CN108853599B

CN108853599B - Oxygen-enriched temperature-sensitive hydrogel and preparation method thereof

Info

Publication number: CN108853599B
Application number: CN201810847002.2A
Authority: CN
Inventors: 王宇清; 刘国辉; 刘长安; 康强军; 王凌云
Original assignee: Individual
Current assignee: 980th Hospital of the Joint Logistics Support Force of PLA
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2021-03-05
Anticipated expiration: 2038-07-27
Also published as: CN108853599A

Abstract

The invention discloses an oxygen-enriched temperature-sensitive hydrogel and a preparation method thereof, wherein the oxygen-enriched temperature-sensitive hydrogel is prepared by mixing a CS acid solution, a beta-GP solution, an HEC solution and a penfluron emulsion; the content of the panflurron in the hydrogel is 5.0vol% to 15.0 vol%. The oxygen-enriched temperature-sensitive hydrogel has no toxic or side effect, has good physicochemical property, oxygen release performance and microstructure, has good histocompatibility, can be completely metabolized and removed, can promote bone defect healing, and is suitable for being used as a slow release carrier of oxygen in the field of bone tissue engineering. The oxygen-enriched temperature-sensitive hydrogel has the basic biological characteristics of CS, has good histocompatibility and degradability proved by experiments, has no obvious stimulation and toxic and side effects on surrounding tissues, and can be gradually and completely metabolized and eliminated; the degradation process can provide a growth space for new bone tissues and is beneficial to bone healing.

Description

Oxygen-enriched temperature-sensitive hydrogel and preparation method thereof

Technical Field

The invention relates to an oxygen-enriched bone filling material, in particular to an oxygen-enriched temperature-sensitive hydrogel and a preparation method thereof.

Background

Bone damage from high-energy trauma has become one of the major disease categories in the trauma area. How to efficiently repair and reconstruct bone defects, bone nonunions and even bone necrosis caused by the injuries becomes a key topic of current orthopedic research. The development of research on the application of bone tissue engineering principles to repair such injuries has become a new hope in the field of bone defect repair research. After bone tissue damage, local partial pressure of oxygen is reduced due to interruption of blood flow or formation of hematomas at the site of the damage. There are studies showing that: after the blood flow of the fracture is interrupted, the oxygen partial pressure of the central part is even reduced to 0 to 2 percent. While hypoxia can lead to abnormal bone metabolism, hypoxia of the bone marrow microenvironment has a great influence on the dynamic balance between bone formation and bone resorption. Therefore, an oxygen-rich material is constructed through research and is compounded with a bone tissue engineering material to be used for repairing bone defects, the internal early-stage anoxic environment after tissue engineering bone transplantation can be improved, the survival and activity of stem cells (seed cells) are improved, and the regeneration and healing of bones are promoted.

Oxygen is an essential substance for life support. The oxygen partial pressure in the normal tissues and the gaps of the tissues of the human body is 24-66 mmHg (1mmHg =0.133kPa), namely 3% -9% (volume fraction). The oxygen partial pressure of the current in vitro cell culture environment is typically 159mmHg, i.e., the volume fraction of oxygen is about 21%. Papandreou et al believe that the external oxygen supply and the oxygen requirements of the cells should be maintained in balance to ensure a good growth environment for the cells. After bone tissue damage, local partial pressure of oxygen is reduced due to interruption of blood flow or formation of hematomas at the site of the damage. There are studies showing that: after the blood flow of the fracture is interrupted, the oxygen partial pressure of the central part is even reduced to 0 to 2 percent. While hypoxia can lead to abnormal bone metabolism, hypoxia of the bone marrow microenvironment has a great influence on the dynamic balance between bone formation and bone resorption. Therefore, maintaining oxygen homeostasis after bone tissue injury, allowing the oxygen concentration to fluctuate within a specific range locally to the injury, is crucial for promoting bone regeneration and healing, where timely and effective reoxygenation has been considered as a critical step.

In addition, when tissue engineering bone inoculated with cells is transplanted into a body at present, the oxygen supply state of early cells is often inferior to that of an in vitro culture environment. Moreover, the more cells are located at the center of the interior of the stent, the more difficult it is to obtain sufficient oxygen. When the cells are in a hypoxic state, their adhesion, proliferation, activity and functional status are greatly compromised and even apoptosis or necrosis occurs. This not only seriously affects the performance of the seed cell repair function, but also the products produced by cell necrosis will further hinder the repair of defects. Therefore, the construction of an oxygen-rich material and the composition of the oxygen-rich material and the tissue engineering bone can improve the early hypoxia environment in the tissue engineering scaffold and promote the survival of transplanted seed cells, thereby improving the repair capability of the functional tissue engineering scaffold. At present, no relevant report is found in the aspect of oxygen-enriched bone filling materials.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the oxygen-enriched temperature-sensitive hydrogel with good repairing effect; the invention also provides a preparation method of the oxygen-enriched temperature-sensitive hydrogel.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the emulsion is formed by mixing a CS acid solution, a beta-GP solution, an HEC solution and a penfluron emulsion; the content of the panflurron in the hydrogel is 5.0vol% to 15.0 vol%.

The hydrogel comprises the following components in percentage by weight: CS 0.010-0.015 g/ml, beta-GP 0.015-0.020 g/ml and HEC 0.003-0.007 g/ml.

The method adopts the component proportion, and the method process comprises the following steps: and (3) mixing the CS acid solution, the beta-GP solution, the HEC solution and the penfluron emulsion, and fully mixing under the ice bath condition to obtain the hydrogel glue solution.

The CS acid solution is prepared from CS powder and a hydrochloric acid solution or an acetic acid solution, and the CS content in the CS acid solution is 0.010-0.050 g/ml.

The beta-GP solution is prepared from beta-GP powder and water, and the content of the beta-GP is 0.10-0.25 g/ml.

The HEC solution is prepared from HEC powder and water, and the content of HEC is 0.01-0.06 g/ml.

The preparation process of the penfluron emulsion comprises the following steps: adding egg yolk lecithin into Tyrode salt buffer solution or PBS salt buffer solution, mixing and emulsifying to obtain basic emulsion; adding a penfluron stock solution into the basic emulsion, mixing and emulsifying to obtain an emulsion; and oxygenating the emulsion in a high-pressure oxygen environment to obtain the penfluron emulsion. The content of the penfluron in the penfluron emulsion is 10-45 vol%. 80-110 mg of egg yolk lecithin is added into 550-900 mu L of Tyrode salt buffer solution or PBS salt buffer solution. The emulsification adopts ultrasonic emulsification.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: the invention has no toxic or side effect, good physicochemical property, oxygen release performance and microstructure, good histocompatibility, can be completely metabolized and removed, can promote bone defect healing, and is suitable for being used as a slow release carrier of oxygen in the field of bone tissue engineering. The invention has the basic biological characteristics of CS, has good histocompatibility and degradability proved by experiments, has no obvious stimulation and toxic and side effects on surrounding tissues, and can be gradually and completely metabolized and eliminated; the degradation process can provide a growth space for new bone tissues and is beneficial to bone healing.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a graph of oxygen evolution rate of 5% and 10% perfluororon concentration oxygen-rich temperature-sensitive hydrogels according to the present invention;

FIGS. 2 and 3 are electron microscope scanning images of the cross section of the oxygen-rich temperature-sensitive hydrogel after freeze-drying;

FIG. 4 is an electron microscope scanning image of tissues when oxygen-rich temperature-sensitive hydrogel is embedded in rabbit thigh muscles for 2 weeks;

FIG. 5 is an electron microscope scanning image of tissues when oxygen-rich temperature-sensitive hydrogel is embedded in rabbit thigh muscles for 4 weeks;

FIG. 6 is an electron microscope scanning image of tissues when oxygen-rich temperature-sensitive hydrogel is embedded in rabbit thigh muscles for 6 weeks;

FIG. 7 is an electron microscope scanning image of tissues when oxygen-rich temperature-sensitive hydrogel is embedded in rabbit thigh muscles for 8 weeks;

FIG. 8 is an X-ray examination of the experimental group at week 4 of the rabbit femoral condyle bone defect repair experiment;

FIG. 9 is an X-ray examination of the control group at week 4 of the rabbit femoral condyle bone defect repair experiment;

FIG. 10 is an X-ray examination of a blank group at week 4 of the rabbit femoral condyle bone defect repair experiment;

FIG. 11 is an X-ray examination of the experimental group at week 12 of the rabbit femoral condyle bone defect repair experiment;

FIG. 12 is an X-ray examination of the control group at week 12 of the rabbit femoral condyle bone defect repair experiment;

FIG. 13 is an X-ray examination of a blank group at week 12 of the rabbit femoral condyle bone defect repair experiment;

FIG. 14 is a three-dimensional Micro-CT image at week 4 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 15 is a pseudo-color Micro-CT image at week 4 of an experiment for repairing bone defects of rabbit femoral condyles;

FIG. 16 is a two-dimensional Micro-CT image at week 4 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 17 is a three-dimensional Micro-CT image at week 6 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 18 is a pseudo-color Micro-CT image at week 6 of an experiment for repairing bone defects of rabbit femoral condyles;

FIG. 19 is a two-dimensional Micro-CT image at week 6 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 20 is a three-dimensional Micro-CT image at week 8 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 21 is a pseudo-color Micro-CT image at week 8 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 22 is a two-dimensional Micro-CT image at week 8 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 23 is a three-dimensional Micro-CT image at week 12 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 24 is a pseudo-color Micro-CT image at week 12 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 25 is a two-dimensional Micro-CT image at week 12 of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 26 is a TMD (transition data model) diagram of Micro-CT data analysis of an experiment for repairing bone defect of rabbit femoral condyle;

FIG. 27 is a BVF (Micro-CT) data analysis chart of a rabbit femoral condyle bone defect repairing experiment;

FIG. 28 is a Tb.N data analysis of Micro-CT data of an experiment for repairing a bone defect of a condyle of a femur of a rabbit;

FIG. 29 is a Tb.Th graph of data analysis of Micro-CT data of an experiment for repairing bone defects of rabbit femoral condyles;

FIG. 30 is a graph of SMI data analysis of Micro-CT data of a rabbit femoral condyle bone defect repairing experiment;

FIG. 31 is a tissue morphology of the experimental group at 4 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 32 is a tissue morphology of a control group at 4 weeks of a rabbit femoral condyle bone defect repair experiment;

FIG. 33 is a tissue morphology of a blank group at 4 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 34 is a photograph of CD31 immunohistochemistry at 4 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 35 is a tissue morphology of the experimental group at 6 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 36 is a tissue morphology of a control group at 6 weeks of a rabbit femoral condyle bone defect repair experiment;

FIG. 37 is a tissue morphology map of a blank group at 6 weeks of a rabbit femoral condyle bone defect repair experiment;

FIG. 38 is a photograph of CD31 immunohistochemistry at 6 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 39 is a tissue morphology of experimental groups at 8 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 40 is a tissue morphology of a control group at 8 weeks of an experiment for repairing a bone defect in a femoral condyle of a rabbit;

FIG. 41 is a tissue morphology of a blank group at 8 weeks of a rabbit femoral condyle bone defect repair experiment;

FIG. 42 is a photograph of CD31 immunohistochemistry at 8 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 43 is a tissue morphology of experimental group at 12 weeks of the experiment for repairing bone defect of rabbit femoral condyle;

FIG. 44 is a tissue morphology of a control group at 12 weeks of a rabbit femoral condyle bone defect repair experiment;

FIG. 45 is a tissue morphology of a blank group at 12 weeks of a rabbit femoral condyle bone defect repair experiment;

FIG. 46 is a photograph of CD31 immunohistochemistry at 12 weeks of the rabbit femoral condyle bone defect repair experiment;

FIG. 47 is a statistical chart of the counts of immunohistochemical microvessels of CD31 in an experiment for repairing a bone defect in a femoral condyle of a rabbit.

Detailed Description

The oxygen-enriched temperature-sensitive hydrogel is formed by mixing a Chitosan (CS) solution, a beta-sodium glycerophosphate (beta-GP) solution, a hydroxyethyl cellulose (HEC) solution and a penfluron emulsion; the content of each component in each milliliter of hydrogel is as follows: 0.010-0.015 g of CS, 0.015-0.020 g of beta-GP, 0.003-0.007 g of HEC and 0.050-0.150 ml.

The treatment mechanism and the component action of the oxygen-enriched temperature-sensitive hydrogel are as follows: hydrogels are three-dimensional porous network stereo-crosslinked structures formed by hydrophilic polymers through physical entanglement or the action of certain chemical bonds, do not dissolve in water but swell to form a solid, "jelly" form by absorbing large amounts of water. Because of the characteristics of water absorption and water retention, the water content of the hydrogel exceeds 95 percent, the hydrogel is similar to human tissues, the surface is soft and moist, the compatibility with tissues is good, the irritation to organisms is small, and the hydrogel serving as a carrier is easy to compound with other substances, so that the hydrogel has very wide application prospects in the medical fields of tissue engineering, carrier drug slow release and the like. Many natural high molecular compounds such as collagen, gelatin, hyaluronic acid, and CS can be used as raw materials for preparing hydrogel, and the preparation of hydrogel using CS as raw material is most widely used.

CS is a product of deacetylation of chitin, and the main repeating unit in the molecule is glucosamine, which is a natural component of mammalian tissues, and the content of CS is in direct proportion to the degree of deacetylation. The hydrogel prepared by using CS as a main raw material has good biocompatibility and no toxic or side effect, and can be gradually metabolized and eliminated in a body. Lysozyme contained in human serum can degrade CS and decompose CS into N-acetylglucosamine and glucosamine which are harmless to human bodies, and the acetylglucosamine is an intermediate product of tricarboxylic acid cycle for generating ATP, which is favorable for improving cell activity and promoting cell growth. The deacetylation degree of CS is very important, and the high deacetylation CS is selected as the hydrogelAt higher time (more than or equal to 90 percent), the degradation speed of CS is slow, the acute inflammatory reaction caused in vivo is also light, and the degree of deacetylation is low (<80%) and has a high degradation rate, and generates a large amount of low molecular weight substances in vivo in a short time to cause acute inflammatory reaction. The degree of deacetylation may also affect the cell adhesion of the CS-based scaffold material and the activity of the adhered cells. In addition, since CS itself has low solubility, its molecular chain-NH-increases as the degree of deacetylation increases₂Increased amount of-NH in hydrochloric acid solvent⁺And H⁺Protonation occurs and the solubility of CS increases dramatically. CS also has biological activity, can promote cell adhesion, proliferation and differentiation, and the monomer can induce the expression of 4 signal transduction genes such as mitogen activated alkaline phosphatase (ALP) and Protein Kinase (PK) genes, and the low-concentration CS monomer can directly improve the level of osteoblast signal transduction mRNA and regulate the cell activity. CS also has the functions of bacteriostasis, sterilization and hemostasis, and the sterilization principle is that the structure with negative charges on the surface of bacteria is combined with the positive charge molecules of CS, so that the cell wall of the bacteria is damaged, and the basic respiratory function is inhibited, thereby causing the death of the bacteria. In addition, CS can also enhance the activity of immune cells, and the higher the degree of deacetylation, the stronger the activating effect of complement. Because neuraminic acid residues on the surfaces of erythrocytes have negative charges and can interact with positive charge molecular chains of CS to mediate the aggregation of erythrocytes, the CS can promote blood coagulation to quickly stop bleeding at injured parts. The hydrogel with CS as the main component can promote wound healing and accelerate granulation tissue growth. In the results of the above muscle embedding experiments, the phenomena of immune cell proliferation, rapid muscle tissue repair, no obvious bleeding and infection, complete metabolic clearance of gel, and the like may be related to the characteristics of CS.

In order to solve the problem, CS with high deacetylation degree and beta-GP are mixed to prepare the temperature-sensitive hydrogel which can be fully filled in the defect part needing to be repaired in a liquid state before gelling and can be gradually gelled in an environment with the body temperature of 37 ℃. The gel principle of such temperature-sensitive hydrogels has not been exactly concluded by researchers to date. It is considered that the solid gel is formed by increasing the hydrophobic effect in the gel liquid due to the increase of molecular force generated between the beta-GP and the CS molecule when the temperature is increased after the beta-GP is added. However, it is thought that the molecular force between the two is rather weakened when the temperature is increased, and the gelation occurs only when the cationic amine group and the negatively charged phosphate ion in CS and β -GP undergo proton conversion. The gelling time and temperature of the CS-beta-GP hydrogel are reduced along with the increase of the pH value, and if the pH value is too low, the gelling time and the gelling temperature can be increased or even can not be gelled. Therefore, in order to neutralize the HCl solution for dissolving CS, the alkaline beta-GP needs to reach a certain concentration to enable the CS-beta-GP mixed solution to be converted into gel at the body temperature, the dosage of the beta-GP needs to be increased in order to further reduce the gel forming time, but when the concentration of the beta-GP is too high, the gel is strong in alkalinity and deviates from the normal pH value of a human body, and in addition, the high-concentration beta-GP can generate toxic and side effects on cells, for example, when the concentration of the beta-GP reaches 800mg/ml, the osmotic pressure of the beta-GP is up to 1080mOsm and is far higher than the physiological osmotic pressure of the human body, so that the normal structure of. Therefore, the addition of the cross-linking agent HEC which is easily soluble in water, non-toxic and harmless can obviously shorten the gelation process of the neutral glue solution at 37 ℃, thereby greatly reducing the dosage of beta-GP. The HEC can promote the gelling process probably because the hydroxyl of the HEC and the amino and hydroxyl on the CS and beta-GP generate hydrogen bond action, and in addition, a HEC macromolecular chain can also form physical entanglement with CS molecules, so that the gelling time of the CS-beta-GP-HEC glue solution in the environment of 37 ℃ is obviously shortened due to the two reasons.

The panflurron (1-bromoheptadecafluorooctane) is a perfluorocarbon compound, is an inert substance, has common physicochemical characteristics due to similar structure, and has stable chemical structure, no toxicity, no color, no odor, lower lipid solubility and water insolubility. More importantly, the composite material is a good oxygen transport medium, and the solubility to oxygen is 20 times of that of water and 2-3 times of that of whole blood. As a main component of the "artificial blood" of the latest generation, its oxygen carrying property and clinical safety have been confirmed.

The oxygen sustained-release by the oxygen-rich gel can improve the anoxic state of the internal environment at the early stage of bone injury repair, and promote the survival of seed cells and the improvement of osteogenesis activity, thereby playing the roles of promoting bone regeneration and improving bone repair capacity. And can also avoid the formation of an excessive oxygen-deficient environment in the early repair stage of the bone defect area, so that residual 'repair cells' around the bone defect area can not lose activity or even die due to excessive oxygen deficiency.

The porous structure of the tissue engineering scaffold can promote the growth of tissues and is beneficial to the adhesion of cells and the growth of blood vessels. Through observation of a scanning electron microscope, the oxygen-enriched temperature-sensitive hydrogel is in a three-dimensional porous reticular microstructure, the pore diameter is about 100-200 mu m, and the pores are compact, so that the oxygen-enriched temperature-sensitive hydrogel is beneficial to release and diffusion of gas molecules to surrounding tissues, and is also beneficial to exchange of surrounding body fluid and active substances and discharge of metabolic wastes. The oxygen carried by the stent material can only relieve the anoxic environment in the early stage, and can only obtain oxygen through the diffusion effect of peripheral new capillary vessels after cells or new tissues grow into the stent in the later stage, so the vascularization of the stent material is very important, and the three-dimensional porous reticular structure can enable the new blood vessels to smoothly grow into the stent through pore channels to promote the vascularization of the stent material. The oxygen-enriched temperature-sensitive hydrogel has the basic biological characteristics of CS, and has good histocompatibility and degradability, no obvious stimulation and toxic and side effects on surrounding tissues, and can be gradually and completely metabolized and eliminated. The degradation process can provide a growth space for new bone tissues and is beneficial to bone healing.

The preparation method of the oxygen-enriched temperature-sensitive hydrogel comprises the following steps: (1) preparing a Chitosan (CS) acid solution: weighing CS powder and a hydrochloric acid solution or an acetic acid solution according to the CS content of 0.01-0.05 g/ml, wherein the concentration of the hydrochloric acid solution or the acetic acid solution is 0.05-0.15 mol/L; mixing, continuously stirring at the speed of 800-1500 r/min for 2-5 h in a room temperature environment until the CS solution is almost clear, refrigerating at 4 ℃ or below overnight, filtering impurities by using a filter membrane with the diameter of 0.5-1.2 mu m to obtain the CS solution finally, sterilizing in a pressure cooker at 100-150 ℃ by using a kraft paper sealing device for 5-15 min, and refrigerating at 4 ℃ or below for later use.

(2) Preparing a solution of beta-sodium glycerophosphate (beta-GP): weighing beta-GP powder and water according to the content of the beta-GP of 0.10-0.25 g/ml; mixing, stirring at 800-1500 r/min for 10-20 min at room temperature for clarification, filtering with 0.15-0.22 μm filter membrane for sterilization, and refrigerating at 4 deg.C or below for storage.

(3) Preparing a hydroxyethyl cellulose (HEC) solution: weighing HEC powder and water according to the HEC content of 0.01-0.06 g/ml; mixing, stirring at 800-1500 r/min for 0.5-2 h at room temperature, filtering with 0.15-0.22 μm filter membrane for sterilization, and refrigerating at 4 deg.C or below for storage.

(4) Preparing a penfluron emulsion: adding egg yolk lecithin into Tyrode salt buffer solution or PBS salt buffer solution according to the proportion of 80-110 mg of egg yolk lecithin and 550-900 mu L of Tyrode salt buffer solution or PBS salt buffer solution, and ultrasonically emulsifying at least twice under the power of 200-500 w, wherein the interval is 1-2 min each time and 10-20 s each time, so as to obtain the basic emulsion. Weighing the mixture according to the content of the panfluron of 10-45 vol%, mixing the basic emulsion with the panfluron stock solution, and ultrasonically emulsifying at least 10 times under 200-500 w of power, wherein the interval is 1-2 min each time and 10-20 s each time; thus obtaining the panflurron emulsion. Sterilizing the panfluron emulsion by using a 0.5-0.8 mu m filter membrane, placing the panfluron emulsion in a high-pressure oxygen chamber, oxygenating for at least 10min, and sealing and storing at the temperature of 4 ℃ or below for later use.

(5) Preparing hydrogel: taking a CS solution, a beta-GP solution, an HEC solution and a penfluron emulsion, and mixing the following components in percentage by weight: 0.010-0.015 g/ml of CS, 0.015-0.020 g/ml of beta-GP, 0.003-0.007 g/ml of HEC and 5.0-15.0 vol% of perfluoromethylen are weighed and mixed. And (3) sealing after mixing, fully mixing by using a vibration instrument in an ice bath environment to obtain the oxygen-enriched temperature-sensitive type hydrogel glue solution, and forming the glue after a plurality of minutes in a 37-DEG environment. The obtained hydrogel glue solution is subpackaged into a plurality of freezing tubes and is hermetically stored at the temperature of 4 ℃ or below.

Example 1: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(1) Preparing a CS acid solution: weighing 0.75g of CS powder, and weighing 25ml of hydrochloric acid solution with the concentration of 0.1mol/L in a beaker; the CS powder was mixed with the hydrochloric acid solution and stirred continuously at 1000r/min for 3h with a magnetic stirrer at room temperature until the CS solution was almost clear. Refrigerating at 4 deg.C overnight, filtering with filter membrane with diameter of 1.2 μm to obtain 0.03g/ml CS acid solution, sterilizing with kraft paper in 120 deg.C pressure cooker for 10min, and refrigerating at 4 deg.C for use.

(2) Preparing a beta-GP solution: weighing 1.725g of beta-GP powder; weighing 10ml of deionized water in a beaker, and adding beta-GP powder; stirring with magnetic stirrer at 1000r/min for 15min at room temperature for clarification, filtering with 0.22 μm filter membrane for sterilization to obtain 0.1725g/ml beta-GP solution, and cold preserving at 4 deg.C for use.

(3) Preparing HEC solution: weighing 0.375g HEC powder; weighing 10ml of deionized water in a beaker, and adding HEC powder; stirring with magnetic stirrer at 1000r/min for 1 hr at room temperature, filtering with 0.22 μm filter membrane to remove bacteria to obtain 0.0375g/ml HEC solution, and cold preserving at 4 deg.C.

(4) Preparing a penfluron emulsion: firstly, 95mg of egg yolk lecithin was added to 850. mu.L of Tyrode salt buffer solution, and ultrasonic emulsification was performed twice at 300w power, with an interval of 1min for 15s each time, to obtain a basic emulsion. This was then mixed with 150. mu.L of the stock solution of penfluron and emulsified 10 times as above to give 1ml of 15vol% penfluron emulsion. This procedure was repeated several times to prepare a suitable amount of the emulsion of penfluron, due to the limited amount of liquid in each phacoemulsification. Sterilizing the penfluron emulsion with 0.8 μm filter membrane, placing in a high pressure oxygen chamber, oxygenating for 10min, and sealing at 4 deg.C for use.

(5) Preparing oxygen-enriched temperature-sensitive hydrogel: mixing 8ml of CS solution and 0.03g/ml with 2ml of beta-GP solution and 0.1725g/ml of beta-GP solution, 2.5ml of HEC solution and 0.0375g/ml of HEC solution, 6.25ml of panflurron emulsion and 15vol% of panflurron emulsion, sealing, fully mixing by using a vibration instrument in an ice bath environment to prepare the oxygen-enriched hydrogel glue solution, subpackaging the oxygen-enriched hydrogel glue solution into a plurality of freezing tubes, and sealing and storing at 4 ℃. The content of each milliliter of hydrogel in the obtained oxygen hydrogel glue solution is as follows: 0.0128g of CS, 0.0184g of beta-GP0, 0.005g of HEC and 0.05ml of panflurron.

Example 2: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

Steps (1) to (3) were the same as in example 1, and Steps (4) and (5) were the same as in example 1 except for the following differences:

(4) 95mg of egg yolk lecithin was mixed with 700. mu.L of tyrode salt buffer and 300. mu.L of penfluron stock solution to prepare a penfluron emulsion with a concentration of 0.30 g/ml.

(5) Preparing oxygen-enriched temperature-sensitive hydrogel: mixing 8ml of CS solution and 0.03g/ml with 2ml of beta-GP solution and 0.1725g/ml of beta-GP solution, 2.5ml of HEC solution and 0.0375g/ml of HEC solution, 6.25ml of panflurron emulsion with the concentration of 30% vol%, sealing, fully mixing by using a vibration instrument under an ice bath environment to prepare the oxygen-enriched hydrogel glue solution, subpackaging the oxygen-enriched hydrogel glue solution into a plurality of freezing tubes, and sealing and storing at 4 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched hydrogel glue solution is as follows: CS0.0128g, beta-GP 0.0184g, HEC 0.005g and panflurron 0.10 ml.

Example 3: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(4) 80mg of egg yolk lecithin, 550 mu of LTyrode salt buffer solution and 450 mu of penfluron stock solution are mixed to prepare penfluron emulsion with the concentration of 45 vol%.

(5) Preparing oxygen-enriched temperature-sensitive hydrogel: mixing 8ml of CS solution and 0.029g/ml with 2ml of beta-GP solution and 0.1725g/ml of beta-GP solution, 2.5ml of HEC solution and 0.0375g/ml of HEC solution, 6.25ml of panflurron emulsion and 45vol% of panflurron emulsion, sealing, fully mixing by using a vibration instrument under an ice bath environment to prepare the oxygen-enriched hydrogel glue solution, subpackaging the oxygen-enriched hydrogel glue solution into a plurality of freezing tubes, and hermetically storing at 4 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched hydrogel glue solution is as follows: 0.0124g of CS, 0.0184g of beta-GP, 0.005g of HEC and 0.15ml of panflurron.

Example 4: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(1) Preparing a CS acid solution: weighing CS powder and a hydrochloric acid solution according to the CS content of 0.015g/ml, wherein the concentration of the hydrochloric acid solution is 0.12 mol/L; mixing, stirring at 900r/min for 4 hr at room temperature until CS solution is almost clear, refrigerating at 4 deg.C overnight, filtering with 1.2 μm filter membrane to obtain CS solution, sterilizing with kraft paper sealing device at 150 deg.C pressure cooker for 12min, and refrigerating at 4 deg.C for use.

(2) Preparing a beta-GP solution: weighing beta-GP powder and water according to the content of the beta-GP of 0.20 g/ml; mixing, stirring at 800r/min for 20min at room temperature to clarify, filtering with 0.22 μm filter membrane for sterilization, and cold preserving at 4 deg.C.

(3) Preparing HEC solution: weighing HEC powder and water according to HEC content of 0.03 g/ml; mixing, stirring at 1500r/min for 1 hr at room temperature, filtering with 0.15 μm filter membrane for sterilization, and cold preserving at 4 deg.C.

(4) Preparing a penfluron emulsion: adding yolk lecithin into Tyrode salt buffer solution according to the mixture ratio of 90mg of yolk lecithin to 600 mu L of Tyrode salt buffer solution, and ultrasonically emulsifying twice under the power of 400w, wherein the interval between every two times is 1.5min, and every time is 10s to obtain the basic emulsion. Then mixing the basic emulsion with the stock solution of the penfluron, and emulsifying for 10 times by the same method; thus obtaining the penfluron emulsion with the penfluron content of 40 vol%. Sterilizing the penfluron emulsion with 0.8 μm filter membrane, placing in a high pressure oxygen chamber, oxygenating for 10min, and sealing at 4 deg.C for use.

(5) Preparing hydrogel: and (3) mixing 16.7ml of CS solution, 2.5ml of beta-GP solution, 2.5ml of HEC solution and 3.3ml of panflurron emulsion, sealing, and fully mixing by using a vibration instrument in an ice bath environment to obtain the oxygen-enriched temperature-sensitive hydrogel. The obtained hydrogel glue solution is subpackaged into a plurality of freezing tubes and is stored in a sealed way at 4 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched temperature-sensitive hydrogel glue solution is as follows: 0.010g of CS, 0.020g of beta-GP, 0.003g of HEC and 0.0528ml of panflurron.

Example 5: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(1) Preparing a CS acid solution: weighing CS powder and a hydrochloric acid solution according to the CS content of 0.04g/ml, wherein the concentration of the hydrochloric acid solution is 0.15 mol/L; mixing, stirring at 1500r/min for 3 hr at room temperature until CS solution is almost clear, refrigerating at 4 deg.C overnight, filtering with 1.0 μm filter membrane to obtain CS solution, sterilizing with kraft paper sealing device at 130 deg.C pressure cooker for 15min, and refrigerating at 4 deg.C for use.

(2) Preparing a beta-GP solution: weighing beta-GP powder and water according to the content of the beta-GP of 0.15 g/ml; mixing, stirring at 1200r/min for 10min at room temperature to clarify, filtering with 0.18 μm filter membrane for sterilization, and cold preserving at 4 deg.C.

(3) Preparing HEC solution: weighing HEC powder and water according to the HEC content of 0.06 g/ml; mixing, stirring at 1200r/min for 0.5h at room temperature, filtering with 0.20 μm filter membrane for sterilization, and cold preserving at 4 deg.C for use.

(4) Preparing a penfluron emulsion: adding egg yolk lecithin into Tyrode salt buffer solution according to the mixture ratio of 95mg of egg yolk lecithin and 750 mu L of Tyrode salt buffer solution, and ultrasonically emulsifying twice at 500w power, wherein the interval between every two times is 2min, and each time is 12s, so as to obtain the basic emulsion. Then mixing the basic emulsion with the stock solution of the penfluron, and emulsifying for 15 times by the same method; thus obtaining the penfluron emulsion, wherein the content of the penfluron is 25 vol%. Sterilizing the penfluron emulsion with 0.8 μm filter membrane, placing in a high pressure oxygen chamber, oxygenating for 10min, and sealing at 4 deg.C for use.

(5) Preparing hydrogel: and mixing 10ml of CS solution, 3ml of beta-GP solution, 3.5ml of HEC solution and 13.5ml of teflon emulsion, sealing, and fully mixing by using a vibration instrument in an ice bath environment to obtain the oxygen-enriched temperature-sensitive hydrogel. The obtained hydrogel glue solution is subpackaged into a plurality of freezing tubes and is stored in a sealed way at 4 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched temperature-sensitive hydrogel glue solution is as follows: CS 0.0133g, beta-GP 0.015g, HEC 0.007g and perfluoron 0.1125 ml.

Example 6: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(1) Preparing a CS acid solution: weighing CS powder and hydrochloric acid solution according to the CS content of 0.025g/ml, wherein the concentration of the hydrochloric acid solution is 0.05 mol/L; mixing, stirring at 1200r/min for 2 hr at room temperature until CS solution is almost clear, refrigerating at 0 deg.C overnight, filtering with 0.5 μm filter membrane to obtain CS solution, sterilizing with kraft paper sealing device at 140 deg.C pressure cooker for 5min, and refrigerating at 0 deg.C for use.

(2) Preparing a beta-GP solution: weighing beta-GP powder and water according to the content of the beta-GP of 0.25 g/ml; mixing, stirring at 1500r/min at room temperature for 12min to clear, filtering with 0.15 μm filter membrane for sterilization, and cold preserving at 0 deg.C.

(3) Preparing HEC solution: weighing HEC powder and water according to HEC content of 0.01 g/ml; mixing, stirring at 800r/min for 2 hr at room temperature, filtering with 0.18 μm filter membrane for sterilization, and cold preserving at 0 deg.C.

(4) Preparing a penfluron emulsion: adding egg yolk lecithin into Tyrode salt buffer solution according to the mixture ratio of 110mg of egg yolk lecithin to 700 mu L of Tyrode salt buffer solution, and ultrasonically emulsifying for three times under 200w of power, wherein the interval is 1.5min every time and 20s every time, so as to obtain the basic emulsion. Then mixing the basic emulsion with the stock solution of the penfluron, and emulsifying for 12 times by the same method; thus obtaining the penfluron emulsion, wherein the content of the penfluron is 30 vol%. Sterilizing the penfluron emulsion with 0.5 μm filter membrane, placing in a high pressure oxygen chamber, oxygenating for 15min, and sealing at 0 deg.C for use.

(5) Preparing hydrogel: and (3) mixing 14ml of CS solution, 2ml of beta-GP solution, 10ml of HEC solution and 7ml of panflurron emulsion, sealing, and fully mixing by using a vibration instrument in an ice bath environment to obtain the oxygen-enriched temperature-sensitive hydrogel. The obtained hydrogel glue solution is subpackaged into a plurality of freezing tubes and is hermetically stored at 0 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched temperature-sensitive hydrogel glue solution is as follows: CS 0.0106g, beta-GP 0.0152g, HEC 0.0030g and panflurron 0.0636 ml.

Example 7: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(1) Preparing a CS acid solution: weighing CS powder and an acetic acid solution according to the CS content of 0.05g/ml, wherein the concentration of the acetic acid solution is 0.08 mol/L; mixing, stirring at 800r/min for 5 hr at room temperature until CS solution is almost clear, refrigerating at 4 deg.C overnight, filtering with 1.2 μm filter membrane to obtain CS solution, sterilizing with kraft paper sealing device in 100 deg.C pressure cooker for 15min, and refrigerating at 4 deg.C for use.

(2) Preparing a beta-GP solution: weighing beta-GP powder and water according to the content of the beta-GP of 0.22 g/ml; mixing, stirring at 1000r/min for 18min at room temperature to clarify, filtering with 0.22 μm filter membrane for sterilization, and cold preserving at 4 deg.C.

(3) Preparing HEC solution: weighing HEC powder and water according to HEC content of 0.02 g/ml; mixing, stirring at 1000r/min for 1.5 hr at room temperature, filtering with 0.22 μm filter membrane for sterilization, and cold preserving at 4 deg.C.

(4) Preparing a penfluron emulsion: adding yolk lecithin into Tyrode salt buffer solution according to the mixture ratio of 100mg of yolk lecithin and 900 mu L of Tyrode salt buffer solution, and ultrasonically emulsifying twice at 450w power, wherein the interval between every two times is 1.5min, and each time is 15s, so as to obtain the basic emulsion. Then mixing the basic emulsion with the stock solution of the penfluron, and emulsifying for 15 times by the same method; thus obtaining the penfluron emulsion with the penfluron content of 10 vol%. Sterilizing the penfluron emulsion with 0.8 μm filter membrane, placing in a high pressure oxygen chamber, oxygenating for 12min, sealing at 4 deg.C, and storing.

(5) Preparing hydrogel: and mixing 9ml of CS solution, 2.5ml of beta-GP solution, 6ml of HEC solution and 18ml of teflon emulsion, sealing, and fully mixing by using a vibration instrument in an ice bath environment to obtain the oxygen-enriched temperature-sensitive hydrogel. The obtained hydrogel glue solution is subpackaged into a plurality of freezing tubes and is stored in a sealed way at 4 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched temperature-sensitive hydrogel glue solution is as follows: CS0.0127g, beta-GP0.0155g, HEC 0.0034g, and panfluron 0.0507 ml.

Example 8: the preparation method of the oxygen-enriched temperature-sensitive hydrogel adopts the following specific process.

(1) Preparing a CS acid solution: weighing CS powder and hydrochloric acid solution according to the CS content of 0.045g/ml, wherein the concentration of the hydrochloric acid solution is 0.10 mol/L; mixing, stirring at 1000r/min for 4 hr at room temperature until CS solution is almost clear, refrigerating at 0 deg.C overnight, filtering with 1.2 μm filter membrane to obtain CS solution, sterilizing with kraft paper sealing device at 150 deg.C pressure cooker for 10min, and refrigerating at 0 deg.C for use.

(2) Preparing a beta-GP solution: weighing beta-GP powder and water according to the content of the beta-GP of 0.10 g/ml; mixing, stirring at 900r/min for 20min at room temperature for clarifying, filtering with 0.22 μm filter membrane for sterilizing, and refrigerating at 0 deg.C for use.

(3) Preparing HEC solution: weighing HEC powder and water according to HEC content of 0.04 g/ml; mixing, stirring at 900r/min for 2 hr at room temperature, filtering with 0.22 μm filter membrane for sterilization, and cold preserving at 0 deg.C.

(4) Preparing a penfluron emulsion: adding yolk lecithin into PBS salt buffer solution according to the mixture ratio of 80mg of yolk lecithin and 650 mu L of PBS salt buffer solution, and ultrasonically emulsifying for four times under 350w of power, wherein the interval between every two times is 1.5min, and each time is 18s, so as to obtain the basic emulsion. Then mixing the basic emulsion with the stock solution of the penfluron, and emulsifying for 12 times by the same method; thus obtaining the penfluron emulsion, wherein the content of the penfluron is 35 vol%. Sterilizing the penfluron emulsion with 0.8 μm filter membrane, placing in a high pressure oxygen chamber, oxygenating for 10min, sealing at 0 deg.C, and storing.

(5) Preparing hydrogel: and mixing 10ml of CS solution, 5ml of beta-GP solution, 5ml of HEC solution and 10ml of panflurron emulsion, sealing, and fully mixing by using a vibration instrument in an ice bath environment to obtain the oxygen-enriched temperature-sensitive hydrogel. The obtained hydrogel glue solution is subpackaged into a plurality of freezing tubes and is hermetically stored at 0 ℃. The content of each milliliter of hydrogel in the obtained oxygen-enriched temperature-sensitive hydrogel glue solution is as follows: CS 0.01.5g, beta-GP 0.0167g, HEC 0.0067g and panflurron 0.1167 ml.

And (3) performance detection: the performance test of the oxygen-rich temperature-sensitive hydrogel containing 5vol% of perfluoron obtained in example 1 and the oxygen-rich temperature-sensitive hydrogel containing 10vol% of perfluoron obtained in example 2 was performed, and the test procedures and results are as follows.

(1) And (3) gelling time: and respectively taking 10 tubes of the two glue solution samples with the concentration of the difluron and placing the two glue solution samples into a constant-temperature water bath at 37 ℃, respectively taking 1 tube of the two glue solution samples with the concentration of 30s at intervals, carrying out inversion observation, taking the time point of taking the sample out as the gelling time if the glue solution does not flow backwards within 30s, and repeating the experiment for 5 times.

The results show that: after the oxygen-rich temperature-sensitive hydrogel with 5vol% and 10vol% of the perfluoroethylene-propylene-based perfluoroethylene copolymer is in an environment at 37 ℃ for a period of time, the light yellow flowing glue solution is changed into a light yellow non-flowable glue solution, the glue forming time is about 4.5 minutes, and no obvious statistical difference exists (P is more than 0.05).

(2) And (3) detection of pH value: the two oxygen-enriched hydrogel glue solutions with the concentration of the difluron are respectively filled in 5 small bottles, the pH value of the glue solution in each small bottle is measured by an electronic pH meter at normal temperature before the solution is gelatinized, and 5 groups of data are recorded.

The results show that: the pH values of the oxygen-enriched hydrogel gels at 5% and 10% penfluron concentrations were close to neutral and there was no significant statistical difference between the two groups (P > 0.05).

(3) Detection of oxygen release performance: respectively mixing two oxygen-enriched hydrogels with different concentrations of the penfluron according to the ratio of 10ml/cm³The leaching solution was prepared by immersing standard (5 samples) in Tyrode salt buffer solution. A small amount of leaching liquor is taken at 0, 1 and 2 … … 10 days respectively, and oxygen partial pressure is measured by a biochemical blood gas instrument to judge the oxygen release capacity of the oxygen-enriched hydrogel with two different concentrations of the penfluron.

The oxygen partial pressure of the leaching solution for continuously soaking the oxygen-enriched temperature-sensitive hydrogel colloid is detected at different time points by using a blood gas analyzer, and the oxygen release rate curve graphs of two kinds of oxygen-enriched hydrogels with different concentrations of teflon are drawn according to the measured data at each time point, and the curve graphs are shown in figure 1. The results show that: the oxygen-enriched hydrogel with the concentration of the perfluoroethyl ketone of 10 percent has larger oxygen release amount and faster oxygen release rate, and has better oxygen release performance than the oxygen-enriched hydrogel of 5 percent; but the oxygen release duration of the two is the same, and lasts for about 7 days.

(4) And (3) detecting the ultrastructure: after the oxygen-enriched hydrogel with the concentration of 10% of the perfluoroethyl ketone is freeze-dried, the cross section of the oxygen-enriched hydrogel is observed by a scanning electron microscope, the scanning images are shown in figures 2 and 3, and as can be seen from figures 2 and 3, the microstructure of the freeze-dried gel is a porous three-dimensional net-shaped framework (the aperture is about 100-200 mu m), the fibers are criss-cross, and the aperture is compact.

(5) And (3) detecting the blood toxicity: the 10% oxygen-rich hydrogel was tested for blood compatibility. 20ml of fresh rabbit blood is taken and added with a proper amount of heparin for standby. 2ml of anticoagulated rabbit blood was taken and diluted with 2.5ml of physiological saline (total 4.5 ml). Three sets of experiments were designed, 5 samples per set, 1, experimental set: mixing 8ml of the extract (prepared by the above method) with 0.2ml of diluted rabbit blood; 2. positive control group: uniformly mixing 8ml of distilled water with 0.2ml of diluted rabbit blood; 3. negative control group: 8ml of normal saline is taken and mixed with 0.2ml of diluted rabbit blood uniformly. The three groups of samples are placed for 1 hour at the constant temperature of 37 ℃, taken out of a test tube centrifuge for centrifugation (2000 r/min, 5 min), and the supernatant is sucked out and placed in a spectrophotometer to measure the OD value (545 nm wavelength).

And (3) detection results: the supernatant after centrifugation of the 10% hydrogel group (experimental group) was colorless and transparent as the negative control group, indicating that the erythrocytes were not destroyed. In contrast, the positive control group appeared as a red liquid, indicating red blood cell rupture. Each group of supernatants was analyzed by a spectrophotometer, and the results of absorbance (OD value) thereof showed: the experimental group has significant difference (P < 0.01) with the positive control group, and has no significant difference (P > 0.05) with the negative control group.

(6) Detecting histocompatibility and degradability by muscle embedding: taking 12 New Zealand white rabbits, randomly dividing into 4 groups of 3 rabbits, anesthetizing and sterilizing experimental rabbits, and adding 1cm of oxygen-rich hydrogel colloid with 10% penflurron concentration³Parallel to the blunt separation of muscle fibers, embedded in the right thigh muscle tissue of all experimental rabbits. The cells are killed at 2, 4, 6 and 8 weeks respectively, muscle tissues at the embedding part are taken out, and paraffin sections are subjected to HE staining to observe tissue reaction and gel metabolism clearance.

And (3) detection results: after operation, all rabbits have stable vital signs, no infection in the incision, normal food intake in the next day after operation and gradually increased weight. After 2 weeks, the incision heals at stage I, and part of the suture automatically falls off. When the oxygen-enriched gel with the concentration of the penflurron of 10 percent is embedded in the thigh muscle of a rabbit for 2 weeks after gelling, a sample is generally complete and milky white and soft, most of the implant is not metabolized and eliminated, and the boundary between the implant and the muscle is clear; under the microscope, the lamellar homogeneous red-stained graft can be seen, and a few inflammatory cells such as neutrophilic granulocytes and lymphocytes permeate into the graft without obvious fibroplasia and tissue necrosis, which is shown in figure 4. At 4 weeks, the specimens were generally visible with significant shrinkage of the graft, a few yellowish white residues, soft texture, and irregular morphology; under the microscope, a large number of inflammatory cells, namely, nucleus lobular neutrophils and lymphocytes, are arranged around the graft, and no tissue necrosis is obvious, as shown in figure 5. At 6 weeks, no significant graft residue was seen in the samples; under the mirror, the graft was almost completely metabolically cleared, with few inflammatory cells between normal muscle fibers and no apparent tissue necrosis, see fig. 6. At 8 weeks, the specimen found substantially no grafts; under the mirror, the inflammatory cells of normal muscle tissue subsided, as shown in fig. 7.

Experiment for repairing rabbit femoral condyle bone defect: an experiment for repairing the bone defect of the femoral condyle of the rabbit is carried out by selecting oxygen-rich temperature-sensitive hydrogel with the volume percent of the perfluoromethyluron 10 obtained in the example 2.

1. The experimental process comprises the following steps:

(1) grouping experimental animals: 72 adult New Zealand white rabbits of 6-9 months old, the weight is 2.5-3.5kg, and the male rabbit and the female rabbit are half in each. The experimental group (filled with oxygen-enriched temperature-sensitive hydrogel), the control group (filled with unoxygenated temperature-sensitive hydrogel) and the blank group (not filled with any material) are randomly divided into 12 groups according to the 4 th, 6 th, 8 th and 12 th weeks, wherein the right side of each rabbit is the operation side. In the experimental group, oxygen-rich temperature-sensitive hydrogel with 10% of perfluoroethylene-propylene-diene monomer concentration is closely filled to the bone defect of the lateral condyle of the femur, in the control group, oxygen-free temperature-sensitive hydrogel which contains oxygen-rich agent perfluoroethylene-propylene-diene monomer but is not oxygenated is filled to the bone defect of the lateral condyle of the femur, and the bone defect of the lateral condyle of the femur in the blank group is not subjected to any filling treatment.

(2) Operation and postoperative treatment: the method comprises the steps of intramuscular injection of 0.3ml/kg of fast dormancy on the thigh of a rabbit, intravenous injection of 2% phenobarbital according to 1.5ml/kg of ear margin, observation of vital signs, slow reduction of respiration, muscle relaxation and no corneal reflection, which prove that anesthesia is successful, fixing a left lateral lying position on an operating table, scraping upper and lower 2cm rabbit hair on a knee joint by using an electric shaver, carrying out iodophor disinfection 2 times in an operation area, paving a sterile hole towel, touching the lateral condyle platform part of the femur, making a longitudinal incision with the length of about 1.5 cm in the center of the platform, sequentially cutting skin and subcutaneous fascia, opening the eyelid to open the incision by using a draw hook, exposing the lateral condyle of the femur and a distal joint gap, taking a drill bit with the diameter of 6mm, marking the drill bit at a position 1mm away from the drill bit, drilling a hole from the outside to the inside by using the position 2-3mm away from the distal joint surface of the femur, paying attention to the peripheral. Successfully drilling a transverse cylindrical bone defect model with the diameter of 6mm and the depth of 1cm, thoroughly scraping periosteum and residual bone tissues at the edge of a hole by using an operation blade, and repeatedly flushing the hole by using an injector containing 20ml of saline water to avoid residual bone fragments. And taking the prepared hydrogel glue solution out of a refrigerator at 4 ℃ for later use. The experimental group rapidly injects the oxygen-enriched temperature-sensitive hydrogel glue solution to the bone defect position by using a 2ml injector to fill the hole, and naturally gelatinizes the glue solution; in the control group, the oxygen-free temperature-sensitive hydrogel glue solution is rapidly injected to the bone defect, the hole is filled, and the gel is formed naturally. The blank set was not filled with any material. The incision was sutured layer by layer and the iodophor re-disinfected the skin margins. After operation, cefuroxime (30 mg/kg) was administered for 3 consecutive days, and the infection was prevented by changing the drug on schedule, and the incision was healed and the suture was dropped after two weeks with regular intake of food.

(3) Material taking: after the operation, after all, after 4, 6, 8, 12 week air embolism method sacrifice the animal, take out the right side thighbone, reject condyle soft tissue carefully, avoid haring the bone defect department callus, cut along femoral shaft and thighbone condyle juncture with the electric saw, remain condyle, arrange 4% paraformaldehyde in and fix.

(4) And (3) detection:

A. gross dissection observation of the specimen: observing the color, texture, metabolic clearance of filling materials and general repair effect of the bone defect.

B. X-ray detection:

the rabbit femoral condyle samples are subjected to X-ray detection (radiation conditions: 50kV, 320mA and 7.5 mS), the change of bone density shadow of the bone defect area is observed, and the healing conditions of the bone defects of all groups are evaluated and compared.

C. And (3) Micro-CT detection: and (3) carrying out Micro-CT detection on the bone defect region row (scanning parameters: 14 mu m in scanning resolution, 360 degrees in rotation angle, 0.4 degree in rotation angle increment, 80kV in voltage, 80 mu A in current and 3000ms in exposure time), carrying out three-dimensional reconstruction by using Micview V2.1.2 three-dimensional reconstruction processing software and ABA special skeleton analysis software, and observing the shape, the quantity and the density of the trabeculae. And further evaluating and comparing the repairing effect of each group of bone defect areas according to the indexes of bone density (TMD), bone mass integral number (BVF), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N) and Structural Model Index (SMI) of the bone defect repairing areas.

(4) Detecting tissue morphology: after rapid decalcification of a bone sample by hydrochloric acid (HCL), HE and Masson staining is carried out on a bone defect area, and the tissue morphological structure of the bone defect area is observed; and (3) carrying out CD31 immunohistochemical staining on the bone defect repair area after decalcification for 2 months by using an EDTA solution, observing and comparing the number of new micro blood vessels in each group of bone defect area, wherein positive staining is brown staining and is positioned in vascular endothelial cells, endothelial cell clusters which are separated from adjacent capillary vessels and are stained and positively expressed are counted as blood vessels, analyzing the blood vessel staining result by referring to a proofreading and counting method such as Chalkley and the like, and repeatedly counting 3 times for each sample to obtain an average value.

(5) The statistical method comprises the following steps: all the measurement data are expressed by +/-s, statistical analysis is carried out by adopting SPSS21.0 software, and if the sample data obeys normal distribution and the variances are uniform, analysis of variance (one-way ANOVA) of a plurality of independent samples is adopted; if the overall variance of the samples is not uniform, a rank-sum test (Kruskai-wallis A test) of multiple independent samples is used, and when the samples are statistically different, the LSD method is used for pairwise comparison. The statistical result is statistically significant with P < 0.05.

2. The experimental results are as follows:

(1) general observations were:

4 weeks after surgery: the blank group has obvious cavities at the bone defects, dark red old blood scabs can be seen in the defects, and no bone defect repair signs are seen; the control group has obvious bone defect, partial metabolism of the filler is eliminated, and the boundary with the bone is clear; the experimental group shows that the bone defect is obvious, most of the fillers are removed in metabolism, and the bone boundary is not clear.

6 weeks after surgery: obvious bone defect can be seen in the blank group, and fibrous tissue can grow into the periphery of the defect wall; the filler in the control group is almost completely metabolized and removed, new bones can be seen at the defect, the semitransparent fibrocartilage on the surface is covered, and cortex defect can still be seen; the experimental group shows that the filler is completely metabolized and removed, the new bone hyperplasia is obvious, the callus is slightly protruded, and no obvious defect exists.

8 weeks after surgery: the blank group of fibrous tissues grows into the bone defect, the pressing is soft, the needle tip can be easily inserted into the defect area, no obvious impedance exists, and no substantial new bone growth is seen on the surface; the control group had new bone growth at the defect, and tiny defect was still seen in cortex; in the experimental group, the bone defect is completely filled by the new bone, and the surface of the cortical bone is rough.

12 weeks after surgery: obvious fibrous tissue hyperplasia can be seen in the blank group, and obvious substantial new bone generation is not seen due to slight resistance to pressing; the bone defect of the control group is completely filled with new bone, the new cortex is thin and slightly protruded, the surface is dark red and hard, and the boundary with normal bone is obvious; the experimental group has the advantages of complete cortex, flat and smooth surface, hard texture and no obvious boundary with normal bone after the bone defect is repaired.

(2) X-ray examination results: at week 4, the X-ray pictures showed that the low density images of the bone defect regions of 3 groups were all obvious, and the newly increased high density images of the bone defect edges of the experimental groups were seen, as shown in fig. 8, 9 and 10. 6. At 8 weeks, the blank group had insignificant decrease in low density shadow over time; the density of the bone defect of the control group gradually increased along with the time; the high density image of the round bone defect of the experimental group is increased rapidly. At week 12, a round low-density image was still visible at the bone defect site of the blank group; fuzzy low-density shadow can be seen at the bone defect part of the control group; the density image of the bone defect of the experimental group has no obvious difference with the density of the surrounding cancellous bone, and is shown in figure 11, figure 12 and figure 13.

Micro-CT imaging results: FIG. 14 is a three-dimensional Micro-CT image at week 4, FIG. 15 is a pseudo-color Micro-CT image at week 4, and FIG. 16 is a two-dimensional Micro-CT image at week 4, wherein a is an experimental group, b is a control group, and c is a blank group. As can be seen from the figure, at 4 weeks, the blank group had few trabeculae formed, and the new trabeculae were tiny and not connected with each other, and the three-dimensional reconstruction showed obvious cavities; a little new bone trabecula is formed in the control group, the new bone trabecula is tiny and is connected with each other, and the three-dimensional reconstruction shows that the bone defect cavity is obvious; the experimental group has a little new bone formation, compared with the control group, the new bone trabecula is thinner and is connected with each other, the three-dimensional reconstruction shows that the cortical bone is not completely repaired, and the cavity is obviously reduced.

FIG. 17 is a three-dimensional Micro-CT image at week 6, FIG. 18 is a pseudo-color Micro-CT image at week 6, and FIG. 19 is a two-dimensional Micro-CT image at week 6, wherein a is an experimental group, b is a control group, and c is a blank group. At 6 weeks, a little new bone trabecula is formed in the blank group, and the three-dimensional reconstruction shows that the cavity is obvious; when the contrast group is in 4 weeks, the number of new bone trabeculae is increased, the number of thin connections of the bone trabeculae is increased, and the three-dimensional reconstruction shows that the cortical bone is discontinuous; compared with the experimental group at 4 weeks, the number of new bone trabeculae is obviously increased, the bone trabeculae are thickened, the number of connections is increased, the three-dimensional reconstruction shows that the cortical bone is incomplete, but the repair condition is better than that of the control group.

FIG. 20 is a three-dimensional Micro-CT image at week 8, FIG. 21 is a pseudo-color Micro-CT image at week 8, and FIG. 22 is a two-dimensional Micro-CT image at week 8, wherein a is an experimental group, b is a control group, and c is a blank group. At 8 weeks, a blank group is formed by new bone trabeculae, the number of new bone trabeculae is slightly increased compared with 6 weeks, the density of the bone trabeculae is poor, the cortex defect is shown by three-dimensional reconstruction, and the cavity is obvious; when the contrast group is 6 weeks later, the number of new bone trabeculae is continuously increased and thickened, the density of the bone trabeculae is increased, and the three-dimensional reconstruction shows that the cortex of the defect part is sunken; compared with the experimental group at 6 weeks, the number and the density of the new bone trabeculae are obviously increased and are mutually connected, the three-dimensional reconstruction shows that the cortex of the bone is continuous, a few of punctiform defects exist, and the repairing effect is better than that of the control group.

FIG. 23 is a three-dimensional Micro-CT image at week 12, FIG. 24 is a pseudo-color Micro-CT image at week 12, and FIG. 25 is a two-dimensional Micro-CT image at week 12, wherein a is an experimental group, b is a control group, and c is a blank group. In 12 weeks, the bone defects in the blank group are not completely repaired, and cavities still exist; the density of the trabecular bone of the control group is obviously increased, the boundary with the peripheral normal trabecular bone is fuzzy, and the new cortex is slightly protruded and continuous; the trabecular bone of the experimental group is thickened, the number and the density of the trabecular bone have no obvious boundary with the surrounding normal trabecular bone, and the cortex lycii radicis is well repaired.

Analysis result of Micro-CT data: fig. 26 TMD analysis chart, fig. 27 BVF analysis chart, fig. 28 tb.n analysis chart, fig. 29 tb.th analysis chart, and fig. 30 SMI analysis chart; as can be seen from the figure, the statistical analysis results show that: three groups of samples at each time point were statistically different (P < 0.05) in bone density (TMD), bone mass integral (BVF), trabecular thickness (tb.th), trabecular number (tb.n) at

weeks

4, 6, 8, 12, and experimental > control > blank. On the aspect of Structural Model Index (SMI), no obvious statistical difference exists between the three groups of data (P is more than 0.05) at the 4 th and 6 th weeks, and the SMI value tends to 3, which indicates that the three groups of new bone trabeculae are all in the shape of young rods; at 8 and 12 weeks, the data of the three groups are statistically different (P < 0.05), and the SMI value of the blank group tends to 3 while the SMI value of the control group and the SMI value of the experimental group tend to 0, which indicates that the new bone trabecula of the blank group is still in the shape of a juvenile rod, while the new bone trabecula of the control group and the experimental group are more in the shape of a mature plate, but the experimental group is superior to the control group.

5. And (3) detecting the tissue morphology: fig. 31 shows the tissue morphology of the experimental group at 4 weeks, fig. 32 shows the tissue morphology of the control group at 4 weeks, and fig. 33 shows the tissue morphology of the blank group at 4 weeks, wherein 1 shows HE staining and 2 shows Masson staining. Fig. 34 is a photograph of CD31 immunohistochemistry at 4 weeks, wherein a is an experimental group, b is a control group, and c is a blank group. As can be seen, at 4 weeks, the blank bone defect area HE staining can show blood scab, contains a large amount of red blood cells and a few inflammatory cells, and immunohistochemistry shows that the new blood vessels are not obvious; the fiber tissue stained by Masson can be seen, and no new bone cell, no new bone formation and no bone cell can be seen in the defect area. The control group HE staining is sparse in visible materials, most of metabolism is eliminated, inflammatory cells penetrate into the filling material, the periphery of the filling material is filled with mesenchymal tissues, and a small amount of new blood vessels are shown by immunohistochemistry; little young new bone scattered in blue is appeared on the edge of the defect area seen by Masson staining, light green collagen is secreted around bone cells in the bone pit, the metabolism is active, and a little osteoblasts are seen. Most of the material is removed in metabolism when HE staining is seen in an experimental group, inflammatory cells penetrate into a gel material, the periphery of new bone is filled with mesenchyme, and more new blood vessels are shown in an immunohistochemical mode compared with a control group; masson staining can show that more blue-stained new and young bones are generated at the edge of the bone defect area, light green new and young collagen is secreted around visible bone cells in the bone pit, the metabolism is active, and a large number of osteoblasts are visible.

Fig. 35 shows the tissue morphology of the experimental group at week 6, fig. 36 shows the tissue morphology of the control group at week 6, and fig. 37 shows the tissue morphology of the blank group at week 6, wherein 1 is HE staining and 2 is Masson staining. Fig. 38 is a photograph of CD31 immunohistochemistry at 6 weeks, in which a is an experimental group, b is a control group, and c is a blank group. As can be seen, at 6 weeks, the blank group of HE staining shows the major organization and absorption of the blood crust, a small amount of red blood cells are seen, the periphery is filled with mesenchyme, and the immunohistochemistry shows a small amount of new blood vessels; the Masson staining can see light blue staining fibrous tissues, a small amount of isolated blue staining immature new bone trabeculae exist at the edge of a bone defect area, bone cells can be seen in a bone pit, light green new collagen can be seen around the cells, the metabolism is active, and a small amount of osteoblasts can be seen. The control group HE staining shows that the filling material is almost completely metabolized and removed, inflammatory cells are obviously reduced compared with 4 weeks, mesenchymal tissues are filled around the filling material, and more new vessels can be seen in immunohistochemistry compared with a blank group; little bluish hyperplastic fibrous tissue and some isolated blue-stained new bone trabeculae can be seen by Masson staining, and more osteoblasts can be seen as the collagen is secreted by the osteocyte in the bone caved-in fossa compared with the blank group. The HE staining of the experimental group shows that the filling material is completely metabolized and removed, a small amount of inflammatory cells are scattered, the rest is filled by the mesenchyme, and the quantity of the new blood vessels is more in the immunohistochemistry than that in the control group; the Masson staining shows that red and blue alternate slightly mature trabeculae of bones exist at the periphery of the bone defect, parts of the trabeculae of bones are connected with each other, osteoblasts in the bone pit secrete fresh collagen, and more osteoblasts are visible.

FIG. 39 shows the tissue morphology of the experimental group at 8 weeks, FIG. 40 shows the tissue morphology of the control group at 8 weeks, and FIG. 41 shows the tissue morphology of the blank group at 8 weeks, wherein 1 shows HE staining and 2 shows Masson staining. Fig. 42 is a photograph of CD31 immunohistochemistry at 8 weeks, wherein a is an experimental group, b is a control group, and c is a blank group. As can be seen, the blank group HE staining shows that the blood scab is completely absorbed and scattered in a large number of fiber cells at 8 weeks, and the immunohistochemistry shows a small amount of new blood vessels which are not obviously increased compared with 6 weeks; the Masson dyeing can see light blue reticular proliferative fibers, the edges of the bone defect area are scattered in red and blue phases to slightly mature trabeculae, the number is increased in 6 weeks, and a small amount of the fibers are connected with each other. The control group HE staining shows that the filling material is completely metabolized and removed, the trabeculae are filled with mesenchyme, and the immunohistochemistry shows that the number of new vessels is increased compared with 6 weeks; masson staining can see that red and blue are alternated in the bone defect area to slightly mature trabeculae, parts are mutually connected, and the number of blank groups is large. The experimental group HE staining shows that the filling material is completely metabolized and removed, the mesenchymal tissue is filled around the filling material, more new vessels are shown by immunohistochemistry, and the number of new vessels is increased compared with 6 weeks; masson staining can see that most of the bone defect areas are red-stained mature plate-shaped trabeculae, which are large in number and thick in shape and are connected with each other mostly.

Fig. 43 shows the tissue morphology of the experimental group at 12 weeks, fig. 44 shows the tissue morphology of the control group at 12 weeks, and fig. 45 shows the tissue morphology of the blank group at 12 weeks, wherein 1 shows HE staining and 2 shows Masson staining. Fig. 46 is a photograph of CD31 immunohistochemistry at 12 weeks, wherein a is the experimental group, b is the control group, and c is the blank group. As can be seen, at 12 weeks, the blank group HE staining shows a large amount of fibrocyte and reticular fibroplasia, and immunohistochemistry shows only a small amount of new vessels; massive blue-stained tiny reticular proliferative fibers can be seen by Masson staining, the blue-stained tiny reticular proliferative fibers are scattered among red and blue phases and slightly mature trabeculae, the number of the mature trabeculae is increased in 8 weeks, the mature trabeculae are respectively connected with each other, and a small amount of light green nascent collagen exists around osteoblasts in a bone pit. The HE staining of the control group can be used for completely removing the metabolism of visible materials, and the immunohistochemistry shows more new blood vessels; masson can see blue dyeing reticular fibers, more red and blue are alternated, the trabecula is slightly mature, part of the trabecula is in a plate layer shape, and a small amount of light green new collagen exists around osteoblasts in the bone pit. The complete metabolic clearance of the filling material can be seen by HE staining of an experimental group, and a large amount of new blood vessels can be shown by immunohistochemistry; massive red staining of plate-shaped mature trabeculae can be seen by Masson staining, the trabeculae are thick, mature bone cells can be seen in bone pits, and light green collagen newly secreted around the cells does not exist.

CD31 immunohistochemical microvascular count results: FIG. 47 is a statistical chart of the counts of CD31 immunohistochemical microvessels, showing that the results of the statistical analysis: in terms of the neovascular counts in the bone defect regions, three groups of samples at each time point were statistically different (P < 0.05) at 4 th, 6 th, 8 th, 12 th week 4 time points, and the experimental group > control group > blank group.

Claims

1. An oxygen-enriched temperature-sensitive hydrogel is characterized in that: the emulsion is formed by mixing a CS acid solution, a beta-GP solution, an HEC solution and a penfluron emulsion; the content of the panflurron in the hydrogel is 5.0vol% to 15.0 vol%.

2. An oxygen-rich temperature-sensitive hydrogel as claimed in claim 1, wherein the hydrogel comprises the following components: CS 0.010-0.015 g/ml, beta-GP 0.015-0.020 g/ml and HEC 0.003-0.007 g/ml.

3. A preparation method of oxygen-enriched temperature-sensitive hydrogel adopts the component proportion of claim 1, and is characterized in that: and (3) mixing the CS acid solution, the beta-GP solution, the HEC solution and the penfluron emulsion, and fully mixing under the ice bath condition to obtain the hydrogel glue solution.

4. The preparation method of the oxygen-rich temperature-sensitive hydrogel according to claim 3, wherein the preparation method comprises the following steps: the CS acid solution is prepared from CS powder and a hydrochloric acid solution or an acetic acid solution, and the CS content in the CS acid solution is 0.010-0.050 g/ml.

5. The preparation method of the oxygen-rich temperature-sensitive hydrogel according to claim 3, wherein the preparation method comprises the following steps: the beta-GP solution is prepared from beta-GP powder and water, and the content of the beta-GP is 0.10-0.25 g/ml.

6. The preparation method of the oxygen-rich temperature-sensitive hydrogel according to claim 3, wherein the preparation method comprises the following steps: the HEC solution is prepared from HEC powder and water, and the content of HEC is 0.01-0.06 g/ml.

7. An oxygen-rich temperature-sensitive hydrogel preparation method as claimed in any one of claims 3 to 6, wherein the preparation process of the emulsion of the panflurron is as follows: adding egg yolk lecithin into Tyrode salt buffer solution or PBS salt buffer solution, mixing and emulsifying to obtain basic emulsion; adding a penfluron stock solution into the basic emulsion, mixing and emulsifying to obtain an emulsion; and oxygenating the emulsion in a high-pressure oxygen environment to obtain the penfluron emulsion.

8. The preparation method of an oxygen-rich temperature-sensitive hydrogel according to claim 7, wherein the content of the perfluoron in the perfluoron emulsion is 10-45 vol%.

9. The method for preparing an oxygen-enriched temperature-sensitive hydrogel according to claim 7, wherein 80-110 mg of egg yolk lecithin is added into 550-900 μ L of Tyrode salt buffer solution or PBS salt buffer solution.

10. The method for preparing an oxygen-enriched temperature-sensitive hydrogel according to claim 7, wherein ultrasonic emulsification is adopted for the emulsification.