Phycomycete symbiotic wound dressing for chronic wound healing

Abstract

According to the preparation method of the phycomycete symbiotic wound dressing for chronic wound healing, microalgae and probiotics are printed in a combined mode through the 3D printing technology, light is fully utilized by the microalgae by changing the spatial structure of dressing hydrogel, the oxygen production efficiency of the microalgae is greatly improved, the long-time survival rate of the microalgae and the probiotics is guaranteed, and the application range of the phycomycete symbiotic wound dressing is widened. Through oxygen supply and antibacterial dual-power treatment, rapid tissue repair of chronic wounds can be realized.

Description

Technical Field

[0002] The invention belongs to the field of medical dressings, and in particular relates to an algae-bacteria symbiotic wound dressing suitable for healing chronic wounds. Background Art

[0003] Traditionally, wound healing is usually divided into four stages: hemostasis, inflammation, proliferation, and remodeling. Oxygen is a key factor in wound healing and tissue regeneration, including inflammation, proliferation, collagen synthesis, and angiogenesis. In particular, adequate oxygen supply or higher than normal oxygen partial pressure has been shown to promote wound healing and tissue regeneration processes. Oxygen-rich conditions can increase intracellular oxygen levels and increase intracellular reactive oxygen and reactive nitrogen levels, thereby promoting the wound healing process. Adequate oxygen partial pressure plays an important role in all stages of wound healing. Hypoxia can lead to apoptosis, tissue necrosis, delayed healing, and bacterial infection. Poor oxygenation in many chronic wounds such as diabetic foot ulcers can cause the wound to be in the inflammatory stage for a long time, which seriously affects the wound healing process.

[0004] Dressings play an important role in wound protection and treatment. Traditional dressings such as gauze and bandages are easy to use and inexpensive, but have a single function and dressing changes may damage the wound. They are not effective for chronic wounds with complex tissue conditions. New wound dressings such as tissue engineered skin, gel dressings, and foam dressings can protect the wound, prevent bacterial infection, and have multiple functions such as good moisturizing and promoting wound healing. However, these dressings can protect the wound, but they cause the wound, especially chronic wounds, to be in a state of hypoxia, and the healing effect is often poor.

[0005] CN110464703A discloses an oxygen-producing hydrogel, which includes photosynthetic microalgae and biodegradable hydrogel. The oxygen-producing hydrogel is formed by encapsulating the photosynthetic microalgae with a hydrogel having good biocompatibility, and the photosynthetic microalgae in the hydrogel photolyze water to produce oxygen, thereby increasing the local oxygen production. The oxygen-producing hydrogel can also be used to improve the hypoxic environment of local hypoxic diseases, such as wound infection, wound healing, arthritis, etc. However, in the absence of mechanical forces such as stirring and shaking, the microalgae in traditional liquid culture will naturally settle, resulting in a self-shading effect, which causes the microalgae in the lower layer to die due to the inability to be exposed to light, resulting in a significant decrease in oxygen production efficiency.

[0006] Therefore, there is an urgent need for a dressing that can ensure the good survival of microalgae and can supply oxygen independently, so as to continuously provide an oxygen-rich environment for the wound, reduce the slow healing of cells due to hypoxia after the capillaries are damaged, and alleviate the pain of patients. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned defects in the prior art and provide an oxygen supply dressing with a good spatial structure, so as to provide a continuous oxygen-rich environment for the wound through the dressing.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] (1) Add microalgae cells (CZ) and probiotics (BS) to the gel solution, mix well, then add colloidal powder, mix well, and let stand for ten minutes to ensure uniformity to form bio-ink.

[0010] (2) transferring the bio-ink into a syringe and loading it into a 3D printer for 3D printing to form a microalgae hydrogel dressing;

[0011] (3) The printed gel dressing was placed in a 100 mM calcium chloride solution for cross-linking.

[0012] Preferably, the gel is one or more of sodium alginate and methacrylated gelatin;

[0013] Preferably, the concentration of the gel in step (1) is 10-50 g / L;

[0014] Preferably, the concentration of the gel in step (1) is 30 g / L;

[0015] Preferably, the colloid in step (1) is one or more of carrageenan and methyl cellulose;

[0016] Preferably, the mass of the colloid in step (1) is 1-10 times the mass of sodium alginate;

[0017] Preferably, the mass of the colloid in step (1) is 3 times the mass of sodium alginate;

[0018] Preferably, the microalgae in step (1) is one or more of Chlorella, Chlorella pyrenoidosa, Chlamydomonas reinhardtii, Euglena, and diatom;

[0019] Preferably, the concentration of the microalgae cells in step (1) is 1.0×10 7 Pieces / mL;

[0020] Preferably, the probiotics described in step (1) are one or more of Bacillus subtilis, Staphylococcus aureus, Bacillus licheniformis, Bacillus brevis, Bacillus coagulans, Bacillus cereus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus plantarum;

[0021] Preferably, the concentration of the probiotics in step (1) is 1.0×10 7 Pieces / mL.

[0022] Preferably, the parameters of the 3D printing in step (2) are set as follows: layer height: 0.27 mm; number of layers: 12 layers; random printing filling, printing outline, and starting point; model: quadrilateral; speed: 8 mm / s; broken wire lifting: 1 mm; air pressure: 0.3 MPa; early wire extraction: 380 ms; early wire closing: 0.2 mm; filling mode: linear filling; filling method: linear filling; filling offset: 0.7 mm; filling spacing: 1.4 mm; rotation angle: 90°; number of rotations: 2 times.

[0023] The present invention has the following beneficial effects:

[0024] (1) Its formula is simple and its design is scientific. It uses microalgae to produce dissolved oxygen through photosynthesis and the antibacterial effect of probiotics. The dissolved oxygen produced by the microalgae promotes wound repair on the one hand, and provides oxygen for the survival of probiotics on the other hand, achieving long-term oxygen supply and antibacterial dual-power treatment, and has a good effect on the healing ability of chronic wounds; (2) It uses 3D printing technology to change the spatial structure of the dressing hydrogel, so as to fully utilize the light of the microalgae, greatly improve the oxygen production efficiency of the microalgae, ensure the long-term survival rate of the microalgae and probiotics, and at the same time meet the customized requirements of the wound such as shape and thickness.

[0025] (3) Through oxygen supply and antibacterial dual-power therapy, tissue repair can be quickly initiated within 3 days, accelerating the rapid control and healing of the initial injury, and restoring 90% of the full cortical structure within 12 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Growth of microalgae (Chlorella vulgaris, CZ) alone in the BBH-CZ dressing of Example 1. (a) Digital image of the hydrogel containing CZ during the culture process; (b) Changes in the amount of CZ during the culture process.

[0027] Figure 2 Growth of probiotics (Bacillus subtilis, BS) in the BBH-BS dressing of Example 2. (a) Changes in the number of BS during the culture process; (b) Fluorescence image of the hydrogel containing BS during the culture process, green represents live BS, and red represents dead BS.

[0028] Figure 3 : Appearance of the BBH-BC dressing product of Example 3;

[0029] Figure 4 Growth of CZ and BS in the BBH-BC group dressing of Example 3. (a) Changes in the number of CZ and BS during the culture process; (b) Fluorescence image of the hydrogel containing CZ and BS during the culture process, green represents BS, and red represents CZ.

[0030] Figure 5. Antibacterial performance of the BBH-BC dressing of Example 3. (a) Sample image of colony forming unit (CFU) experiment; (b) Image of CFU formed by untreated Staphylococcus aureus; (c) Image of CFU formed by BBH-BC; (d) CFU statistics of untreated and BBH-BC.

[0031] Figure 6 .Dissolved oxygen (DO) production of the BBH-BC dressing of Example 3. (a) Schematic diagram of the DO test method; (b) Dissolved oxygen production of BBH-BC.

[0032] Figure 7 . Wound healing of diabetic rats treated with the wound dressing of Example 4. (a) Digital image of the wound healing process; (b) Ratio of healed and unhealed wounds.

[0033] Figure 8 . Data statistics of wound healing in diabetic rats treated with the wound dressing of Example 4. (a) Change in wound area during wound recovery; (b) Proportion of healed wounds.

[0034] Fig. 9 .Images of the heart, liver, spleen, lungs, and kidneys of diabetic rats treated with the wound dressing of Example 4. DETAILED DESCRIPTION

[0035] Embodiment 1:

[0036] Add 300 mg of methacryloyl gelatin to 15 mL of sterile water and dissolve it. 7 Chlorella cells were added to the gel, and 1000 mg of methyl cellulose was added after being fully stirred and mixed. After mixing, the hydrogel was allowed to stand for 10 minutes to be fully uniform. The hydrogel was then placed in a 10 mL syringe and the grid hydrogel dressing was printed using 3D printing. The hydrogel was then placed in a 100 mM calcium chloride solution for cross-linking for 10 minutes to impart mechanical properties, which was marked as the BBH-CZ group dressing.

[0037] Digital photos and algae counts were taken on the 0th, 1st, 2nd, 3rd, 4th, and 5th days of the BBH-CZ group dressing culture process. Figure 2 As shown, Chlorella microalgae cells (CZ) grew fourfold in 5 days.

[0038] Embodiment 2:

[0039] Add 800 mg of methacryloyl gelatin to 30 mL of sterile water and dissolve it. 7Bacillus subtilis (BS) cells were added to the gel, and 2000 mg of carrageenan was added after being fully stirred and mixed. After being mixed and mixed, it was allowed to stand for 10 minutes to be fully uniform. The hydrogel was then placed in a 10 mL syringe and a grid hydrogel dressing was printed using 3D printing. The hydrogel was then placed in a 100 mM calcium chloride solution for cross-linking for 1 minute to impart mechanical properties. This is marked as the BBH-BS group dressing.

[0040] Digital photos and fluorescence images were taken on the 0th, 1st, 2nd and 3rd days of the BBH-BS dressing culture process to count the number of probiotics. Figure 3 As shown, the number of Bacillus subtilis (BS) increased to 2.5 times within 1 day, but then Bacillus subtilis (BS) died and the number dropped back to about 2 times, indicating that the dressing in the BBH-BS group was in a state of hypoxia, causing the death of Bacillus subtilis (BS).

[0041] Embodiment 3:

[0042] Add 450 mg of sodium alginate to 15 mL of sterile water and dissolve it. 7 Chlorella cells and 1.5×10 7 Staphylococcus aureus was added to the gel, and 1350 mg of methylcellulose was added after being fully stirred and mixed. After being mixed, it was allowed to stand for 10 minutes to be fully uniform. The hydrogel was then placed in a 10 mL syringe and a grid hydrogel dressing was printed using 3D printing. The hydrogel was then placed in a 100 mM calcium chloride solution for cross-linking for 5 minutes to impart mechanical properties. It is marked as the BBH-BC group dressing.

[0043] Digital photos and fluorescence images were taken on the 0th, 1st, 2nd and 3rd days of the BBH-BC dressing culture process to count the number of Chlorella and Staphylococcus aureus. Figure 4 As shown, the number of Chlorella and Staphylococcus aureus increased rapidly and proportionally within 3 days, indicating that the unique 3D printing structure enables Chlorella to fully utilize light. At the same time, Chlorella provides sufficient oxygen for Bacillus licheniformis, ensuring the long-term survival rate of microalgae and probiotics. Figure 5 The results showed that the number of colony forming units (CFU) of Staphylococcus aureus in the BBH-BC group dressings was reduced to one third compared with that in the control group, indicating that the BBH-BC group dressings had excellent antibacterial properties. Figure 6 The oxygen content of the BBH-BC group dressing increased rapidly after 24 hours, reaching 10 mg / L after 36 hours and 20 mg / L after 72 hours. The oxygen content in the control group was basically maintained at 7 mg / L after 72 hours. This shows that the algae-bacteria symbiotic wound dressing can improve oxygen efficiency.

[0044] Embodiment 4:

[0045] In order to prove the therapeutic effect of algae-fungus symbiotic wound dressing, the rats were divided into four groups, and the dermis, epidermis and superficial parts of the central back were removed to remove the full-thickness wound. Blank: diabetic rats, without any additional treatment; BBH-BS: diabetic rats, treated with 3D printed sodium alginate + carrageenan + probiotic hydrogel dressing; BBH-CZ: diabetic rats, treated with 3D printed sodium alginate + methacryloyl gelatin + microalgae hydrogel dressing; BBH-BC: diabetic rats, treated with 3D printed sodium alginate + carrageenan + microalgae + probiotic hydrogel dressing. The wound healing condition was photographed and the proportion of healed wounds was calculated on day 0, day 1, day 3, day 5, day 7, day 10, and day 12. Figure 7 and Figure 8 As shown, compared with the diabetic rats without any treatment, the wound appearance of the rats in the BBH-BS group, BBH-CZ group, and BBH-BC group improved significantly. The wound healing rate of the rats in the BBH-BC group reached 50% on the 5th day, while the wound healing rate of the rats in the Blank group was less than 20% during the same period, indicating that the use of 3D printed sodium alginate + carrageenan + microalgae + probiotics hydrogel dressings has obvious advantages in wound healing in diabetic rats. Fig. 9 The images of the heart, liver, spleen, lung, and kidney of diabetic rats treated with hydrogel dressings in the BBH-BS group, BBH-CZ group, and BBH-BC group show that the hydrogel dressings have no toxic side effects and do not damage the internal organs.

Claims

1. A method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing, characterized in that The following steps are involved: (1) Add microalgae cells and probiotics to the gel solution, mix well, then add colloidal powder, mix well, and let stand for ten minutes to ensure uniformity, thereby forming bio-ink. (2) transferring the bio-ink into a syringe and loading it into a 3D printer for 3D printing to form a microalgae hydrogel dressing; (3) The printed gel dressing was placed in a 100 mM calcium chloride solution for cross-linking.

2. The method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to claim 1, characterized in that: The gel is one or more of sodium alginate and methacrylated gelatin.

3. The method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to claim 2, characterized in that: The concentration of the gel in step (1) is 10-50 g / L.

4. The method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to claim 3, characterized in that: The mass of the colloid is 1-10 times the mass of sodium alginate.

5. A method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to any of claims 1 to 4, characterized in that: The colloid is one or more of carrageenan and methyl cellulose.

6. A method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to any one of claims 1 to 5, characterized in that: The microalgae are one or more of Chlorella, Chlorella pyrenoidosa, Chlamydomonas reinhardtii, Euglena and diatom.

7. A method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to any one of claims 1 to 6, characterized in that: The concentration of the microalgae cells is 1.0-5.0×10 7 Pieces / mL.

8. A method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to any one of claims 1 to 7, characterized in that: The probiotics are one or more of Bacillus subtilis, Staphylococcus aureus, Bacillus licheniformis, Bacillus brevis, Bacillus coagulans, Bacillus cereus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum.

9. The method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to claim 8, characterized in that: The concentration of the probiotics is 1.0-5.0×10 7 Pieces / mL.

10. The method for preparing an algae-bacteria symbiotic wound dressing for chronic wound healing according to claim 1, characterized in that: The 3D printing parameters of step (2) are set as follows: layer height: 0.27 mm; number of layers: 12 layers; print fill, print outline, and start point are random; model: quadrilateral; Speed: 8mm / s; Broken wire lifting: 1mm; Air pressure: 0.3MPa; Early wire extraction: 380ms; Early wire closing: 0.2mm; Fill Mode: Linear filling; Filling method: linear filling; Filling offset: 0.7mm; Filling spacing: 1.4mm; Rotation angle: 90°; Number of rotations: 2 times.

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