CN116772523B - Freeze-drying method of collagen sponge - Google Patents
Freeze-drying method of collagen sponge Download PDFInfo
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Abstract
The invention relates to a freeze-drying method of collagen sponge, which specifically comprises the following steps: after the animal-derived collagen is extracted and purified, preparing a collagen solution with the concentration of 0.1% -0.6%, and preprocessing the obtained collagen solution, wherein the preprocessing steps are as follows: after the collagen solution is filled into a mould or a freeze-drying plate, carrying out cyclic vacuumizing treatment in a freeze dryer, wherein the cyclic vacuumizing procedure is vacuumizing-air inlet normal pressure-vacuumizing, the cyclic vacuumizing times are 2-5 times, the vacuum degree during vacuumizing is-25 to-45 Pa, the holding time during vacuumizing is 30-90 min/each time, and the set temperature during vacuumizing is 5-15 ℃; and freeze-drying the pretreated collagen solution. The collagen sponge prepared by the freeze-drying process has stable biological activity, more uniform pores, better hydrophilicity and good hemostatic effect in clinical use.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a freeze-drying method of collagen sponge.
Background
Collagen is the most abundant protein in human body, accounting for 25% -30% of the protein content in human body, the collagen is mainly used for wound hemostasis, subcutaneous tissue filling and the like in the field of medical instruments, the main product form of the collagen for intraoperative hemostasis is spongy, and the collagen sponge has good water absorption and liquid absorption properties, can absorb a large amount of water in blood, and forms scab to block blood vessels; and simultaneously, after the angiogenesis injury, collagen can be combined with platelet receptors, so that the adhesion aggregation of platelets is promoted, and then an endogenous hemostasis mechanism of a human body is started, and the two organic combinations realize the rapid hemostasis process of the wound. Research shows that collagen can promote migration of fibroblasts and macrophages, stimulate regeneration of collagen and clearance of cell debris in an organism, and promote repair and healing of tissues. The collagen sponge can effectively promote the healing of wounds while playing a good hemostatic property, so that the collagen sponge has a good application prospect in clinical hemostasis.
The forms of the hemostatic products of collagen are various, wherein the collagen sponge is paid attention to due to the strong mechanical properties, and the existing preparation method of the collagen sponge comprises the following steps: the collagen from animal sources is extracted by an acid enzyme combination method, and then is subjected to salting-out purification to obtain collagen solution with higher purity, and the collagen solution is obtained by a freeze-drying process.
The freeze-drying process is to pre-freeze the collagen solution, and as the temperature decreases, the solution forms ice crystals, and simultaneously, the collagen solution is subjected to negative pressure vacuum drying treatment, and in the drying process, the ice crystals are gradually sublimated to enable the inside of the collagen to be porous. The parameter conditions of the freeze-drying process have great influence on the performance of the collagen, and the problems commonly existing at present are that the collagen sponge obtained in the freeze-drying process has poor water absorption and poor hemostatic effect in the clinical application process.
For example, chinese patent No. CN1416907a discloses a method for preparing a medical collagen sponge, wherein the freeze-drying process is as follows: freezing at-10 to-80 deg.c for 1-5 days. The above method has a large temperature variation range in the freezing process, and is easy to cause the nucleation and growth of ice crystals to become uneven, so that the pore size of the formed collagen sponge is uneven, thereby affecting the activity of cells and the capability of the cells to generate uniform extracellular matrixes. At the same time, glycerol is also required.
Chinese patent No. CN104922732B relates to a preparation method of oral cavity biomembrane, which comprises adding type I collagen into acetic acid solution to prepare collagen acetic acid swelling solution; adding chondroitin sulfate into the mixture, and stirring the mixture to prepare collagen-chondroitin sulfate slurry; vacuum freeze drying; pressing to form a collagen composite film; spraying collagen-chondroitin sulfate slurry into the two layers of collagen composite films, and performing vacuum freeze drying; then high-temperature vacuum crosslinking is carried out; and (3) sterilizing after crosslinking to obtain the collagen-chondroitin sulfate composite oral biological membrane. The collagen-chondroitin sulfate composite membrane adopting the mode of twice vacuum freeze drying is required, and the operation is relatively complex.
US patent US20200121826A1 relates to a high strength collagen sponge comprising the steps of: (1) A step of stirring and degassing a collagen solution obtained by mixing collagen and a solvent; (2) a step of subjecting the collagen solution to a freeze-drying treatment. And (3) a step of insolubilizing the collagen dried product after the freeze-drying treatment. The step (1) comprises the following steps (1-1) and (1-2): (1-1) a step of preparing a collagen solution by adding collagen to a solvent, followed by stirring and degassing treatment; and (1-2) a step of stirring and deaerating the collagen solution injected into the container to reduce bubbles in the collagen solution. The stirring and degassing treatment is performed under a rotational centrifugal force of 1G to 600G, and a rotational centrifugal force of 0.1G to 80G. The degassing treatment efficiency of this patent is relatively low and the effect is not good.
In view of this, it is desirable to provide a new lyophilization process to produce collagen sponge with uniform pores, good water absorption and good hemostatic properties.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a brand-new freeze-drying process, realizes the advantages of simplicity in operation, no freeze-drying protective agent, energy conservation and environmental protection on industrial production, and simultaneously produces the collagen sponge with good hemostatic performance.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a freeze-drying method of collagen sponge, which comprises the following steps:
s1: the animal-derived collagen is prepared into a collagen solution with the mass concentration of 0.1% -0.6% after being extracted and purified;
s2: pretreating the collagen solution obtained in the step S1, wherein the pretreatment steps are as follows:
after the collagen solution is filled into a mould or a freeze-drying disc, carrying out cyclic vacuumizing treatment in a freeze dryer, wherein the cyclic vacuumizing procedure is vacuumizing-air inlet normal pressure-vacuumizing, the number of times of cyclic vacuumizing is 2-5, the vacuum degree during vacuumizing is-25 to-45 Pa, the holding time during vacuumizing is 30-90 min/time, and the temperature during vacuumizing is 5-15 ℃;
S3: and (3) freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, vacuumizing after pre-freezing, and performing the sublimation drying and the analysis drying under vacuum, wherein the specific parameter conditions are as follows:
the pre-freezing temperature is minus 35 ℃ to minus 45 ℃, the cooling rate of the pre-freezing is 1-2 ℃/min, and the pre-freezing time is 1-3 h; the negative pressure vacuum degree of sublimation drying is minus 25 Pa to minus 45Pa; the temperature of sublimation drying is-10 to-25 ℃, and the period of sublimation drying is 12-18 hours; the temperature of the analysis drying is 30-40 ℃, and the time of the analysis drying is 1-3 hours;
the total time of lyophilization, including the time of the pretreatment and the time of the lyophilization, is 14-40 hours.
As a further improvement of the invention, the mass concentration of the collagen solution in the S1 is 0.3% -0.4%.
As a further improvement of the invention, the number of times of circulating vacuumizing in the step S2 is 3, the vacuum degree during vacuumizing is-35 Pa, the holding time during vacuumizing is 60 min/time, and the temperature during vacuumizing is 10 ℃.
As a further improvement of the invention, the pre-freezing temperature is minus 35 ℃, and the pre-freezing cooling rate is 1 ℃/min.
As a further improvement of the invention, the pre-freezing time is 3 hours.
As a further improvement of the invention, the temperature of the sublimation drying is-10 ℃.
As a further improvement of the invention, the period of the sublimation drying is 15 hours.
As a further improvement of the invention, the negative pressure vacuum degree of the sublimation drying under vacuum is-30 Pa.
As a further improvement of the present invention, the temperature of the analytical drying is 30 ℃.
As a further improvement of the invention, the duration of the analytical drying is 1h.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
the freeze-drying method provided by the invention adopts proper freeze-drying parameters, and is matched with reasonable concentration of collagen solution, so that the prepared collagen sponge has more uniform pores, better hydrophilicity and good hemostatic effect in clinical use.
According to the invention, no freeze-drying protective agent is used, and the concentration and parameters are researched, so that the biological activity of the collagen sponge is maintained, the ideal effect is achieved, and the introduction of impurities is avoided.
The process of the invention uses the freeze dryer to carry out bubble removal treatment on the collagen solution, thereby reducing the cost, avoiding homogenization procedures such as stirring, centrifugation and the like, reducing the damage to the collagen structure and protecting the bioactivity of the collagen.
The process fully utilizes the synergistic effect among the processes, and based on the selection of specific process parameters of collagen solution concentration, degassing bubble treatment and freeze drying, the microstructure of the collagen sponge is fully optimized and regulated as a whole, a pore-forming template is not adopted, the uniformity of the pore diameter of the pore-forming template at 100-200 mu m reaches 95%, compared with the uniformity of the common collagen sponge, the uniformity of the pore-forming template is greatly improved, and meanwhile, from the aspect of the flatness of the outer surface, the surface of the collagen sponge prepared by the process has no granular bulges. On the basis of a high-performance microstructure, the collagen sponge has excellent water absorption performance and tensile resistance, and has wide application prospects in various fields. Especially has good hemostatic effect in clinical use.
The process is suitable for industrial application, and the prepared collagen sponge has the advantages of clean appearance, uniform sponge pores and good liquid absorption and hemostasis performances.
Drawings
FIG. 1 is a photograph of a pre-processed portion of the lyophilization process described in example 2;
FIG. 2 is a photograph of a homogenized sample of comparative example 1 during a lyophilization process;
FIG. 3 is a view showing the appearance of a collagen sponge according to the lyophilization process described in example 2;
FIG. 4 is an external view of a collagen sponge of the lyophilization process described in comparative example 1;
FIG. 5 is an electron microscopy image of a collagen sponge of the lyophilization process described in example 2;
FIG. 6 is an electron micrograph of a collagen sponge of the lyophilization process described in comparative example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be clearly and completely described in connection with the following specific embodiments.
Unless otherwise indicated, the equipment used in this embodiment is conventional, the reagents used are conventional, all commercially available, and the methods of operation used are described in textbooks in the field.
EXAMPLE 1 preparation of collagen solution
Cleaning animal origin tissue bovine Achilles tendon, putting into a reaction kettle, adding 0.15% pepsin and 0.5mol/L acetic acid solution, stirring for reaction for 24 hours, and centrifuging to obtain a supernatant to obtain a collagen crude solution; adding saturated sodium chloride solution into the solution, separating out solid collagen, washing with purified water to obtain collagen, dissolving the collagen in 0.5mol/L acetic acid solution, dialyzing to obtain collagen solution, diluting the collagen solution after dialyzing and purifying with buffer salt, and preparing into 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6% collagen solutions respectively.
The viscosities of the collagen solutions at different concentrations at 15 ℃ are shown in the following table:
TABLE 1 example 1 different concentration-viscosity comparison Table (15 ℃ C.)
As can be seen from the above table, the difficulty in homogenization at and above 0.6% concentration is extremely high, while the viscosity at 0.1% concentration is too low, preferably 0.3% -0.5%.
EXAMPLE 2 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the pre-freezing time is 3h, the sublimation drying temperature is-10 ℃, and the sublimation drying period is 15h; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 3 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.4% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 2 times, the vacuum degree is-40 Pa, the maintaining time of each vacuumizing is 50min, and the vacuumizing temperature is 12 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 2 ℃/min, the pre-freezing time is 3h, the sublimation drying temperature is-15 ℃, and the sublimation drying period is 14h; the negative pressure vacuum degree of sublimation drying under vacuum is-25 Pa; the analytic drying temperature is 32 ℃, and the analytic drying time is 2 hours; the total time of lyophilization was 30h.
EXAMPLE 4 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.5% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-45 Pa, the maintaining time of each vacuumizing is 45min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the pre-freezing time is 3h, the sublimation drying temperature is-15 ℃, and the sublimation drying period is 15h; the negative pressure vacuum degree of sublimation drying under vacuum is-40 Pa; the analytic drying temperature is 34 ℃, and the analytic drying time is 1h; the total lyophilization time was 26h.
EXAMPLE 5 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.2% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 6 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.4% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 7 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.5% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 8 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.6% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 9 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 3mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 10 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 8mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 11 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 2 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 12 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the glue solution of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 4 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 13 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 5 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 14 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 45min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 15 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 75min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 16 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-15 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
EXAMPLE 17 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-45 pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 1 preparation of collagen sponge
The collagen solution with the collagen concentration of 0.3% obtained in the example 1 is selected for homogenization treatment, the homogenization rate is 5000rpm, the stirring rate is 40rpm, the vacuum degree is-80 Pa, and the holding time is 60min; after finishing, placing the mixture into a freeze dryer die through a peristaltic pump, wherein the thickness of the collagen solution is 5mm; after the collagen solution is tiled uniformly, starting a freeze dryer, and freeze drying the pretreated collagen solution, wherein the freeze drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after the pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is minus 35 ℃, the cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 28 h.
Comparative example 2 preparation of collagen sponge
The collagen solution with the collagen concentration of 0.4% obtained in example 1 was selected for homogenization treatment, the homogenization rate was 6000rpm, the stirring rate was 50rpm, the vacuum degree was-80 Pa, and the holding time was 50min; after finishing, placing the mixture into a freeze dryer die through a peristaltic pump, wherein the thickness of the collagen solution is 5mm; starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, vacuumizing after pre-freezing, and performing sublimation drying and analysis drying under vacuum, wherein the specific parameter conditions are as follows: the pre-freezing times are 2 times, the pre-freezing temperature is minus 35 ℃, the cooling rate is 2 ℃/min, the 2 times of pre-freezing time are 3 hours respectively, the drying temperature is minus 15 ℃, and the duration of the drying stage is 14 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-25 Pa; the analytic drying temperature is 32 ℃, and the analytic drying time is 2 hours; the total time of lyophilization was 30h.
Comparative example 3 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.05% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 4 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.7% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 5 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 1 time, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 6 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 6 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 7 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 30min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 8 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 90min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 9 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-60 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing speed is 1 ℃/min, the 2 times of pre-freezing time is 3h, the drying temperature is-10 ℃, and the drying period is 15h; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 10 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-5 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 11 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing temperature is-55 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period time is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 12 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is-15 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is-10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 13 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 5 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 14 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 0.5 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 15 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 30 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 16 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 5 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analytic drying temperature is 30 ℃, and the analytic drying time is 1h; the total lyophilization time was 27h.
Comparative example 17 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 Pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analysis drying temperature is 20 ℃, and the analysis drying time is 1h; the total lyophilization time was 27h.
Comparative example 18 preparation of collagen sponge
Placing the collagen solution with the concentration of 0.3% obtained in the example 1 into a mold of a freeze dryer, wherein the thickness of the collagen solution is 5mm; after the flat spreading, repeated vacuumizing treatment is carried out for 3 times, the vacuum degree is-35 pa, the maintaining time of each vacuumizing is 60min, and the vacuumizing temperature is 10 ℃. After removing bubbles in the glue solution, starting a freeze dryer, and freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, and vacuumizing after pre-freezing, and the sublimation drying and the analysis drying are carried out under the vacuum, wherein the specific parameter conditions are as follows: the pre-freezing time is 2 times, the pre-freezing temperature is minus 35 ℃, the pre-freezing cooling rate is 1 ℃/min, the 2 times of pre-freezing time is 3 hours, the drying temperature is minus 10 ℃, and the drying period is 15 hours; the negative pressure vacuum degree of sublimation drying under vacuum is-30 Pa; the analysis drying temperature is 50 ℃, and the analysis drying time is 1h; the total lyophilization time was 27h.
Examples and comparative examples results and characterization thereof:
(1) Test 1: collagen sponge technological process and finished product appearance observation
By observing the processes and products of example 2 and comparative example 1, it can be seen from comparison of fig. 1 and fig. 2 that the surface of the collagen solution subjected to repeated vacuuming treatment by the process is substantially free of bubbles, and fig. 2 is a stock solution treated by a homogenizer, and then injected into a mold of a freeze dryer by a peristaltic pump, and a large amount of bubbles still appear.
Comparison of fig. 3 and fig. 4 shows that the collagen sponge prepared by the process of the present invention has a flat outer surface, and fig. 4 is the collagen sponge obtained in comparative example 1, and has granular ridges on the surface and uneven pore diameters.
(2) Test 2: electron microscopic examination of collagen sponge finished product
The collagen sponges obtained in example 2 and comparative example 1 were subjected to electron microscopy, and as shown in fig. 5 and 6, the collagen sponges obtained in example 2 had a more uniform pore size of 100 to 200 μm; the sponge obtained in comparative example 1 has poor pore diameter uniformity.
(3) Test 3: liquid absorption performance test of collagen sponge finished product
The collagen sponges obtained in examples and comparative examples were subjected to liquid absorption performance test as follows:
square with collagen sponge size of 2cm was taken, the weight was measured and recorded as m1, the square was put into water, the square was gently and completely soaked with fingers, the forceps were held gently and drained on the water surface for 1min, the weight was recorded as m2, the water absorption performance= (m 2-m 1)/m 1, and the specific test method and specific water absorption performance results were shown in tables 2 and 3 below, taking example 2 and comparative example 1 as an example:
TABLE 2 data for testing the Water absorption Properties of collagen sponges obtained in example 2
TABLE 3 data for testing the Water absorption Properties of collagen sponge obtained in comparative example 1
Test data analysis in table 2: 10 times of test data show that the collagen sponge obtained in the example 2 can absorb 48-70 times of water of the weight of the collagen sponge; analysis of test data in table 3: the collagen sponge obtained in comparative example 1 can absorb 27-41 times of water of its own weight; therefore, the collagen sponge finished product prepared by the freeze-drying process has good water absorption performance.
The average value of the water absorption performance test data of the collagen sponge of all the examples and the comparative examples in the invention is as follows:
table 4 average value of data for testing water absorption properties of collagen sponge obtained in examples and comparative examples
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(4) Test 4: collagen sponge tensile property test
The collagen sponges obtained in the examples and comparative examples were subjected to tensile property test, the sponges were formed into 1cm wide strips, and a tensile force of 0.5N was applied thereto for 1min without breaking, and the test results are shown in Table 5 below:
TABLE 5 collagen sponge tensile test results
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The collagen sponge prepared by the process has high tensile property, and bubbles in the production process of the comparative example lead to poor tensile property of the final product.
(5) Test 5: hemostatic property test of collagen sponge finished product
The collagen sponge obtained in example 2 was subjected to a hemostatic performance test as follows:
A small pig is used as an animal model and is divided into a collagen sponge group and a gauze control group; after the skin preparation of the experimental animal, the femoral artery/jugular vein is exposed, an incision with the length of 1cm is made, and after the incision is sutured, the experimental animal is free to bleed for 2 seconds, thus completing the model establishment. Then covering the bleeding points with collagen sponge and hemostatic gauze, and recording the hemostatic time and the hemostatic amount; the specific data are as follows:
table 6 collagen sponge hemostatic Performance test data
As can be seen from the experimental result data, compared with the gauze control group, the collagen sponge group has obviously reduced hemostatic time and obviously reduced bleeding amount; the collagen sponge produced by the process has better hemostatic performance.
The hemostatic properties of the collagen sponges of all examples and comparative examples in the present invention are as follows:
table 7 hemostatic performance data for collagen sponges of examples and comparative examples
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Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art may modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A method for freeze-drying collagen sponge, which is characterized by comprising the following steps:
s1: the animal-derived collagen is prepared into a collagen solution with the mass concentration of 0.3% -0.4% after being extracted and purified;
s2: the collagen solution obtained in the step S1 is pretreated, and the pretreatment steps are as follows:
after the collagen solution is filled into a mould or a freeze-drying disc, carrying out cyclic vacuumizing treatment in a freeze dryer, wherein the cyclic vacuumizing procedure is vacuumizing-air inlet normal pressure-vacuumizing, the number of times of cyclic vacuumizing is 3, the vacuum degree during vacuumizing is-35 Pa, the holding time during vacuumizing is 60 min/time, and the temperature during vacuumizing is 10 ℃;
s3: and (3) freeze-drying the pretreated collagen solution, wherein the freeze-drying comprises pre-freezing, sublimation drying and analysis drying, vacuumizing after pre-freezing, and performing the sublimation drying and the analysis drying under vacuum, wherein the specific parameter conditions are as follows:
the pre-freezing temperature is-35 ℃ to-45 ℃, the pre-freezing cooling rate is 1-2 ℃/min, and the pre-freezing time is 1-3 h; the negative pressure vacuum degree of the sublimation drying is minus 25 Pa to minus 45Pa; the temperature of sublimation drying is-10 to-25 ℃, and the period of the sublimation drying is 12-18 hours; the temperature of the analytic drying is 30-40 ℃, and the time of the analytic drying is 1-3 h;
The total time of lyophilization, including the time of the pretreatment and the time of the lyophilization, is 15-40 hours.
2. The method for lyophilizing a collagen sponge according to claim 1, wherein the pre-frozen temperature is-35 ℃, and the pre-frozen temperature-reducing rate is 1 ℃/min.
3. The method of claim 1, wherein the pre-freezing time is 3 hours.
4. The method of claim 1, wherein the temperature of sublimation drying is-10 ℃.
5. The method of claim 1, wherein the sublimation drying period is 15 hours.
6. The method for lyophilizing a collagen sponge according to claim 1, wherein the negative pressure vacuum degree of the sublimation drying under vacuum is-30 Pa.
7. The method of claim 1, wherein the temperature of the analytical drying is 30 ℃.
8. The method of claim 1, wherein the analytical drying is performed for a period of 1h.
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