Recombinant human type i / iii collagen microgel containing micro-nano bubbles and preparation method and application thereof

Abstract

The application discloses a kind of recombinant human Ⅰ / Ⅲ type collagen microgel containing micro-nano bubble and its preparation method and application, belong to the field of biomedical materials.In recombinant human Ⅰ / Ⅲ type collagen, methyl methacrylate is added to obtain methyl methacrylate anhydride recombinant human Ⅰ type collagen, then dissolved in EDC solution to obtain a sponge-like solid sample, then dissolved in PBS solution containing photoinitiator and dispersant to obtain RHC I-MA-EDC solution and RHC III-MA-EDC solution, oxygen is introduced into the mixed solution in the form of nano-bubbles after mixing, and then the micro-nano bubble-containing recombinant collagen microgel structure is constructed by emulsification method.The micro-nano bubble-containing recombinant human Ⅰ / Ⅲ type collagen microgel prepared by the application has the ability to carry sufficient oxygen, which is beneficial to cell survival, proliferation and metabolism, and can also promote tissue repair and regeneration.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical materials, and particularly relates to preparation and application of recombinant human type I / III collagen microgel containing micro-nano bubbles. BACKGROUND

[0002] As a new type of biomaterial, recombinant human type I / III collagen has attracted extensive attention in the field of biomedicine in recent years. Type I collagen mainly exists in adult skin, tendon, bone tissue and other parts, has good support, can provide a solid structural basis for tissue, and maintain the morphology and stability of tissue. Type III collagen is rich in infant skin, which can keep the skin tender, delicate and elastic. In wound repair, type III collagen plays an important role in promoting wound recovery and reducing scar formation. In the process of wound healing, it can participate in the basic activities of cell adhesion, proliferation, migration and differentiation, and provide strong support for rapid wound healing. The recombinant human type I / III collagen microgel microspheres not only can fully exert the advantages of the two types of collagen, but also can realize effective encapsulation and protection of cells by using the unique structure of microspheres, promote cell proliferation and differentiation, and provide an ideal cell carrier for tissue repair. The current recombinant human type I / III collagen microspheres still have certain limitations. In some tissue repair processes, especially in the hypoxic environment, the metabolism and function of cells will be seriously affected, resulting in poor repair effect.

[0003] Micro-nano bubbles have the advantages of large specific surface area, high mass transfer efficiency and good stability. By using the micro-nano bubble generator on the gel, the gel microspheres can carry sufficient oxygen supply. In the use process, through the slow release of oxygen, the metabolic environment of cells can be improved, and the problem of slow cell growth caused by hypoxia in deep parts of tissue engineering repair can be further promoted, and a more suitable living environment for cells can be provided.

[0004] The common methods for feeding microgels at present include using chemical oxygen release agents, biological oxygen production systems and enzyme catalytic systems. Using chemical oxygen release agents to supply oxygen is to wrap solid peroxides such as calcium peroxide (CaO2) in the form of nanoparticles in microgels, which react with water to release oxygen. The disadvantage is that the chemical oxygen release agent may produce alkaline by-products after reaction, which has potential toxicity to cells. The biological oxygen production system mainly encapsulates microalgae (such as Chlorella) in microgels to produce oxygen by photosynthesis. Biocompatible hydrogels such as sodium alginate and silk fibroin are commonly used carriers. The disadvantage is that the oxygen production of microalgae is heavily dependent on external light, which is difficult to achieve in vivo application, limiting its clinical application scenarios. The enzyme catalytic system supplies oxygen by immobilizing catalase (CAT) in microgels. It can catalyze the decomposition of by-product H2O2 into water and oxygen, which can not only remove harmful substances but also supplement oxygen. The defect is that the enzyme is a protein, and its activity is easily affected by environmental factors. SUMMARY

[0005] The purpose of the present application is to provide a kind of micro-nano bubble-containing recombinant human Ⅰ / Ⅲ collagen microgel and its preparation method and application.

[0006] The present application prepares micro-nano bubble-containing recombinant human Ⅰ / Ⅲ collagen microgel systems with different particle sizes, different oxygen contents and different mechanical properties by adjusting the content and concentration of recombinant human Ⅰ / Ⅲ collagen, the ventilation flow rate, ventilation time in the preparation process of micro-nano bubbles and the stirring rate in the preparation of microgels, which are used for tissue engineering.

[0007] The preparation method of the gel microspheres is to modify the recombinant human Ⅰ / Ⅲ collagen with methacrylic acid. The modified gel has better biocompatibility and biodegradability. The micro-nano bubble generator makes the prepared gel microspheres have better oxygen carrying capacity, which can be used for tissue repair in deep hypoxic sites.

[0008] A kind of micro-nano bubble-containing recombinant human Ⅰ / Ⅲ collagen microgel preparation includes the following steps:

[0009] (1) First, mix methacrylic acid (MAA) with recombinant human Ⅰ / Ⅲ collagen (RHCⅠ / RHC Ⅲ) solution, centrifuge, microfiltration, dialysis and freeze-drying to prepare porous sponge-like RHCⅠ-MA and RHCⅢ-MA solids, respectively; then, dissolve them in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, further activate the carboxyl group and strengthen the intermolecular crosslinking by using EDC, and perform secondary crosslinking and purification treatment on the two solids to obtain RHCⅠ-MA-EDC and RHCⅢ-MA-EDC solids with high crosslinking degree;

[0010] (2) Dissolve the two solids in PBS solution containing a photoinitiator and a dispersant respectively, form a homogeneous solution, and then mix them in a volume ratio of 1:10 to 10:1;

[0011] (3) Place the aeration head of the micro-nano bubble generator in the mixed solution obtained in step (2), and control the type, pressure and aeration time of the gas of the micro-nano bubble generator to accurately control the size and content of the micro-nano bubbles in the recombinant human collagen gel;

[0012] (4) Under a certain rotating speed, slowly drop the recombinant collagen solution containing micro-nano bubbles prepared in step (3) into the mixed solution of oil phase and surfactant through a syringe, stir for 5-10 min, irradiate under 405 nm ultraviolet light for 3-10 min, and then obtain the solidified recombinant collagen microgel; remove the supernatant by centrifugation, add PBS and shake to remove the remaining oil phase, finally wash with PBS buffer solution, and then collect the recombinant human type I / III collagen microgel through filtration.

[0013] Further, the biomaterial used in step (1) is selected from recombinant human type I / III collagen, gelatin and chitosan, and preferably recombinant human type I / III collagen.

[0014] Further, the recombinant human type I collagen solution in step (1) is prepared by dissolving recombinant human type I collagen powder in PBS buffer solution, and the prepared solution has a concentration of 10%-20% (w / v).

[0015] Further, the concentration of methacrylic acid (MAA) in step (1) is 3%-12% (V / V), preferably 8% (V / V).

[0016] Further, the concentration of EDC in step (1) is 0.1 M-1 M, preferably 0.1 M.

[0017] Further, the concentration of the photoinitiator in step (2) is 0.1%-2% (w / v), preferably 0.5% (w / v).

[0018] Further, the photoinitiator in step (2) can be selected from lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or irgacure 2959, preferably LAP.

[0019] Further, the concentration of the dispersant in step (2) is 0.2%-1% (w / v), preferably 0.5% (w / v).

[0020] Further, the dispersing agent in step (2) can be selected from Tween 80 or Pluronic F-127, preferably Pluronic F-127.

[0021] Further, the volume ratio of the mixed RHC I-MA-EDC solution and RHC III-MA-EDC solution dissolved in the photoinitiator and the dispersing agent in step (2) is 1:10-10:1, further preferably 9:1, 7:3, 5:5, preferably 7:3.

[0022] Further, the oxygen-containing gas in step (3) is selected from a gas containing 20%-100% oxygen; preferably, the gas is a gas containing 40% oxygen and 60% nitrogen.

[0023] The pressure is adjusted to 0.05-1 MPa and the aeration time is 10-60 seconds; the pressure is preferably 0.05-0.15 MPa; the aeration time is preferably 20-40 seconds, more preferably the pressure is 0.1 MPa; the aeration time is 30 seconds.

[0024] Further, the oil phase in step (4) can be selected from at least one of liquid paraffin, rapeseed oil, mineral oil or soybean oil, preferably rapeseed oil, which is a natural vegetable oil; the surfactant can be selected from lecithin, Tween or Span 80, preferably Span 80, which is a high-efficiency synthetic emulsifier.

[0025] Further, the volume ratio of the oil phase to the surfactant in step (4) is 10:1-10:5, preferably 10:2.

[0026] Further, the rotation speed in step (4) is selected to be 400-1000 rpm, at a rotation speed of 400 rpm, larger emulsion droplets can be formed, which is beneficial for preparing larger microspheres; at 1000 rpm, the emulsion droplet size becomes smaller, and the prepared microspheres also correspondingly decrease in size; the rotation speed is preferably 800 rpm.

[0027] In the preparation method, first, a mixed solution of RHC I-MA-EDC and RHC III-MA-EDC is prepared, then a micro-nano bubble generator is started to inject micro-nano bubbles into the recombinant human collagen solution, and finally, a microsphere of the recombinant human type I / III collagen microgel containing micro-nano bubbles is prepared by emulsification and ultraviolet curing. In the prepared microsphere, the diameter of the micro-nano bubbles is 1-15 μm, is concentrated at about 6 μm, the oxygen content is 6-12 mg / mL, and the stress value under the same strain gradually increases as the concentration of the recombinant human type I / III collagen microgel containing micro-nano bubbles increases from 10% to 30%. The higher the concentration, the greater the Young's modulus (10%<20%<30%). As the concentration increases, the maximum stress value of the material increases (10%<20%<30%).

[0028] The recombinant human type I / III collagen microgel containing micro-nano bubbles prepared in the application can realize accurate control of the particle size of the microgel by adjusting the size of the rotating speed, and can prepare microspheres with different particle sizes to meet the needs of different application scenarios. By accurately regulating the aeration time, gas pressure and gas type of the micro-nano bubble generator, the oxygen content of the micro-nano bubbles in the microgel can be accurately controlled. The microspheres prepared in the application encapsulate nano-sized bubbles invisible under a microscope and micron-sized bubbles visible under a microscope, the micron-sized bubbles have a diameter concentrated at about 6 μm, and the overall oxygen content of the microspheres can be stably controlled in the range of 6-12 mg / L, thereby constructing a microgel system with different numbers of micro-nano bubbles and different oxygen enrichment levels. By adjusting the concentration of the mixed solution of RHC I-MA and RHC III-MA, the mechanical strength of the microgel can be regulated, so that microgel carriers with a series of different mechanical properties (such as elastic modulus and compression strength) are obtained.

[0029] In the application, a dispersing agent is added to the mixed solution of RHC I-MA-EDC and RHC III-MA-EDC. The addition of the dispersing agent can reduce the size of the bubbles, the dispersing agent can reduce the surface tension of the solution, so that smaller bubbles are more easily generated under the same aeration pressure and aeration time. The addition of the dispersing agent can also increase the stability of the bubbles, so that the bubbles are more uniformly distributed. The molecules of the dispersing agent are adsorbed on the surface of the bubbles, forming a steric hindrance or electrostatic repulsion barrier, which effectively prevents the collision and merging of the bubbles.

[0030] Application of the recombinant human type I / III collagen microgel containing micro-nano bubbles in tissue repair and regeneration.

[0031] The recombinant human type I / III collagen used in the application has many advantages compared with traditional animal-derived collagen. Because its amino acid sequence is highly consistent with human body's own collagen, it greatly reduces the immunogenicity and the probability of rejection reaction in the human body, providing higher protection for the user. The recombinant human type I / III collagen has good water solubility, can be more conveniently mixed with other ingredients, and prepared into products of various dosage forms. Its uniform dispersion in solution helps to improve the stability and effectiveness of the product. During preparation, by accurately controlling the gene expression and synthesis conditions, the structure and performance of the collagen can be accurately regulated.

[0032] The recombinant human type I / III collagen microgel containing micro-nano bubbles has great application potential in tissue repair and regeneration. In the future biomedical field, this microgel is expected to be applied in skin wound repair, burn treatment, chronic wound healing and other aspects. In skin wound repair, the microgel containing micro-nano bubbles can accelerate wound healing, reduce scar formation and improve the quality of life of patients; in burn treatment, it can provide a good repair environment for burn wounds, promote tissue regeneration at the burn site and reduce the risk of infection; in chronic wound healing, such as diabetic foot ulcers, the oxygen-rich properties and biocompatibility of the microgel can improve the local hypoxic microenvironment of the wound, promote cell proliferation and repair, and solve the problem of difficult healing of chronic wounds. With the continuous deepening of research and the continuous progress of technology, the recombinant human type I / III collagen microgel containing micro-nano bubbles is expected to bring new breakthroughs and developments to the biomedical field and provide more effective treatment methods for patients.

[0033] Beneficial effects:

[0034] The present application first realizes the preparation of microgel containing micro-nano bubbles by using a micro-nano bubble generator. By adjusting the content and concentration of recombinant human type I / III collagen, the air flow and air time during the preparation of micro-nano bubbles, and the stirring rate during the preparation of microgel, micro-nano bubble-loaded recombinant human type I / III collagen microgel with different particle sizes, different micro-nano bubble contents, and different mechanical properties can be prepared. The prepared microgel containing micro-nano bubbles can provide sufficient oxygen supply for cells and effectively improve the hypoxic microenvironment of tissues.

[0035] In skin wound repair, microgel containing micro-nano bubbles can accelerate wound healing and reduce scar formation; in burn treatment, it can provide a good repair environment for burn wounds, promote tissue regeneration at the burn site, and reduce the risk of infection; in chronic wound healing, the oxygen-rich properties and biocompatibility of microgel can improve the local hypoxic microenvironment of the wound, promote cell proliferation and repair, and solve the problem of difficult healing of chronic wounds; recombinant human Ⅰ / Ⅲ collagen microgel containing micro-nano bubbles is expected to bring new breakthroughs and developments to the biomedical field and provide more effective treatment for patients. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The flow chart of the preparation method of the recombinant human Ⅰ / Ⅲ collagen microgel containing micro-nano bubbles in the application.

[0037] Figure 2 The impact of dispersant pluronic F-127 on the recombinant human Ⅰ / Ⅲ collagen microgel containing micro-nano bubbles is shown in the comparison chart; a) adding dispersant pluronic F-127, b) without adding dispersant pluronic F-127.

[0038] Figure 3 The bubble content comparison chart of RHCMA (recombinant human Ⅰ / Ⅲ collagen) and GELMA before and after oxygenation.

[0039] Figure 4 The bubble size imaging chart of RHCMA (recombinant human Ⅰ / Ⅲ collagen) and GELMA under an optical microscope.

[0040] Figure 5 The bubble size distribution chart of RHCMA (recombinant human Ⅰ / Ⅲ collagen) and GELMA at one minute.

[0041] Figure 6 The oxygen concentration distribution chart of RHCMA (recombinant human Ⅰ / Ⅲ collagen) after standing for 1, 5 and 10 minutes after aeration.

[0042] Figure 7 The oxygen concentration chart of the dissolved oxygen detector (HQ40d) detecting the effect of different pressures and different aeration times on the oxygen-containing performance of recombinant human Ⅰ / Ⅲ collagen gel microspheres; (a) the oxygen concentration chart of the effect of different aeration times on the oxygen-containing performance of recombinant human Ⅰ / Ⅲ collagen gel microspheres, (b) the oxygen concentration chart of the effect of different pressures on the oxygen-containing performance of recombinant human Ⅰ / Ⅲ collagen gel microspheres.

[0043] Figure 8 The imaging chart of recombinant human Ⅰ / Ⅲ collagen gel microspheres under an optical microscope.

[0044] Figure 9The size distribution of the recombinant human collagen type I / III gel microspheres at different rotation speeds.

[0045] Figure 10 The oxygen content of the recombinant human collagen type I / III microgel in PBS at different rotation speeds.

[0046] Figure 11 The mechanical property analysis diagram of the recombinant human collagen type I / III gel microspheres, wherein: (a) the stress-strain diagram of the recombinant human collagen type I / III gel microspheres (b) the Young's modulus diagram of the recombinant human collagen type I / III gel microspheres (c) the maximum stress value of the recombinant human collagen type I / III gel microspheres.

[0047] Figure 12 The ATDC5 cell activity fluorescence distribution diagram of the recombinant human collagen type I / III gel microspheres at different aeration times and the analysis diagram of the effect of the recombinant human collagen type I / III gel microspheres at different aeration times on the activity of ATDC5 cells; (a) the ATDC5 cell activity fluorescence distribution diagram of the recombinant human collagen type I / III gel microspheres at different aeration times (b) the effect diagram of the recombinant human collagen type I / III gel microspheres at different aeration times on the survival of ATDC5 cells.

[0048] Figure 13 The HSFs cell activity fluorescence distribution diagram of the recombinant human collagen type I / III gel microspheres at different concentrations and the analysis diagram of the effect of the recombinant human collagen type I / III gel microspheres at different concentrations on the activity of HSFs cells; (a) the HSFs cell activity fluorescence distribution diagram of the recombinant human collagen type I / III gel microspheres at different concentrations (b) the effect diagram of the recombinant human collagen type I / III gel microspheres at different concentrations on the survival of HSFs cells. DETAILED DESCRIPTION

[0049] The preparation method of RHC I-MA and RHC III-MA is referred to CN119055827A.

[0050] Example 1

[0051] The purpose of this example is to prepare recombinant human collagen type I / III microgel containing micro-nano bubbles, and the preparation process is as shown in Figure 1 :

[0052] The 1 g of recombinant human type I collagen powder was accurately weighed and dissolved in 10 mL of PBS buffer to prepare a 10% (w / v) recombinant human type I collagen solution. Similarly, 1 g of recombinant human type III collagen powder was accurately weighed and dissolved in 10 mL of PBS buffer to prepare a 10% (w / v) recombinant human type III collagen solution. Then, 6% (V / V) of methacrylic acid was added to the recombinant human type I collagen solution and the recombinant human type III collagen solution, respectively, and reacted for 3 hours under magnetic stirring to prepare RHC I-MA and RHC III-MA solutions. After the reaction, the solutions were centrifuged at 6000 rpm for 4 minutes, and the supernatant was collected and diluted 2-fold with the corresponding solvent. Next, the diluted solution was filtered through a 0.22-micron disposable needle filter, and then dialyzed for 3 to 4 days using a dialysis bag. After dialysis, the solution was freeze-dried to obtain two types of sponge-like solid samples of RHC I-MA and RHC III-MA, respectively;

[0053] An appropriate amount of EDC was accurately weighed and dissolved in PBS buffer to prepare a 0.1 M EDC solution. The solution was filtered through a 0.22-micron disposable needle filter and then used. The freeze-dried RHC I-MA and RHC III-MA samples were dissolved in the above-mentioned EDC solution and reacted for 3 hours under magnetic stirring to obtain RHC I-MA-EDC and RHC III-MA-EDC solutions. After the reaction, the two solutions were again filtered through a 0.22-micron disposable needle filter, and then dialyzed for 3 to 4 days using a dialysis bag. After dialysis, the solution was freeze-dried to obtain two types of sponge-like solid samples of RHC I-MA-EDC and RHC III-MA-EDC, respectively;

[0054] 0.5 g of the RHC I-MA-EDC freeze-dried sample was accurately weighed and dissolved in 5 mL of a PBS solution containing 0.5% (w / v) of a photoinitiator LAP and 0.5% (w / v) of a dispersant (Pluronic F-127) to obtain a 10% (w / v) RHC I-MA-EDC solution. Similarly, 0.5 g of the RHC III-MA-EDC freeze-dried sample was accurately weighed and dissolved in a PBS solution of 0.5% (w / v) of a photoinitiator LAP and 0.5% (w / v) of a dispersant Pluronic F-127 to obtain 5 mL of a 10% (w / v) RHC III-MA-EDC solution. Next, the RHC I-MA-EDC solution and the RHC III-MA-EDC solution were mixed in a volume ratio of 9:1, 7:3, and 5:5 to ensure uniform mixing;

[0055] The aeration head of the micro-nano bubble generator was placed in the mixed solution of RHC I-MA-EDC and RHC III-MA-EDC, the oxygen-containing gas containing 40% oxygen and 60% nitrogen was selected, the pressure was adjusted to 0.1 MPa, and the aeration time was 30 seconds, so as to accurately control the size and oxygen-containing performance of the nano bubbles;

[0056] Then, the mixed and uniform photosensitive recombinant human type I / III collagen solution was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 in batches, and a syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started, and the mixed solution was continuously stirred at a speed of 800 rpm for 5 minutes to fully dissolve and mix. Then, the curing treatment was carried out under light (usually ultraviolet light), and the light was kept for 5 minutes to complete the crosslinking reaction.

[0057] Example 2

[0058] The purpose of this example is to study the effect of adding the dispersant pluronic F-127 on the bubbles in the recombinant human type I / III collagen microgel containing micro-nano bubbles when preparing the mixed solution of RHC I-MA-EDC and RHC III-MA-EDC:

[0059] The recombinant human type I / III collagen solution (with the addition of the dispersant pluronic F-127) with a volume ratio of 7:3 of RHC I-MA-EDC solution and RHC III-MA-EDC solution was prepared according to steps (1)-(3) in Example 1;

[0060] According to steps (1)-(2) in Example 1, two types of sponge-like solid samples of RHC I-MA-EDC and RHC III-MA-EDC were prepared, 0.5 g of the freeze-dried sample of RHC I-MA-EDC was accurately weighed and dissolved in 5 mL of a 0.5%(w / v) photoinitiator LAP solution to obtain a 10%(w / v) RHC I-MA-EDC solution. Similarly, 0.5 g of the freeze-dried sample of RHC III-MA-EDC was accurately weighed and dissolved in a 0.5%(w / v) photoinitiator LAP solution to obtain 5 mL of a 10%(w / v) RHC III-MA-EDC solution. Next, the RHC I-MA-EDC solution and the RHC III-MA-EDC solution were mixed in a volume ratio of 7:3 to ensure uniform mixing;

[0061] The aeration head of the micro-nano bubble generator was placed in the RHC I-MA-EDC solution mixed with the RHC III-MA-EDC mixed solution to which the dispersing agent Pluronic F-127 was added and the RHC I-MA-EDC solution mixed with the RHC III-MA-EDC mixed solution to which the dispersing agent Pluronic F-127 was not added, the gas was a gas containing 40% oxygen and 60% nitrogen, the pressure was adjusted to 0.1 MPa, and the aeration time was 30 seconds, so as to accurately control the size and oxygen-containing performance of the nano bubbles;

[0062] Subsequently, the mixed and uniformly mixed photosensitive micro-nano bubble-containing recombinant human type I / III collagen solution to which the dispersing agent Pluronic F-127 was added was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 in several times, and a syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started, and the mixed solution was continuously stirred at a speed of 800 rpm for 5 minutes to fully dissolve and mix. Subsequently, curing treatment was performed under light (usually ultraviolet light), and the light was kept on for 5 minutes to complete the crosslinking reaction.

[0063] The mixed and uniformly mixed photosensitive micro-nano bubble-containing recombinant human type I / III collagen solution to which the dispersing agent Pluronic F-127 was not added was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 in several times, and a syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started, and the mixed solution was continuously stirred at a speed of 800 rpm for 5 minutes to fully dissolve and mix. Subsequently, curing treatment was performed under light (usually ultraviolet light), and the light was kept on for 5 minutes to complete the crosslinking reaction.

[0064] The experimental results are shown in Figure 2 The micro-nano bubble-containing microgel to which the dispersing agent Pluronic F-127 was added has more uniform bubble size, more uniform bubble distribution, and more stable bubbles.

[0065] Example 3

[0066] The purpose of this example is to compare the characteristics of oxygen-containing bubbles of GELMA and recombinant human collagen

[0067] Firstly, 40 mg of the photoinitiator LAP and 100 mg of the dispersant Pluronic F-127 were weighed, and then dissolved in PBS buffer solution by stirring. The total volume was made up to 20 ml, and a PBS solution with a concentration of 0.2% LAP and 0.5% (w / v) Pluronic F-127 was prepared. Subsequently, 1.2 g of GELMA was dissolved in the above-mentioned LAP and Pluronic F-127 PBS solution and made up to 20 mL, and a 6% GELMA solution was obtained, which was stored in the dark. A recombinant human collagen solution (RHC I-MA-EDC and RHC III-MA-EDC mixed solution with a ratio of 7:3) with a concentration of 6% was prepared by the above-mentioned method; similarly, 40 mg of the photoinitiator LAP and 100 mg of the dispersant Pluronic F-127 were weighed, and then dissolved in PBS buffer solution by stirring. The total volume was made up to 20 ml, and a PBS solution with a concentration of 0.2% LAP and 0.5% (w / v) Pluronic F-127 was prepared. Subsequently, 1.2 g of the RHC I-MA-EDC and RHC III-MA-EDC mixed solution (RHC I-MA-EDC and RHC III-MA-EDC ratio of 7:3) was dissolved in the above-mentioned LAP and Pluronic F-127 mixed solution and made up to 20 mL, and a RHCMA solution with a concentration of 6% was obtained, which was stored in the dark.

[0068] The aeration head of the micro-nano bubble generator was placed in the 6% GELMA solution, and a gas containing 40% oxygen and 60% nitrogen was selected. The pressure was adjusted to 0.1 MPa, and the aeration time was 30 seconds. The 6% recombinant human collagen solution was oxygenated by the above-mentioned method;

[0069] The 6% GELMA and 6% recombinant human collagen solution after oxygenation were irradiated with ultraviolet light for 45 s to solidify them, and then they were placed under an optical microscope for photography. Three non-overlapping imaging areas of GELMA and recombinant human collagen solution were selected respectively. The solution characteristics were recorded, and the particle size, average particle size and distribution density were measured using ImageJ.

[0070] As shown in Figure 3 , the bubble density of GELMA and RHCMA was different under different aeration times, and after standing for 1 to 5 minutes, it was found that the bubbles in GELMA mostly floated on the surface in the form of foam, while the bubbles in RHCMA were mostly stored in the gel in the form of micro-nano bubbles.

[0071] As shown in Figure 4 , it was found by using an optical microscope that the oxygen bubble particle size of GELMA was larger than that of RHC, indicating that the oxygen bubbles in RHCMA were mostly stored in the form of micro-nano bubbles.

[0072] As shown in Figure 5 : The bubble diameter of GELMA is mainly concentrated in 17 μm, and the distribution is more concentrated. The bubble diameter of RHCMA is smaller, concentrated in 6 μm. It is further illustrated that the bubbles in RHCMA are mostly stored in the form of micro-nano bubbles. The bubble particle size of GELMA is too large to be made into microspheres, and the bubbles in RHCMA mostly exist in the form of micro-nano bubbles, which are more suitable for making gel microspheres of different sizes.

[0073] As shown in Figure 6 : The oxygen content of RHCMA is still high after standing for 1, 5, 10 min, indicating that the bubbles in RHCMA mostly exist in the form of micro-nano bubbles.

[0074] Example 4

[0075] The purpose of this embodiment is to compare the oxygen content of recombinant human type I / III collagen microgels under different pressures

[0076] Recombinant human type I / III collagen microgels containing micro-nano bubbles were prepared under pressures of 0.05 MPa, 0.1 MPa, and 0.15 MPa. The specific steps are as follows:

[0077] According to steps (1)-(3) in Example 1, a recombinant human type I / III collagen solution with a volume ratio of RHC I-MA-EDC solution to RHC III-MA-EDC solution of 7:3 was prepared;

[0078] The aeration head of the micro-nano bubble generator was placed in the mixed solution of RHC I-MA-EDC solution and RHC III-MA-EDC solution. The gas type was selected to be a gas containing 40% oxygen and 60% nitrogen. The pressure was adjusted to 0.05 MPa, 0.1 MPa, and 0.15 MPa, and the aeration time was 30 seconds. The size and oxygen content of the nano bubbles were precisely controlled;

[0079] Subsequently, the uniformly mixed photosensitive recombinant human type I / III collagen solution was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span80 in several times. A syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started, and the stirring speed was maintained at 800 rpm for 5 minutes to fully dissolve and mix. Subsequently, the curing treatment was carried out under light (usually ultraviolet light), and the light was maintained for 5 minutes to complete the crosslinking reaction. Finally, the oxygen content of the hydrogel microspheres was detected in the dissolved oxygen detector;

[0080] The oxygen content of the microgels detected by the dissolved oxygen detector (HQ40d) under different pressures is shown in Figure 7b shows: As the pressure rises from 0 MPa to 0.15 MPa, the oxygen content in the microgel generally shows an upward trend, and the oxygen content tends to be stable and relatively high from 0.1 MPa to 0.15 MPa. At 0.1 MPa, the oxygen content has reached a higher and relatively stable level, so the pressure of 0.1 MPa is the most suitable.

[0081] Example 5

[0082] The purpose of this example is to compare the effects of different aeration times on the oxygen content in the gel

[0083] Under the oxygen pressure of 0.1 MPa, the aeration time of 10 seconds, 30 seconds, 60 seconds, the micro-nano bubble-containing recombinant human collagen type I / III microgel was prepared to reflect the effect of aeration time on the oxygen content in the gel. The specific steps are as follows:

[0084] According to steps (1)-(3) in Example 1, the RHC I-MA-EDC solution and the RHC III-MA-EDC solution were prepared in a volume ratio of 7:3 to form a recombinant human collagen type I / III solution;

[0085] The aeration head of the micro-nano bubble generator was placed in the mixed solution of RHC I-MA-EDC and RHC III-MA-EDC, the gas type was selected to be a gas containing 40% oxygen and 60% nitrogen, the pressure was adjusted to 0.1 MPa, and the aeration time was 10 seconds, 30 seconds and 60 seconds to accurately control the size and oxygen content of the nano bubbles;

[0086] Then the uniformly mixed photosensitive recombinant human collagen type I / III solution was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span80 in several times, and a syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started to continuously stir the mixed solution at a speed of 800 rpm for 5 minutes to fully dissolve and mix. Then, the curing treatment was carried out under light (usually ultraviolet light) for 5 minutes to complete the crosslinking reaction. Finally, the oxygen content of the hydrogel microspheres was detected in the dissolved oxygen detector.

[0087] The oxygen content of the microgel detected by the dissolved oxygen detector (HQ40d) under different aeration times is shown in Figure 7 a shows: As the aeration time increases from 0 s to 60 s, the oxygen content in the microgel continues to rise. This shows that the oxygen content in the microgel can be regulated by prolonging the aeration time, and the oxygen content has reached a higher level at 30 s, and the error bar at 30 s shows that the oxygen content at this time is more stable, so the aeration time is preferably 30 s.

[0088] Example 6

[0089] The purpose of the present embodiment is to prepare different sizes of micro-nano bubble-containing recombinant human type I / III collagen microgels by adjusting the rotation speed (400 rpm, 800 rpm, 1000 rpm), and the specific steps are as follows:

[0090] The RHC I-MA-EDC solution and the RHC III-MA-EDC solution were prepared according to steps (1)-(3) in Example 1, and the volume ratio of the two solutions was 7:3;

[0091] The aeration head of the micro-nano bubble generator was placed in the mixed solution of the RHC I-MA-EDC solution and the RHC III-MA-EDC solution, the gas type was selected to be a gas containing 40% oxygen and 60% nitrogen, the pressure was adjusted to 0.1 MPa, and the aeration time was 30 seconds, so as to accurately control the size and oxygen content of the nano bubbles;

[0092] Subsequently, the uniformly mixed photosensitive recombinant human type I / III collagen solution was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 in several times, and a syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started, and the mixed solution was continuously stirred at a rotation speed of 400 rpm, 800 rpm or 1000 rpm for 5 minutes to fully dissolve and mix. Subsequently, a curing treatment was performed under light (usually ultraviolet light), and the light was maintained for 5 minutes to complete the crosslinking reaction. Finally, the oxygen content of the hydrogel microspheres was detected in the dissolved oxygen detector. In order to investigate whether the rotation speed has an effect on the oxygen content of the microspheres, the microspheres were collected and then placed in a PBS solution, and the oxygen content of the PBS was measured after the microspheres were allowed to stand for 30 minutes.

[0093] The rotation speed affects the particle size of the microgels, as shown in Figure 8 , Figure 9 : the results show that the larger the rotation speed, the smaller the particle size of the microspheres. When the rotation speed is 400 rpm, the particle size of the microspheres is about 28 μm; when the rotation speed is 800 rpm, the particle size of the microspheres is about 13 μm; and when the rotation speed is 1000 rpm, the particle size of the microspheres is about 7 μm. When the rotation speed is 800 rpm, the particle size of the microspheres is appropriate and uniform.

[0094] The rotation speed affects the oxygen content of the microgels, as shown in Figure 10 : the results show that the different rotation speeds do not affect the change in the oxygen content in the microspheres, because the stirring is performed in the oil phase, and the oil phase and the water phase are not compatible with each other, and the oxygen in the microgels is not easy to lose.

[0095] Example 7

[0096] The purpose of this embodiment is to compare the effects of different concentrations of recombinant human type I / III collagen solution on the mechanical properties of microgels.

[0097] Two types of sponge-like solid samples of RHC I-MA-EDC and RHC III-MA-EDC were prepared according to steps (1)-(2) in Example 1;

[0098] A PBS solution of 0.5 (w / v) photoinitiator LAP and 0.5 (w / v) dispersant (Pluronic F-127) was prepared, and 0.7 g of RHC-I-MA-EDC and 0.3 g of RHC-III-MA-EDC sponge-like solid samples were dissolved in 10 ml of the PBS solution of 0.5 (w / v) photoinitiator LAP and 0.5 (w / v) dispersant to obtain a 10% RHC I-MA-EDC and RHC III-MA-EDC mixed solution. In the same way, 20% and 30% RHC I-MA-EDC and RHC III-MA-EDC mixed solutions were prepared;

[0099] The aeration head of the micro-nano bubble generator was placed in the RHC I-MA-EDC solution and the RHC III-MA-EDC mixed solution, and a mixed gas containing 40% oxygen and 60% nitrogen was selected as the gas, the pressure was adjusted to 0.1 MPa, and the aeration time was 30 seconds to accurately control the size and oxygen content of the nano bubbles;

[0100] Then the uniformly mixed photosensitive recombinant human type I / III collagen solution was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 in several portions, and a syringe was used to control the dripping speed to avoid splashing of the solution. After the dripping was completed, the stirring device was quickly started, and the mixture was continuously stirred at a speed of 800 rpm for 5 minutes to fully dissolve and mix. Then, the curing process was carried out under light (usually ultraviolet light), and the light was kept on for 5 minutes to complete the crosslinking reaction;

[0101] The compression modulus was tested by compression test using a universal material testing machine, and the strain rate was set to 1 mm / min. The stress-strain curve of each sample at different compression rates was recorded. The compression modulus was calculated according to the stress-strain curve.

[0102] The results of the mechanical property analysis are shown in Table 1: Figure 11 As the RHC concentration increased from 10% to 30%, the stress value at the same strain gradually increased. The higher the RHC concentration, the greater the Young's modulus (10% < 20% < 30%). With the increase of RHC concentration, the maximum stress value of the material increased (10% < 20% < 30%).

[0103] Example 8

[0104] Mouse chondrocytes (ATDC5) were cultured on the surface of recombinant human type I / III collagen microgels with a concentration of 5 mg / ml in micro / nanobubbles at different ventilation times.

[0105] ATDC5 cells were resuspended in complete culture medium (components: 500 mL basal medium, 25 mL fetal bovine serum, 5 mL mouse chondrocyte growth supplement, 5 mL penicillin / streptomycin solution) and cultured at 10 mL per culture dish. 5 Cells were seeded at a density of [number] cells. The experimental groups were prepared by uniformly mixing 5 mL of the above-described complete culture medium with the microgel prepared in Example 4, with aeration times of 10 seconds, 30 seconds, and 60 seconds. The control group used 5 mL of complete culture medium. After seeding, the cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. At the end of the culture, the cells were stained using a live / dead cell staining kit and observed under a fluorescence inverted microscope.

[0106] Results of biocompatibility cell experiments as follows Figure 12 As shown, the microgel resulted in the highest survival rate of ATDC5 cells after 30 seconds of ventilation.

[0107] Example 9

[0108] Human skin fibroblasts (HSFs) were cultured on the surface of recombinant human type I / III collagen microgels with different concentrations of micro / nanobubbles after 30 seconds of ventilation.

[0109] HSFs cells were resuspended in complete culture medium (components: 500 mL basal medium, 25 mL fetal bovine serum, 5 mL fibroblast growth supplement, 5 mL penicillin / streptomycin solution) and cultured at 10 mL per culture dish. 5 Cells were seeded at a density of [number] cells. Experimental group 1 consisted of 5 mL of the complete culture medium described above, mixed with the 1.0 mg / mL, 2.5 mg / mL, and 5.0 mg / mL microgels prepared in Example 6. The control group used 5 mL of complete culture medium. After seeding, cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. At the end of the culture, live / dead cell staining was performed, and the cells were observed under a fluorescence inverted microscope.

[0110] Results of biocompatibility cell experiments as follows Figure 13 As shown: HSFs cells showed the highest survival rate at a microgel concentration of 5 mg / ml.

Claims

1. A method for preparing a microgel of recombinant human type I / III collagen containing micro-nano bubbles, characterized in that, The method comprises the following steps: 1) mixing methacrylic acid with a solution of recombinant human type I collagen and a solution of recombinant human type III collagen respectively to prepare porous sponge-like RHC I-MA and RHC III-MA solids; then dissolving the RHC I-MA and RHC III-MA solids in a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution to obtain RHC I-MA-EDC and RHC III-MA-EDC solids with high cross-linking degree; 2) dissolving the RHC I-MA-EDC and RHC III-MA-EDC solids in a PBS solution containing a photoinitiator and a dispersant respectively to form homogeneous solutions, and then mixing the homogeneous solutions in a volume ratio of 1:10 to 10:1 to obtain a mixed solution; 3) placing an aeration head of a micro-nano bubble generator in the mixed solution obtained in step 2), and passing an oxygen-containing gas at an aeration pressure of 0.05-1 MPa for 10-60 s to enrich the RHC I-MA-EDC and RHC III-MA-EDC solution prepared with micro-nano oxygen bubbles, thereby obtaining an oxygen-containing mixed solution; 4) slowly adding the oxygen-containing mixed solution prepared in step 3) to a mixed solution of an oil phase and a surfactant, stirring, irradiating under ultraviolet light for 3-10 min, removing the supernatant by centrifugation, adding PBS and shaking to remove the remaining oil phase, finally adding a PBS buffer to wash clean, and then collecting by filtration to obtain a recombinant human type I / III collagen microgel containing micro-nano oxygen bubbles.

2. The method for preparing recombinant human type I / III collagen microgels containing micro / nanobubbles according to claim 1, characterized in that, In step 2), the dispersant is Tween-80 or pluronic F-127.

3. The method for preparing recombinant human type I / III collagen microgels containing micro / nanobubbles according to claim 1, characterized in that, In step 2), the PBS solution containing a photoinitiator and a dispersant has a photoinitiator concentration of 0.1%-2%(w / v) and a dispersant concentration of 0.2%-1%(w / v).

4. The method for preparing recombinant human type I / III collagen microgels containing micro / nanobubbles according to claim 1, characterized in that, In step 3), the oxygen-containing gas has an oxygen concentration of 20%-100%.

5. The method for preparing recombinant human type I / III collagen microgel containing micro / nanobubbles according to claim 1, characterized in that, In step 3), the aeration pressure is 0.05-0.15 MPa, and the aeration time is 20-40 s. 6.The method for preparing the micro-nano bubble-containing recombinant human type I / III collagen microgel according to claim 1, characterized in that, In step 4), the stirring speed is 400-1000 rpm, and the stirring time is 5-10 min.

7. The method for preparing recombinant human type I / III collagen microgels containing micro / nanobubbles according to claim 4, characterized in that, In step 3), the oxygen-containing gas is a gas containing 40% oxygen and 60% nitrogen.

8. The recombinant human type I / III collagen microgel containing micro-nano bubbles prepared by the method of any one of claims 1 to 7. 9.The micro-nano bubble-containing recombinant human type I / III collagen microgel of claim 8, wherein, The micro-nano bubbles have a diameter of 6 μm, and an oxygen content of 6-12 mg / mL.

10. Use of the recombinant human type I / III collagen microgel containing micro-nano bubbles of claim 8 in the preparation of a tissue repair and regeneration material.

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