Disclosure of Invention
The invention provides a lubricating liquid containing recombinant type III human collagen, a filling agent and application thereof for overcoming the defects, and aims to solve the problem that the prior art cannot quickly repair damage of human tissues caused by crosslinking sodium hyaluronate gel in the injection process.
In order to achieve the aim of the invention, the invention is realized by the following technical scheme:
a lubricating fluid containing recombinant III type human collagen,
the sodium hyaluronate gel lubricating liquid comprises recombinant type III human collagen.
Some existing studies indicate that in normal skin tissue, collagen exists mainly in the form of I, III type collagen fibers. Among them, type III collagen is closely related to the skin injury repair process and repair quality, and in general, the higher the content of type III collagen, the stronger the repair ability to skin tissues. Type III collagen accounts for 60% of normal infant skin, and as the infant grows, type III collagen is continuously reduced and type I collagen is continuously increased. Therefore, increasing the content of type III collagen at the sodium hyaluronate gel injection section is a great help to shorten the wound healing time.
The invention is different from the prior art in that the lubricating substance selected in the sodium hyaluronate gel lubricating liquid is not conventional non-crosslinked sodium hyaluronate, but recombinant type III human collagen. Compared with non-crosslinked sodium hyaluronate, the recombinant human Collagen (Reallagen) also called RHC (Recombinant Human-source Collagen) is a high-molecular biological protein produced by a microorganism (such as active yeast) high-density fermentation and green separation and purification process according to the structural characteristics of the human Collagen and by utilizing advanced biological science technology and international leading fermentation technology, and is highly similar to the human Collagen. Therefore, the invention can effectively supplement the III type collagen in the skin by adding the recombinant III type human collagen into the lubricating liquid.
Therefore, after the lubricating fluid containing the recombinant type III human collagen is compounded with the sodium hyaluronate gel particles, the effects of repairing the appearance and correcting the contour by injecting and filling the micro-crosslinked sodium hyaluronate gel and the dermal tissue can be achieved, the skin regeneration speed can be effectively improved, the wound healing time is shortened, the skin barrier function is recovered, and the product quality is improved
Preferably, it is obtained by dissolving recombinant type III human collagen in a buffer solution.
The preparation method of the lubricating fluid containing the recombinant type III human collagen is simple, and the lubricating fluid can be obtained by only dissolving the recombinant type III human collagen in a buffer solution according to the measured parts, so that the subsequent use and operation are simplified.
Preferably, the buffer solution contains sodium chloride, a phosphate buffer system and water for injection.
Preferably, the buffer solution contains 0.7% -1.0% sodium chloride, 0.56% disodium hydrogen phosphate, 0.04% sodium dihydrogen phosphate and water for injection.
Preferably, the concentration of the recombinant type III human collagen in the lubricating liquid is 15-24 mg/ml.
The concentration of the recombinant type III human collagen in the lubricating fluid has a great influence on the actual performance of the lubricating fluid.
When the concentration of the recombinant III type humanized collagen is lower than 15mg/ml, the skin repairing effect is not obvious through practical tests, and meanwhile, the pushing force of the lubricating liquid is lower, so that the hand feeling of injection is avoided;
when the concentration of the recombinant III type humanized collagen is higher than 24mg/ml, the pushing force in the injection process is greatly improved, the injection is not facilitated, and meanwhile, the cost of the product is greatly improved.
When the concentration of the recombinant type III human collagen is 15-24 mg/ml, the skin repair effect and the injection pushing force can be considered, and the pushing force of the product can be at a proper level (10-20N), so that the hand feeling in the injection process is better.
A sodium hyaluronate gel filler,
comprises crosslinked sodium hyaluronate gel particles; the method comprises the steps of,
a lubricating fluid comprising recombinant type III human collagen as described above.
Preferably, the preparation method of the sodium hyaluronate gel filler comprises the following steps:
(1) Dissolving: completely dissolving sodium hyaluronate raw material with sodium hydroxide solution;
(2) Crosslinking reaction: adding a cross-linking agent into the sodium hyaluronate dissolved in the step (1), uniformly mixing, and carrying out heat preservation in a water bath for cross-linking reaction to obtain micro-crosslinked sodium hyaluronate gel;
(3) Preparing an eluent: preparing an eluent containing sodium chloride and a phosphate buffer system for later use;
(4) Preparing a lubricant: preparing a lubricating liquid containing recombinant type III human collagen, wherein other components of the lubricating agent comprise sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate;
(5) Eluting: eluting the micro-crosslinked gel prepared in the step (2) by using the eluent prepared in the step (3), dispersing the micro-crosslinked gel into particles with the size of 0.5-2 cm in the process, and simultaneously replacing the eluent until the micro-crosslinked sodium hyaluronate gel swells to the required weight;
(6) Homogenizing: homogenizing the micro-crosslinked sodium hyaluronate gel subjected to the dialysis in the step (5) to prepare particles with the particle diameter of 150-400 mu m;
(7) And (3) sterilization: carrying out wet heat sterilization on the micro-crosslinked sodium hyaluronate gel particles homogenized in the step (6) by pure steam to achieve a sterile state;
(8) Sterile mixing: mixing the homogenized micro-crosslinked sodium hyaluronate gel particles in the step (7) with the lubricant prepared in the step (4) according to a certain proportion;
(9) And (3) filling: uniformly mixing the micro-crosslinked sodium hyaluronate gel particles prepared in the step (8) with a lubricant containing recombinant type III human collagen to obtain the micro-crosslinked sodium hyaluronate gel containing recombinant type III human collagen, and filling the micro-crosslinked sodium hyaluronate gel into a pre-filling and sealing syringe.
Preferably, in the step (1), the sodium hyaluronate raw material is prepared by a fermentation method or an extraction method, and the molecular weight is 120-230 ten thousand.
Preferably, in the step (1), the concentration of the sodium hydroxide-containing solution is 0.85% to 1.25%.
Preferably, in the step (2), the crosslinking agent is one or a mixture of two or more of 1, 4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), polyethylene glycol, genipin, and carbodiimide.
Preferably, in the step (2), the crosslinking reaction temperature is 35 ℃ to 58 ℃.
Preferably, in the step (2), the crosslinking reaction is carried out for a period of time of 60 to 150 minutes.
Preferably, in the step (2), the mass ratio of the cross-linking agent to the sodium hyaluronate raw material is 0.05-0.12.
Preferably, in the step (3), the content of sodium chloride in the eluent is 0.7% -1.0%.
Preferably, in the step (4), the concentration of the recombinant type III human collagen in the lubricant is 15-24 mg/ml.
Preferably, the mass of the eluent is 50-80 times of the gel mass after the crosslinking is completed.
Preferably, the first elution time in the eluting step is 2-4 hours, and the total eluting times are 3-6 times.
Preferably, in the step (5), the concentration of the sodium hyaluronate at the elution end point is 5mg/ml to 8mg/ml.
Preferably, in the step (6), the particle diameter of the micro-crosslinked sodium hyaluronate gel after being homogenized is 150-400 μm.
Preferably, in the step (6), the homogenization frequency of the micro-crosslinked sodium hyaluronate gel is 2-4 times.
Preferably, in the step (7), the sterilization temperature of the micro-crosslinked sodium hyaluronate gel is 115 ℃ to 125 ℃.
Preferably, in the step (7), the sterilization F0 value of the micro-crosslinked sodium hyaluronate gel is 8 to 12.
Preferably, in the step (8), the mass ratio of the crosslinked sodium hyaluronate gel particles to the lubricating liquid is (1 to 10): 1.
therefore, the invention has the following beneficial effects:
according to the invention, through designing reaction conditions and controlling the degree of crosslinking reaction of HA and BDDE, a low-crosslinking-degree sodium hyaluronate gel is obtained, and then a homogenization-sterilization process is carried out to obtain a sterile micro-crosslinking sodium hyaluronate gel, which is mixed with a lubricant of recombinant type III human collagen tissue to obtain the micro-crosslinking sodium hyaluronate gel containing recombinant type III human collagen. Because the final product has proper rheology and low viscoelastic performance, the micro-crosslinked sodium hyaluronate gel injection filling and dermal tissue are used for repairing the appearance and correcting the contour so as to achieve satisfactory effects, and meanwhile, the recombinant type III human collagen with high bioactivity can effectively improve the skin regeneration speed and shorten the wound healing time, thereby recovering the skin barrier function and improving the product quality.
Detailed Description
The invention is further described below in connection with specific embodiments. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
Description of the invention
1: the sodium hyaluronate raw material selected in the following examples was produced by fermentation and was the same batch.
2: the crosslinker used in the following examples was 1, 4-butanediol diglycidyl ether (BDDE)
3: the examples are not limited to the raw materials prepared by the fermentation method, but the effectiveness and versatility of the present invention will also be illustrated by taking the raw materials prepared by the extraction method as an example.
3: the recombinant type III human collagen raw material selected in the following examples is a freeze-dried sponge produced by a fermentation method, and is the same batch.
Example 1
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 5g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing a lubricant with the concentration of 20mg/ml for later use.
50ml of 1.2% sodium hydroxide solution was prepared, and 5g of sodium hyaluronate raw material having a molecular weight of 180 ten thousand was added thereto and stirred to dissolve until no white undissolved HA was visible to the naked eye. After dissolution, 0.35ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 48 ℃, placing the gel into the water bath, and preserving the heat for 90min. And taking out the gel after crosslinking, adding 3.5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 3.5L of eluent after the second elution is finished, and adding 3.5L of eluent again for the third elution until the final weight of the gel is 1kg, stopping the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 5mg/ml after the time of 6 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 90g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 10g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant =9:1, and finally a micro-crosslinked sodium hyaluronate gel containing recombinant type III human collagen was obtained, and filled into 1.0ml pre-filled syringes, sample No. a.
Example 2
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 5g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing a lubricant with the concentration of 20mg/ml for later use.
50ml of 1.2% sodium hydroxide solution was prepared, and 5g of sodium hyaluronate raw material having a molecular weight of 230 ten thousand was added thereto and stirred to dissolve until no white undissolved HA was visible to the naked eye. After dissolution, 0.60ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 55 ℃, placing the gel into the water bath, and preserving the heat for 60min. And taking out the gel after crosslinking, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is finished, and adding 5L of eluent again to perform the third elution until the final weight of the gel is 1kg, stopping the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 5mg/ml after 10 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 80g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 20g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant And (2) obtaining the micro-crosslinked sodium hyaluronate gel containing the recombinant type III human collagen, filling the micro-crosslinked sodium hyaluronate gel into a 1.0ml pre-filling and sealing syringe, and obtaining a sample number B.
Example 3
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 5g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing a lubricant with the concentration of 20mg/ml for later use.
50ml of 1.0% strength sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 150 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.40ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 50 ℃, placing the gel into the water bath, and preserving the heat for 120min. Will be crosslinked completelyTaking out the gel, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is finished, and adding 5L of eluent again to perform the third elution until the final weight of the gel is 0.62kg, and stopping the elution until the final weight of the gel reaches the end point of the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 8mg/ml after 6 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 80g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 20g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant And (2) obtaining the micro-crosslinked sodium hyaluronate gel containing the recombinant type III human collagen, filling the micro-crosslinked sodium hyaluronate gel into a 1.0ml pre-filling and sealing syringe, and obtaining a sample number C.
Example 4
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 5g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing a lubricant with the concentration of 20mg/ml for later use.
50ml of 1.0% strength sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 150 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.40ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 50 ℃, placing the gel into the water bath, and preserving the heat for 120min. And taking out the gel after crosslinking, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is finished, and adding 5L of eluent again to perform the third elution until the final weight of the gel is 0.62kg, and stopping the elution until the final weight of the gel reaches the end point of the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 8mg/ml after 6 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 60g of gel, sterilizing under the conditions of 121deg.C and F0=12 by wet heat, addingAdding 40g of lubricant, stirring and mixing uniformly, MMicro-crosslinked sodium hyaluronate ：MLubricant =6:4, and finally a micro-crosslinked sodium hyaluronate gel containing recombinant type III human collagen was obtained, and filled into 1.0ml pre-filled syringes, sample No. D.
Example 5
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 5g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing a lubricant with the concentration of 20mg/ml for later use.
50ml of 1.2% sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 200 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.48ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 48 ℃, placing the gel into the water bath, and preserving the heat for 75 minutes. And taking out the gel after crosslinking, adding 3.5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 3.5L of eluent after the second elution is finished, adding 3.5L of eluent again for the third elution until the final weight of the gel is 0.62kg, stopping the elution, and taking 9h until the final concentration of the micro-crosslinked sodium hyaluronate is 8mg/ml. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 90g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 10g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant =9:1, and finally a micro-crosslinked sodium hyaluronate gel containing recombinant type III human collagen was obtained, and filled into 1.0ml pre-filled syringes, sample No. E.
The finished products obtained in the above examples are inspected, the inspection indexes are the pushing force (27G disposable sterile injection needle), BDDE residues, particle size distribution, degradation performance, viscoelasticity modulus and other indexes, and the specific results are shown in the attached table:
table 1
Table 2:
from the above table, it can be concluded that the proportion of the crosslinking agent in the crosslinking process of sodium hyaluronate, the crosslinking reaction temperature, the dialysis end point (final product concentration) all have a great influence on the indexes of the final product, and directly influence the clinical use effect, wherein the push force (N), the viscoelastic modulus and the degradation performance (%) are particularly important, and the sample C process in example 3 is the preferred option by integrating the evaluation of each index.
Thus, we continued to use the process of example 3 with a configuration of lubricating fluids of varying concentrations of recombinant type III human collagen. The method comprises the following steps:
example 6
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 3.75g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing the lubricant with the concentration of 15mg/ml for later use.
50ml of 1.0% strength sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 150 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.40ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 50 ℃, placing the gel into the water bath, and preserving the heat for 120min. And taking out the gel after crosslinking, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is completed, adding 5L of eluent again for the third elution until the final weight of the gel is 0.62kg, stopping the elution until the final weight of the gel reaches the end point of the elution, and using the solution for 6h to achieve the final concentration of the micro-crosslinked sodium hyaluronate8mg/ml. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 80g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 20g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant And (2) obtaining the micro-crosslinked sodium hyaluronate gel containing the recombinant type III human collagen, filling the micro-crosslinked sodium hyaluronate gel into a 1.0ml pre-filling and sealing syringe, and obtaining a sample number F.
Example 7
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 6g of recombinant III type human collagen raw material, adding 250ml of eluent for dissolution, and preparing a lubricant with the concentration of 24mg/ml for later use.
50ml of 1.0% strength sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 150 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.40ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 50 ℃, placing the gel into the water bath, and preserving the heat for 120min. And taking out the gel after crosslinking, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is finished, and adding 5L of eluent again to perform the third elution until the final weight of the gel is 0.62kg, and stopping the elution until the final weight of the gel reaches the end point of the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 8mg/ml after 6 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 80g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 20g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant And (2) obtaining the micro-crosslinked sodium hyaluronate gel containing the recombinant type III human collagen, filling the micro-crosslinked sodium hyaluronate gel into a 1.0ml pre-filling and sealing syringe, and obtaining a sample number G.
Comparative example 1
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
Weighing 2.5g of recombinant type III human collagen raw material, adding 250ml of eluent for dissolution, and preparing the lubricant with the concentration of 10mg/ml for later use.
50ml of 1.0% strength sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 150 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.40ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating the temperature of the water bath to 50 ℃, placing the gel into the water bath, and preserving the heat for 120min. And taking out the gel after crosslinking, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is finished, and adding 5L of eluent again to perform the third elution until the final weight of the gel is 0.62kg, and stopping the elution until the final weight of the gel reaches the end point of the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 8mg/ml after 6 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 80g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 20g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant And (2) obtaining the micro-crosslinked sodium hyaluronate gel containing the recombinant type III human collagen, filling the micro-crosslinked sodium hyaluronate gel into a 1.0ml pre-filling and sealing syringe, and carrying out sample number H.
Comparative example 2
Preparing 20L of eluent (sodium chloride-containing, sodium dihydrogen phosphate, disodium hydrogen phosphate and water for injection) for later use.
7.5g of recombinant III-type humanized collagen raw material is weighed, 250ml of eluent is added for dissolution, and the lubricant with the concentration of 30mg/ml is prepared for standby.
50ml of 1.0% strength sodium hydroxide solution was prepared, and 5g of a sodium hyaluronate raw material having a molecular weight of 150 ten thousand was added thereto and dissolved by stirring until no white undissolved HA was visible to the naked eye. After dissolution, 0.40ml of 1, 4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred again and mixed well. Regulating water bath temperature to 50deg.C, placing the above gel in water bathIn the process, the temperature is kept for 120min. And taking out the gel after crosslinking, adding 5L of eluent, stirring at a low speed for swelling for 3 hours, removing the eluent, and scattering the swelled gel, wherein the size is about 0.5-2 cm. Continuing the second elution for 16h, adding 5L of eluent after the second elution is finished, and adding 5L of eluent again to perform the third elution until the final weight of the gel is 0.62kg, and stopping the elution until the final weight of the gel reaches the end point of the elution, wherein the final concentration of the micro-crosslinked sodium hyaluronate is 8mg/ml after 6 h. And (3) taking the dialyzed gel, and continuously homogenizing for 2 times by using a screen to obtain the gel with the particle diameter of 150-400 mu m. Taking 80g of gel, sterilizing under the conditions of 121 ℃ and F0=12 by moist heat, adding 20g of lubricant, stirring and mixing uniformly, and MMicro-crosslinked sodium hyaluronate ：MLubricant And (2) obtaining the micro-crosslinked sodium hyaluronate gel containing the recombinant type III human collagen, filling the micro-crosslinked sodium hyaluronate gel into a 1.0ml pre-filling and sealing syringe, and obtaining a sample number I.
The finished products obtained in the examples and the comparative examples are inspected, the inspection indexes are the indexes of pushing force (27G disposable sterile injection needle), viscoelasticity modulus and the like, and the specific results are shown in the attached table:
table 3
[ data analysis ]
From the data, when the concentration of the recombinant type III human collagen is 15-24 mg/ml, the pushing force of the product can be at a proper level (10-20N), so that the hand feeling in the injection process is better.
The above description of the embodiments is only intended to assist in understanding the method of the invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.