuge at 3500 rpm, 4 ℃, for 30 min, and collect the bacterial cells.

[0051] 4. Purification of recombinant humanized type I collagen

[0052] The bacterial cells were dissolved in buffer solution (20 mM sodium phosphate buffer, 20 mM imidazole, 0.5 M sodium chloride, pH 7.4) at a ratio of 1:10. Cell disruption was performed using a high-pressure homogenizer. The disrupted suspension was centrifuged again, and the supernatant (crude protein solution) was collected. This solution was further purified using a nickel affinity chromatography column to obtain precursor collagen. Thrombin was added to a final concentration of 8 U / mL to treat the precursor collagen, and the enzyme digestion products were removed to obtain recombinant humanized type I collagen (RCI).

[0053] Example 2: Preparation, characterization, and printing performance verification of recombinant type I collagen bio-ink (RCIMA)

[0054] 1. Preparation of recombinant type I collagen bio-ink

[0055] Recombinant type I collagen bio-ink (RCIMA) is prepared by chemically modifying the primary amine groups of lysine residues in recombinant humanized type I collagen (RCI) using methacrylic anhydride (MA). 1 g of recombinant type I collagen (RCI) was dissolved in 10 mM PBS buffer (pH=7.4), and 1 mL of methacrylic anhydride (MA) was slowly added dropwise at 4 °C, reacting for 24 hours with continuous stirring. Throughout the reaction, the pH was adjusted to 8-9 using NaOH solution. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8-14 kDa and dialyzed in 5 L of ultrapure water for 3-5 days to remove unreacted MA. After dialysis, the dialyzed sample was freeze-dried to obtain RCIMA. Figure 1 As shown in Figure A.

[0056] 2. Proton nuclear magnetic resonance spectrum (NMR) 1 H NMR characterization

[0057] Lyophilized RCI and RCIMA samples were dissolved in deuterium water to prepare a solution with a mass concentration of 15 mg / mL, and nuclear magnetic resonance spectroscopy was used to collect samples at 25 °C. 1 The 1H NMR spectrum was processed and analyzed using MestReNova software.

[0058] The results are as follows Figure 1 As shown in Figure B, compared with RCI, RCIMA detected characteristic peaks of vinyl (-CH2) at chemical shifts of 5.3 ppm and 5.6 ppm, and characteristic peaks of methyl (-CH3) at chemical shift of 1.8 ppm, indicating that the methacryloyl group has been successfully modified into the RCI molecule.

[0059] 3. Fourier Transform Infrared Spectroscopy (FTIR) Characterization

[0060] 2 mg of lyophilized RCI and RCIMA samples were weighed separately, thoroughly mixed with 200 mg of KBr, and ground until homogeneous. Transparent tablets with a diameter of 10 mm were prepared using a hydraulic tablet press. The samples were then analyzed using Fourier transform infrared spectroscopy (FTIR) with a wavenumber range of 4000–400 cm⁻¹. -1 .

[0061] The results are as follows Figure 1 As shown in C, RCI and RCIMA are at 3323 cm. -1 and 3072 cm -1 Characteristic absorption peaks corresponding to amide A and amide B bands can be observed at 1655, 1548, and 1240 cm⁻¹. -1 Characteristic absorption peaks belonging to amide bands I, II, and III can be observed. Compared to RCI, RCIMA shows a peak at 1056 cm⁻¹. -1 A new absorption peak appears at the point, which can be attributed to the in-plane bending vibration of C=CH, further proving that RCI has been successfully modified by methacrylation.

[0062] 4. Circular dichroism (CD) characterization

[0063] Weigh 2 mg of RCI and RCIMA samples respectively and dissolve them in 2 mL of 10 mM PBS solution (pH=7.4). The samples were tested using a circular dichroism chromatograph equipped with a PeltIer temperature control. The test temperature was set to 4 ℃, the wavelength scan range was 190-260 nm, and the step size was 1.0 nm.

[0064] Scan results as follows Figure 1As shown in Figure D, both RCI and RCIMA exhibit a characteristic positive absorption peak at 220 nm and a characteristic negative absorption peak at 190 nm. The results indicate that both maintain the typical triple helix structure of collagen, and MA modification does not significantly affect the triple helix structure of RCI.

[0065] 5. Characterization of photocuring performance

[0066] RCIMA was dissolved in ultrapure water to prepare a 100 mg / mL solution, which was then thoroughly mixed with phenyl-2,4,6-trimethylbenzoyl lithium phosphine (50 mg / mL) at a volume ratio of 25:1. The resulting solution was quantitatively transferred to a 2 mL transparent glass vial and then heated to a 405 nm light source (25 mW / cm²). 2 Observe its photocrosslinking behavior under irradiation.

[0067] Experimental results are as follows Figure 1 As shown in E, after adding the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphinate and irradiating with 405 nm blue light, the RCIMA bio-ink changed from a flowing state to a stable hydrogel state, indicating that it has the ability to photocrosslink and form gels.

[0068] 6. Rheological property testing

[0069] RCIMA was dissolved in ultrapure water to prepare a 100 mg / mL solution, and 50 mg / mL of phenyl-2,4,6-trimethylbenzoyl lithium phosphine was added and mixed thoroughly. The RCIMA bio-ink was then characterized in situ using a rheometer for photocuring rheology. The test conditions were set as follows: no blue light irradiation for the first 15 seconds, followed by blue light irradiation after 15 seconds, and the changes in storage modulus (G') and loss modulus (G'') over time were recorded in real time. Amplitude scanning was performed at a fixed angular frequency of 1 rad / s, with a strain range of 0.1-100%, testing the G' and G'' of the samples. Frequency scanning was performed under a constant shear strain of 1%, with a scanning frequency range of 0.1-100 rad / s.

[0070] In-situ photocuring rheological results showed that the storage modulus (G') of the RCIMA bio-ink gradually increased with prolonged irradiation time, reaching its maximum value and stabilizing after 30 seconds of irradiation, indicating that it can complete photocrosslinking and curing in a short time. Figure 1 F). Amplitude scanning results show that within the strain range of 0.1-10%, G' remains consistently higher than the loss modulus (G'') and remains relatively stable, indicating that the material exhibits good structural stability within this strain range. As the strain increases, G' gradually decreases while G'' gradually increases, indicating that its internal network structure begins to deteriorate. Figure 1Furthermore, frequency scanning results show that within the frequency range of 1-100 rad / s, G' of RCIMA is consistently higher than G'', indicating that the hydrogel formed exhibits stable viscoelastic characteristics. Figure 1 H).

[0071] 7. Printability Performance Study

[0072] A 50 mg / mL photoresist (UA) solution was added to RCIMA bio-ink at a volume ratio of 100:1, followed by a 50 mg / mL lithium phenyl-2,4,6-trimethylbenzoylphosphine solution at a volume ratio of 25:1. After mixing, the mixture was transferred to the feed tank of a DLP 3D printer. Printing parameters such as layer height, light intensity, and exposure time were set in the control software. A pre-built 3D model was imported into the control software, sliced, and then printed. Finally, a honeycomb structure, an ear, and an octopus model were fabricated using the DLP 3D printer.

[0073] The results are as follows Figure 1 As shown in Figure IK, the successful printing of the honeycomb structure demonstrates that RCIMA bio-ink possesses stable forming capabilities. Further printing of complex models such as ears and octopuses yielded constructs with complete morphology, clear boundaries, and essentially consistent with the pre-designed structure. These results indicate that RCIMA bio-ink has good printability and can be used for the fabrication of complex three-dimensional structures.

[0074] Example 3: Characterization of the physicochemical properties of RCIMA stents

[0075] 1. Morphological characteristics

[0076] To further evaluate the physicochemical properties of the RCIMA scaffold, this embodiment used methacrylic anhydride-modified yak type I collagen bio-ink (ColMA) as a control. Considering the solubility characteristics of ColMA, its concentration was set at 10 mg / mL. Cylindrical scaffolds with a diameter of 10 mm and a height of 2 mm were printed using a DLP 3D printer and then freeze-dried. The freeze-dried scaffolds were fixed onto a silicon wafer, sputter-coated with gold, and their microstructure was observed using a scanning electron microscope at an accelerating voltage of 5.0 kV.

[0077] The microstructure of freeze-dried RCIMA and ColMA scaffolds was observed using scanning electron microscopy (SEM).

[0078] The results showed that both scaffolds formed a porous and interconnected three-dimensional network structure, with average pore sizes of 175.8 μm and 242.8 μm for RCIMA and ColMA, respectively. Figure 2These results indicate that RCIMA possesses a suitable porous structure, providing a favorable microenvironment for cell growth.

[0079] 2. Swelling performance test

[0080] Weigh the printed RCIMA and ColMA cylindrical supports separately and record the initial mass m0. Immerse the samples in ultrapure water at room temperature, and remove them after 1, 2, 4, 8, 16, and 24 hours. After removing excess surface moisture, weigh the samples and record the mass m. t Calculate the swelling ratio (SR) of the material according to Formula 1.

[0081] (1)

[0082] The results are as follows Figure 2 As shown in Figure E, ColMA exhibits a lower swelling rate, while RCIMA reaches swelling equilibrium within 4 hours with a swelling rate of 130%, indicating a stronger water absorption capacity. This property facilitates the material's absorption of wound exudate and maintenance of a moist local environment, thereby providing conditions for wound healing.

[0083] 3. Evaluation of Degradation Performance

[0084] Take the printed RCIMA and ColMA cylindrical scaffolds and determine their initial mass, denoted as m0. Place the samples in TES buffer (pH 7.4) containing 5 U / mL collagenase and 1 mM CaCl2 and incubate at 37 °C. Weigh the samples again at preset time points and record the remaining mass m. t The remaining mass fraction was calculated according to Formula 2 to assess its in vitro degradation behavior.

[0085] (2)

[0086] The degradation behavior of RCIMA and ColMA is as follows: Figure 2 As shown in Figure F, there was no significant difference in the residual mass fraction of RCIMA and ColMA during the first 9 days; however, on day 14, the residual mass fraction of RCIMA was higher than that of ColMA, at 75.1% ± 7.5% and 56.2% ± 4.5%, respectively. These results indicate that RCIMA possesses excellent resistance to enzymatic hydrolysis.

[0087] 4. Compression performance test

[0088] RCIMA and ColMA cylindrical supports with a diameter of 10 mm and a height of 2 mm were fabricated using a DLP 3D printer, and compression tests were performed on them using a universal testing machine with a compression rate of 10 mm / min.

[0089] The results showed that the critical strain values ​​of ColMA and RCIMA during the compression process were 40.4% and 49.2%, respectively, and the maximum average compressive strengths were 35.0 kPa and 284.2 kPa, respectively. Figure 2 Based on the slope of the linear elastic region of the stress-strain curve, the average compressive moduli of ColMA and RCIMA are calculated to be 14.0 kPa and 57.9 kPa, respectively. Figure 2 I). These results show that RCIMA has superior mechanical properties compared to ColMA in both compressive strength and compressive modulus.

[0090] Example 4: Cell viability of RCIMA scaffold

[0091] 1. Cell adhesion

[0092] Human foreskin fibroblasts (HFF-1) and immortalized keratinocytes (HaCaT) were purchased from the Cell Bank of the Chinese Academy of Sciences. Both cell types were cultured in DMEM as the basal medium, supplemented with 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, at 37 ℃ in a 5% CO2 incubator. HFF-1 or HaCaT cells were seeded into 6-well plates. When cell confluence reached approximately 90%, a linear scratch was made on the cell surface using a sterile 20 μL pipette tip, followed by gentle rinsing with PBS to remove detached cells. Cells were then treated with either RCIMA or ColMA scaffolds, with the DMEM-treated group serving as a blank control. After 24 hours of further culture, the closure of the scratches was observed and photographed using an inverted fluorescence microscope. Cell migration area was quantitatively analyzed using ImageJ software, and cell migration rate was calculated according to Formula 3.

[0093] (3)

[0094] Where A0 represents the initial scratch area, A t This indicates the area of ​​the scratch after 24 hours.

[0095] The results are as follows Figure 3 AB and Figure 4As shown in Figures AB, in HFF-1 cells, the cell migration rates of the ColMA group and the RCIMA group were 66.7 ± 12.1% and 72.1 ± 11.2%, respectively, both significantly better than the control group's 31.7 ± 4.5%. In HaCaT cells, the cell migration rates of the ColMA group and the RCIMA group were 77.9 ± 14.8% and 81.4 ± 13.2%, respectively, also significantly higher than the control group's 35.5 ± 4.4%. These results indicate that the RCIMA scaffold can effectively promote the migration of HFF-1 cells and HaCaT cells, providing experimental evidence for their application in wound repair.

[0096] 2. Cell proliferation

[0097] Add 100 μL of cells at a density of 1 × 10⁶ cells to a 96-well plate. 5 HFF-1 or HaCaT cell suspensions were added at a density of 1 × 10⁶ cells / mL. After cell adhesion, RCIMA or ColMA scaffolds were added to each well, with a DMEM-only cell group serving as a blank control. Cells were cultured for 1, 4, and 7 days. At each predetermined time point, the culture medium was discarded, and after washing with PBS, CCK-8 working solution was added to each well, which was then incubated at 37 °C for 1 hour. Finally, the absorbance at 450 nm was measured using a microplate reader. The RCIMA or ColMA scaffolds were placed in confocal culture dishes, and cells were added at a density of 1 × 10⁶ cells / mL. 5 HFF-1 cells or HaCaT cells per mL were cultured for 1, 4, and 7 days. At the preset time points, the scaffolds were stained for 1 hour under light-protected conditions using a live / dead cell staining kit, and the cell viability was observed using a laser confocal microscope.

[0098] The results are as follows Figure 3 CD and Figure 4 As shown in CD, after HFF-1 cells and HaCaT cells were seeded onto ColMA and RCIMA scaffolds, the number of viable cells on the scaffold surface gradually increased with prolonged culture time. CCK-8 assay results further indicated that the cell viability of both cell types gradually increased with prolonged culture time in both the ColMA and RCIMA groups. By day 7, the cell viability of HFF-1 cells and HaCaT cells in the RCIMA group were 285.0 ± 11.9% and 223.8 ± 11.8%, respectively, both significantly higher than those in the ColMA group (261.7 ± 6.3% and 163.4 ± 5.6%). These results indicate that the RCIMA scaffold can significantly promote the proliferation of HFF-1 cells and HaCaT cells.

[0099] 3. Gene expression analysis

[0100] The inoculation density in the 6-well plate was 1 × 10⁻⁶. 5 HFF-1 cells or HaCaT cells were cultured for 7 days using RCIMA or ColMA scaffolds, respectively. After culture, total RNA was isolated using a total RNA extraction kit, and cDNA was further prepared using a PrimeScript RT kit. The obtained cDNA was then used as a template for amplification and detection on an Agilent Mx3005P system using TB Green Premix Ex Taq II. GAPDH was used as an internal control gene, and 2... -ΔΔCt The relative expression levels of genes were calculated using the method described in Table 1.

[0101] Table 1 Primer sequences of differentiation-related genes in HFF-1 cells and HaCaT cells

[0102] α-Smooth muscle actin (α-SMA) is an important marker of fibroblast differentiation into myofibroblasts, which play a crucial role in wound contraction and repair. Compared with the control group, the expression levels of α-SMA in the ColMA and RCIMA groups were significantly increased, upregulated by 1.61-fold and 3.23-fold, respectively. Type I collagen and type III collagen are the main structural proteins in the extracellular matrix (ECM) of the dermis, jointly participating in ECM remodeling. The experimental results showed that the expression of Collagen I gene was upregulated by 7.97-fold and 9.84-fold, respectively, and the expression of Collagen III gene was upregulated by 1.29-fold and 1.58-fold, respectively, in the ColMA and RCIMA groups. Figure 3 E). Filaggrin, Involucrin, and Loricrin are all important markers related to terminal differentiation of keratinocytes and epidermal barrier formation. Compared with the control group, the Filaggrin gene was upregulated by 1.34-fold and 1.48-fold in the ColMA and RCIMA groups, respectively; the Involucrin gene was upregulated by 1.57-fold and 1.70-fold in the ColMA and RCIMA groups, respectively; and the Loricrin gene was upregulated by 1.29-fold and 2.28-fold in the ColMA and RCIMA groups, respectively. Figure 4 E). Experimental results show that the RCIMA scaffold can upregulate the expression of genes related to HFF-1 cell and HaCaT cell differentiation, and has the potential to promote dermal matrix remodeling and epidermal function reconstruction.

[0103] Example 5: Bioactivity of RCIMA Cell-Printed Scaffolds

[0104] RCIMA bio-ink was mixed with HaCaT cells or HFF-1 cells, and heart-shaped and star-shaped cell-borne models were prepared using DLP 3D printing technology. Cell viability of the printed constructs was evaluated using live / dead cell staining. Figure 5 Aa and Figure 5 Ba).

[0105] The results are as follows Figure 5 Ab and Figure 5 As shown in Figure Bb, green viable cell signals predominated in both the heart-shaped and star-shaped cell constructs printed by DLP 3D. Magnified local images and 3D imaging further demonstrate that both cell types were uniformly distributed within the constructs; the cell number gradually increased with prolonged culture time, indicating that the cells not only maintained a high survival rate after printing but also continued to grow within the scaffold. Furthermore, compared to day 1, the relative viability of HaCaT cells and HFF-1 cells increased significantly to 115.6 ± 3.5% and 131.5 ± 2.9% on day 7, respectively. Figure 5 Ac and Figure 5 The results show that RCIMA bio-ink can provide a suitable environment for cell survival and growth while meeting the requirements for printing.

[0106] Example 6: Study on cell-borne scaffolds promoting wound repair in diabetic patients

[0107] A cylindrical bilayer cell-carrying scaffold with a diameter of 10 mm and a height of 2 mm was fabricated using a DLP 3D printer. During printing, the lower layer was first printed using RCIMA bio-ink loaded with HFF-1 cells. Once the printing height reached 1.8 mm, the printing process was paused, and the bio-ink in the ink tank was replaced with RCIMA bio-ink loaded with HaCaT cells to continue printing the upper layer. After printing, the bilayer cell-carrying scaffold was transferred to complete culture medium and incubated at 37 °C in a 5% CO2 incubator for later use.

[0108] The control group used RCIMA bio-ink without cell loading, and the printing process was the same.

[0109] A streptozotocin (STZ)-induced full-thickness skin defect model was established in diabetic rats to evaluate the repair effect of a double-layer cell-loaded scaffold (RCIMA-Cell) on diabetic wounds. Figure 6A) After creating a 10 mm diameter full-thickness skin defect on the back of STZ-induced diabetic rats, the rats were treated with RCIMA and RCIMA-Cell, respectively. The model group received no treatment. A control group of normal rats that had not received STZ-induced diabetes had the same full-thickness skin defect created. The wounds were photographed at predetermined time points, and tissue samples were collected to analyze wound healing.

[0110] General observation results are as follows Figure 6 As shown in Figure B, from day 4 onwards, the wound area in each group gradually decreased, with the healing speed of the blank group, RCIMA group, and RCIMA-Cell group all being faster than that of the model group. By day 21, the wound closure rate of the RCIMA-Cell group reached 98.1 ± 0.7%, significantly higher than that of the model group (68.8 ± 7.1%) and the RCIMA group (84.8 ± 3.5%). Figure 6 C). These results indicate that RCIMA-Cell can significantly promote wound healing in diabetic patients.

[0111] Example 7: Histological evaluation of wound tissue

[0112] To further evaluate the wound-healing promoting effect of RCIMA-Cell, hematoxylin and eosin (H&E) staining was used to observe wound tissue morphology and measure wound length. H&E staining results showed that by day 14, incompletely sloughed scabs were still visible in the control group; the model group showed significant inflammatory cell infiltration at the wound site, and incomplete epidermal formation; both the RCIMA and RCIMA-Cell groups achieved complete epidermal regeneration. On day 21, a significant gap was still visible at the epidermal-dermal interface in the model group, indicating a lack of continuous and tight tissue connection, while the other groups had achieved relatively complete epidermal reconstruction. Figure 7 A). Furthermore, wound length measurements showed that on day 14, compared to the model group (4.25 ± 0.18 mm), the wound lengths in the blank group, RCIMA group, and RCIMA-Cell group were significantly shorter, at 2.43 ± 0.02 mm, 3.39 ± 0.08 mm, and 2.02 ± 0.44 mm, respectively. By day 21, the wound length in the model group was 3.23 ± 0.52 mm, while the wound lengths in the blank group, RCIMA group, and RCIMA-Cell group decreased to 1.16 ± 0.21 mm, 1.17 ± 0.04 mm, and 0.86 ± 0.14 mm, respectively. Figure 7C). Epidermal thickness is also an important indicator reflecting the wound healing process. It usually increases gradually during the inflammatory and proliferative phases, and decreases gradually during the remodeling phase. Epidermal thickness analysis showed that on day 14, the model group had the lowest epidermal thickness, only 28.7 ± 2.8 μm; the blank group, RCIMA group, and RCIMA-Cell group had thicknesses of 76.8 ± 14.5 μm, 75.2 ± 9.7 μm, and 81.2 ± 7.6 μm, respectively, all significantly higher than the model group. On day 21, the epidermal thickness in the model group increased significantly, while the thicknesses in the other groups showed a decreasing trend. The blank group, RCIMA group, and RCIMA-Cell group had thicknesses of 45.3 ± 7.6 μm, 44.7 ± 6.2 μm, and 25.4 ± 5.3 μm, respectively. Figure 7 D). The above results indicate that the RCIMA-Cell group wounds enter the remodeling stage earlier, which is beneficial for accelerating the wound healing process.

[0113] Masson staining was used to assess collagen deposition and remodeling at the wound site. Results showed that, compared to the model group, collagen deposition was significantly increased in the blank group, RCIMA group, and RCIMA-Cell group at both 14 and 21 days. Furthermore, at day 21, the collagen fibers in the RCIMA-Cell group exhibited a coarser bundle structure and a more pronounced wavy characteristic, indicating that it effectively promoted collagen deposition and remodeling at the wound site. Figure 7 B, E).

[0114] Sirius red staining was further used to analyze collagen deposition types. The ratio of type I to type III collagen is an important indicator for evaluating wound repair quality and scarless healing potential. Yellow-orange mainly represents more mature and coarser type I collagen fibers, while green mainly represents finer type III collagen fibers. Compared with the control group, the ratio of type I to type III collagen in both the model group and the RCIMA group was significantly increased; the ratio in the RCIMA-Cell group was similar to that in the control group, indicating that it helps to reduce scar formation during wound repair. Figure 7 F, G).

[0115] Reactive oxygen species (ROS) levels are a key indicator for evaluating the microenvironment of diabetic wounds. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence staining was used to evaluate ROS levels in the wound area. The results showed that on day 14, the model group still had a large amount of ROS fluorescence signal in the wound area, with a positive area ratio of 31.2 ± 6.2%; the blank group, RCIMA group, and RCIMA-Cell group had ratios of 5.4 ± 0.6%, 7.2 ± 1.9%, and 5.8 ± 1.0%, respectively. Figure 8A, B). The above results indicate that RCIMA-Cell treatment helps reduce ROS levels in diabetic wounds.

[0116] To evaluate the immunomodulatory effects of RCIMA-Cell in vivo, immunofluorescence staining analysis was performed on the expression of CD86 (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages) in wound tissue on day 7. Compared with the control group, the positive signal of CD86 was significantly enhanced and the positive signal of CD206 was significantly reduced in the model group, indicating that macrophages in the model group were more inclined to polarize towards M1. After treatment with RCIMA and RCIMA-Cell, CD86 expression was significantly decreased, CD206 expression was significantly upregulated, and the M2 / M1 ratio increased, indicating that it can regulate the local macrophage polarization state. Figure 8 The above results indicate that RCIMA-Cell can regulate the polarization state of macrophages in the wound and promote the transformation of macrophages from M1 type to M2 type, which is beneficial to the transition of diabetic wounds from an inflammatory state to a repair state.

[0117] Angiogenesis is a crucial foundation for dermal regeneration in diabetic wounds. This study evaluated the angiogenesis-promoting ability of RCIMA-Cell by immunofluorescence staining of CD31 and α-SMA in wound tissue. CD31, a marker of vascular endothelial cells, is primarily used to identify new blood vessels; α-SMA mainly marks vascular smooth muscle cells and is often used to assess vascular maturation. On day 21, the model group showed the lowest proportions of positive areas for both CD31 and α-SMA, indicating significant inhibition of angiogenesis under diabetic conditions. In contrast, the RCIMA-Cell treatment group showed positive areas of 8.8 ± 0.9% and 1.4 ± 0.2% for CD31 and α-SMA, respectively, significantly higher than the control group, model group, and RCIMA group. These results indicate that RCIMA-Cell can significantly promote both neovascularization and mature angiogenesis in wounds. Figure 8 FI).

[0118] In summary, this invention, by modifying recombinant type I collagen with methacrylylation, introduces photocurable functional groups while preserving the biological properties of collagen, enabling the resulting bio-ink to meet DLP (Digital Perceptual Technology) requirements. 3D printing demands rapid prototyping and structural stability. This invention, by combining RCIMA with lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator and tartrazine light absorber, effectively controls the photocuring process, improving the controllability of photocuring during printing, thus facilitating the acquisition of well-formed, clearly defined 3D printed structures. The recombinant type I collagen scaffold prepared by this invention exhibits excellent biological properties, significantly promoting the migration, proliferation, and differentiation of HaCaT and HFF-1 cells. This invention constructs a biomimetic bilayer cell-carrying scaffold by loading HaCaT and HFF-1 cells separately into bio-ink. This scaffold effectively mimics the layered structure of natural skin and demonstrates good effects in promoting epidermal regeneration, dermal remodeling, reducing oxidative stress levels, regulating the local immune microenvironment, and promoting angiogenesis. The cell-carrying scaffold provided by this invention offers a novel tissue engineering approach for the repair of chronic diabetic wounds, possessing high application value and promising clinical prospects.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant humanized type I collagen, characterized in that, The amino acid sequence of the recombinant humanized type I collagen is shown in SEQ ID NO.

1.

2. A recombinant vector or recombinant genetically engineered bacterium carrying a gene sequence encoding the amino acid sequence of the recombinant humanized type I collagen as described in claim 1.

3. A recombinant type I collagen bio-ink for DLP 3D printing, characterized in that, The bio-ink comprises the following components: 50-150 mg / mL methacrylamide recombinant humanized type I collagen, 2.0-5.0 mg / mL photoinitiator, and 0.1-0.5 mg / mL light absorber; wherein the methacrylamide recombinant humanized type I collagen is a photocrosslinkable collagen derivative obtained by chemically modifying the primary amine groups in the recombinant humanized type I collagen molecule as described in claim 1 with methacrylic anhydride.

4. The bio-ink as described in claim 3, characterized in that, The photoinitiator is one or more of phenyl-2,4,6-trimethylbenzoylphosphinate lithium and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone; the light absorber is one or more of tartrazine and brilliant blue.

5. The method for preparing the bio-ink according to any one of claims 3-4, characterized in that, Includes the following steps: (1) Methacrylamide recombinant humanized type I collagen was dissolved in PBS buffer to obtain a collagen solution; (2) Add a photoinitiator and a light absorber to the collagen solution described in step (1); (3) After mixing evenly, a recombinant humanized type I collagen bio-ink for DLP 3D printing is obtained; The methacrylamide recombinant type I collagen described in step (1) is prepared by the following method: recombinant humanized type I collagen is dissolved in PBS buffer; Methacrylic anhydride was added to a recombinant humanized type I collagen solution at 4 ℃ and reacted under alkaline conditions for 12-24 hours. After the reaction, the solution was purified by dialysis and then freeze-dried to obtain methacrylamide recombinant humanized type I collagen.

6. The recombinant humanized type I collagen 3D scaffold prepared by DLP 3D printing technology using the bio-ink as described in claim 3.

7. The bracket as described in claim 6, characterized in that, The scaffold is a double-layered cell-carrying scaffold, with both the upper and lower layers printed using RCIMA bio-ink. The upper and lower layers of the scaffold are loaded with different live cells, which are selected from one or more of keratinocytes, fibroblasts, vascular endothelial cells, and mesenchymal stem cells.

8. The use of the bio-ink as described in claim 4 or the 3D scaffold as described in claim 7 in the manufacture of medical devices.

9. The use of the bio-ink as described in claim 4 or the 3D scaffold as described in claim 7 in the preparation of skin repair dressings or implants.

10. The use of the bio-ink as described in claim 4 or the 3D scaffold as described in claim 7 in the preparation of diabetic wound repair dressings.

Images