CN117860959A - Repair-promoting recombinant humanized collagen gel and preparation method thereof - Google Patents
Repair-promoting recombinant humanized collagen gel and preparation method thereof Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a repair-promoting recombinant humanized collagen gel, which comprises the following components in percentage by mass: 0.05% of recombinant III type humanized collagen; 0.1 to 1.0 percent of carbomer; 0-20% of glycerol; tween 80 0.1% -0.5%; 1.0 to 1.5 percent of triethanolamine; d-mannose 0.1-2.0%; the balance being water. The invention changes the viscosity of the gel by adding the proportion of carbomer and triethanolamine, thereby leading the gel to have excellent adhesiveness and ductility; the glycerol is added to ensure that the wound has strong moisture retention, and a gel film can be formed to cover the wound, so that the wound can be protected from infection; the D-mannose is added to ensure that the gel has certain anti-inflammatory activity, so that the microenvironment of the wound is improved, and the repair speed of the wound surface is further increased.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a repair-promoting recombinant humanized collagen gel and a preparation method thereof.
Background
Collagen is the main component of extracellular matrix, is formed by winding three L-polypeptide alpha-chains, is mainly stored in human skin, can form a collagen film on the skin cuticle, prevents internal moisture from evaporating to the outside, plays a role in moisturizing, improves the moisture content of the cuticle, and maintains the normal function of keratinocytes, thereby protecting the skin from damage. Collagen can be divided into 28 collagen molecules according to the composition of tripeptide chains. The collagens in the skin are mainly type i, iii and v collagens present in the dermis layer, and type iv, vii and xvii collagens in the basement membrane. Most commonly, the type I and type III collagens are coarse, the type I collagen fibers are abundant in the dermis layer, and the type III collagen fibrils are fine, so that the ordered arrangement of the type I collagen can be guided, and the skin is rich in elasticity.
In recent years, collagen is increasingly used in wound dressings, and as an important component of extracellular matrix, collagen can promote migration and proliferation of various cells such as fibroblasts and endothelial cells, promote production of cytokines such as growth factors, and promote wound healing.
Animal-derived collagen has high extraction cost and relatively high safety risk due to heterology, so that the recombinant humanized collagen becomes the first choice of collagen products. The recombinant collagen is prepared from genetically engineered bacteria, has no virus hidden trouble, high biocompatibility and excellent water solubility, and is widely used in the fields of tissue engineering, biomedicine, medicine release and the like. The recombinant collagen has high biological activity and good tissue compatibility, can promote hyperplasia of dermal fibroblasts, improve cell activity, is absorbed by fibroblasts as raw materials for synthesizing collagen, stimulates more synthetic collagen of cells, and fills and repairs damaged skin. The recombinant III type humanized collagen (Recombinant human collagen III, rhc-III) is obtained by optimizing and recombining and expressing the original gene sequence of the human III type collagen by utilizing a genetic engineering technology, has the structure characteristics of the III type collagen and has the molecular weight of 30-130 kDa, and the amino acid sequence of the recombinant III type humanized collagen is highly consistent with the amino acid sequence of the human natural collagen. Rhc-III contains abundant hydrophilic amino acid residues, is favorable for adhesion and proliferation of fibroblasts, accelerates synthesis and deposition of collagen, and shortens wound healing time.
Skin tissues are relatively fragile, and can not be damaged to different degrees in daily life, and mechanical wounds, burns, scalds, scratches, postoperative wounds, diabetic wounds and the like are common. Wound healing is a process of skin tissue regeneration, mainly comprising bleeding, inflammatory, cell proliferation and tissue remodeling phases. If no intervention is performed, local blood flow is reduced, inflammatory cells infiltrate along with the local blood flow, muscle spasm is induced, wound healing is seriously affected, chronic wounds are developed, and how to accelerate wound healing becomes a global common treatment problem. Therefore, good pro-repair products are needed to not only promote skin cell proliferation, but also to inhibit the occurrence and progression of inflammation. The recombinant human III type collagen gel is externally applied with collagen, provides a moist growth environment for facial cells of a patient, can effectively remove unhealthy tissues of skin, inhibit bacterial reproduction, enhance the cell molecular growth capacity, and plays an important role in enhancing the treatment effect of the patient.
The gel is a semisolid preparation prepared by preparing the medicine and proper auxiliary materials into a uniform or suspension, and is often used as a wound dressing due to the advantages of high bioadhesion, large contact area with skin and high local concentration of the medicine. Gel matrices are generally classified into aqueous and oily matrices, and common aqueous gel matrices include carbomers, HPMC, CMC-Na, PVA, etc., and oily gel matrices include liquid paraffin, polyethylene, etc. The aqueous gel is easy to spread, has low viscosity and is favorable for drug release and widely applied.
The current medical dressing applied to non-chronic wounds (chronic wounds are refractory wounds with no healing tendency or no healing tendency for more than one month) has relatively little research, and the recombinant III type humanized collagen gel which can prevent inflammation (i.e. prevent infection) and inhibit inflammation development so as to synergistically promote rapid wound repair is more rarely reported. Therefore, developing a stable collagen dressing has wide application prospect.
Disclosure of Invention
In order to solve the technical problems, the invention provides a repair-promoting recombinant humanized collagen gel and a preparation method thereof.
The technical scheme of the invention is as follows:
the repair-promoting recombinant humanized collagen gel comprises the following components in percentage by mass:
0.05% of recombinant III type humanized collagen;
0.1 to 1.0 percent of carbomer;
0-20% of glycerol;
tween 80 0.1% -0.5%;
1.0 to 1.5 percent of triethanolamine;
d-mannose 0.1-2.0%;
the balance being water.
The molecular weight of the recombinant III type humanized collagen gel is 55kDa.
The repair-promoting recombinant humanized collagen gel is characterized in that the carbomer comprises one or more of carbomer 940, carbomer 941, carbomer 980, carbomer 981, carbomer 934P, carbomer 971P, carbomer 974P, carbomer 71G, carbomer 5984 and carbomer 1342.
The weight ratio of carbomer to triethanolamine in the repair-promoting recombinant humanized collagen gel is 1-10:10-15.
Preferably, the weight ratio of carbomer to triethanolamine in the repair-promoting recombinant humanized collagen gel is 5:12.
The mass ratio of the glycerol to the tween 80 to the D-mannose is 0-200:1-5:1-20.
Preferably, the mass ratio of the glycerol to the tween 80 to the D-mannose is 75:2:1.
Preferably, the repair-promoting recombinant humanized collagen gel comprises the following components in percentage by mass:
0.05% of recombinant III type humanized collagen;
carbomer 0.5%;
15% of glycerol;
tween 80.4%;
triethanolamine 1.2%;
d-mannose 0.2%;
the balance being water.
The preparation method of the repair-promoting recombinant humanized collagen gel comprises the following steps:
1) Uniformly mixing carbomer with water, and stirring for a certain time to obtain a first solution;
2) Uniformly mixing glycerol, tween 80, D-mannose and water to obtain a second solution;
3) Adding the second solution into the first solution, and uniformly stirring to obtain a third solution;
4) Adding recombinant III type humanized collagen and triethanolamine into the third solution, regulating pH value, stirring for a certain time, discharging, and sterilizing with ethylene oxide after filling.
Preferably, in step 1), the stirring speed is 400 r.min -1 Stirring time is 12h; in the step 2), the stirring speed is 300 r.min -1 Stirring for 30min; in the step 3), the stirring speed is 300 r.min -1 Stirring for 30min; in the step 4), the stirring speed is 600 r.min -1 Stirring for 1h.
The beneficial effects of the invention are as follows:
1) The invention changes the viscosity of the gel by adding the proportion of carbomer and triethanolamine, thereby leading the gel to have excellent adhesiveness and ductility; the glycerol is added to ensure that the wound has strong moisture retention, and a gel film can be formed to cover the wound, so that the wound can be protected from infection; the D-mannose is added to ensure that the gel has certain anti-inflammatory activity, so that the microenvironment of the wound is improved, and the repair speed of the wound surface is further increased.
2) According to the invention, a certain proportion of glycerol is added to regulate the moisture retention of the gel, so that the gel can quickly form a protective film on a wound surface, and a wet healing environment is provided for the wound surface; the gel has obvious anti-inflammatory activity, resists wound infection and synergistically promotes the repairing effect of collagen on the wound surface by adding the D-mannose with proper concentration.
3) The preparation method of the humanized collagen gel adds glycerol and D-mannose into the gel, and uniformly mixes the glycerol and the D-mannose, so that the humanized collagen gel plays a synergistic effect on promoting wound repair of the recombinant III type humanized collagen.
Drawings
FIG. 1 is a graph showing the effect of recombinant humanized collagen III gels of each formulation on HaCat cell and HSF cell growth, wherein FIG. A shows the effect of collagen gel of each formulation on HaCat cell growth, and FIG. B shows the effect of collagen gel of each formulation on HFS cell growth;
FIG. 2 shows the result of SDS-PAGE of recombinant type III humanized collagen;
FIG. 3 shows the HPLC results for recombinant type III humanized collagen.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments.
Example 1
The embodiment is a synergistic repair-promoting recombinant III type humanized collagen gel, which comprises the following components in percentage by mass: 0.05% of recombinant III type humanized collagen; 0.1 to 1.0 percent of carbomer; 0-20% of glycerol; tween 80 0.1% -0.5%; 1.0 to 1.5 percent of triethanolamine; d-mannose 0.1-2.0%; the balance being water. The viscosity of the gel is changed by adding carbomer and triethanolamine and changing the proportion of carbomer and triethanolamine; the gel has strong moisture retention property by adding glycerol, and can form gel film to cover the wound, thereby protecting the wound from infection; the D-mannose is added to ensure that the gel has certain anti-inflammatory activity, so that the microenvironment of the wound is improved, and the repair speed of the wound surface is further increased.
The recombinant III type humanized collagen gel changes the viscosity of the gel by changing the proportion of carbomer and triethanolamine, so that the collagen is more tightly connected. However, too high a viscosity may affect the solvation of the collagen, thereby destroying its structure and exposing the non-polar groups of the collagen, which may lead to its denaturation and inactivation. Therefore, the ratio of carbomer to triethanolamine in the collagen gel described in this example is 1-10:10-15; preferably 5:12. In this example, the molecular weight of the recombinant type III humanized collagen is 55kDa; the carbomers include one or more of carbomers 940, carbomer 941, carbomer 980, carbomer 981, carbomer 934P, carbomer 971P, carbomer 974P, carbomer 71G, carbomer 5984, and carbomer 1342.
The recombinant III humanized collagen gel has good moisture retention through glycerin, so that the gel can form a film protection at a skin injury part and can form a wet healing environment for a wound surface rapidly; meanwhile, the gel has a certain anti-inflammatory activity by adding D-mannose; the active ingredient in the gel was homogeneously dispersed by adding tween 80. The proportion of the glycerol, the Tween 80 and the D-mannose in the collagen gel is 0-200:1-5:1-20; preferably 75:2:1. A preferred formulation for the gel is: 0.05% of recombinant III type humanized collagen; carbomer 0.5%; 15% of glycerol; tween 80.4%; triethanolamine 1.2%; d-mannose 0.2%; the balance being water.
The preparation method of the synergistic repair-promoting recombinant III type humanized collagen gel comprises the steps of firstly, uniformly mixing carbomer and water in a first stage, and stirring for a certain time to obtain a first solution; uniformly mixing glycerol, tween 80, D-mannose and water, and stirring to obtain a second solution; adding the second solution into the first solution, and uniformly stirring to obtain a third solution; and finally, adding the recombinant III type humanized collagen and triethanolamine into the third solution, adjusting the pH value, stirring for a certain time, and discharging to obtain the finished product. The preparation method has the advantages that the recombinant III type humanized collagen gel prepared by the preparation method has good moisture retention and stability. The preparation method comprises the following specific steps:
s1: mixing carbomer with water to obtain a mixture of 400 r.min -1 Stirring for 12h to obtain a first solutionA liquid;
s2: mixing glycerol, tween 80, D-mannose and water uniformly, and concentrating at 300 r.min -1 Stirring for 30min to obtain a second solution;
s3: adding the second solution into the first solution, 300 r.min -1 Stirring for 30min to obtain a third solution;
s4: adding recombinant III-type humanized collagen and triethanolamine into the third solution, and adjusting pH value to 600 r.min -1 Stirring for 1h and discharging. And (5) sterilizing the filled ethylene oxide.
Example 2
In this example, recombinant type III humanized collagen gels of different proportions of carbomers and triethanolamine were prepared, and observation of gel properties and measurement of viscosity were performed. The specific formulation is shown in table 1:
TABLE 1 different collagen gel formulations
And (3) performing property evaluation and viscosity detection on the recombinant III type humanized collagen gel prepared by the formulas 1-16. The evaluation method comprises the following steps: the gel form and texture of each formulation were observed, and the gel was optimal (gel transparency, fine and uniform texture, and excellent viscosity), excellent (gel transparency, fine and uniform texture, and thick (thin) viscosity), excellent (gel transparency, uniform texture, and slightly thick (thin)) and slightly inferior (gel transparency, non-uniform texture (local), and slightly thick (thin)) were observed. The results are shown in Table 2:
table 2 gel property evaluation and viscosity number for each formulation
| Formulation of | Evaluation | Viscosity (mpa.s) | Formulation of | Evaluation | Viscosity (mpa.s) |
| Formulation 1 | Slightly inferior | 31778 | Formulation 9 | Slightly inferior | 64527 |
| Formulation 2 | Slightly inferior | 36106 | Formulation 10 | Preferably is good | 71263 |
| Formulation 3 | Slightly inferior | 38055 | Formulation 11 | Optimum for the production of a product | 84934 |
| Formulation 4 | Slightly inferior | 40571 | Formulation 12 | Excellent and excellent properties | 95008 |
| Formulation 5 | Slightly inferior | 56148 | Formulation 13 | Slightly inferior | 69127 |
| Formulation 6 | Slightly inferior | 57297 | Formulation 14 | Preferably is good | 74182 |
| Formulation 7 | Preferably is good | 74439 | Formulation 15 | Optimum for the production of a product | 93463 |
| Formulation 8 | Optimum for the production of a product | 85101 | Formulation 16 | Slightly inferior | >100000 |
Example 3
This example is to examine the effect of adding glycerol in different proportions on the moisturizing properties of collagen gels. The preparation method comprises collecting recombinant III-type humanized collagen, carbomer and D-mannoseMixing sugar, tween 80, triethanolamine and water uniformly, and controlling the flow rate to 300 r.min -1 Stirring for 30min; then adding glycerol, 300 r.min -1 Stirring for 10min,600 r.min -1 Homogenizing for 5 min. The specific formulation is shown in Table 3:
TABLE 3 different collagen gel formulations
The moisturizing property of the recombinant III-type humanized collagen gel prepared by each formula is evaluated by the following method: each 0.5g sample was smeared on a glass plate surface (10×10 cm), the smeared area was (2×2 cm), 3 samples were parallel, and the samples were left to stand at room temperature, and were observed at 0, 1, 2, 4, and 8 hours, respectively, and the observed phenomena included gel morphology (gel-like, dry-out), texture (uniform, partially dry-out), and water wettability (moist, general, dry-out). The specific results are shown in Table 4:
table 4 gel moisturizing results for each formulation
The results in Table 4 show that the recombinant type III humanized collagen gels prepared from each formulation maintained good moisture retention at room temperature for 1h, and maintained relatively good gel state when left at room temperature for 8h only when the percentage of glycerol in the formulation was not less than 15%, such as formulation 23, formulation 24, formulation 25, wherein the percentage of glycerol in formulation 23 was minimal, as the preferred formulation.
Example 4
This example is an examination of anti-inflammatory activity of the recombinant type III humanized collagen gel prepared in formulation 23 of example 3. D-mannose with different percentage contents is added into the gel of the formula 23, and the preparation method is as followsThe method comprises mixing recombinant III humanized collagen, carbomer, D-mannose, tween 80, triethanolamine and water uniformly, and mixing at 300 r.min -1 Stirring for 30min; then adding glycerol and D-mannose, 300 r.min -1 Stirring for 10min,600 r.min -1 Homogenizing for 5 min. The in vitro anti-inflammatory activity of the collagen gel of each formulation was studied by establishing an inflammatory cell model by Lipopolysaccharide (LPS) intervention in mouse macrophage RAW264.7, and the specific formulation is shown in Table 5:
TABLE 5 different collagen gel formulations
The experimental method comprises the following steps: the concentration range of the test object is determined by cytotoxicity detection, and the concentration with the cell activity being more than 90% is selected. Then taking RAW264.7 cells in logarithmic growth phase, counting after digestion, 1000 r.min -1 Centrifuging for 5min, discarding supernatant, and re-suspending with culture medium; according to 5X 10 4 cells/well were inoculated into 96-well plates, 200. Mu.L per well, and placed in an incubator for 24 h.+ -. 2h. The medium was discarded and induction and administration was started. Adding a culture medium containing a certain concentration of a test substance and LPS (1 mug/mL) into a test substance hole; adding a negative control into a cell culture medium containing LPS; the positive control wells were added with medium containing positive control (dexamethasone 100. Mu.g/mL) and LPS; cell culture medium was added to the blank/solvent control wells, 200 μl per well, and incubated for 24h±2h. After the incubation, 150. Mu.L of the cell culture supernatant was collected in a 1.5mL sterile centrifuge tube and stored in a-80℃refrigerator for further use. The content of TNF-alpha and IL-6 was detected by ELISA detection kit. The results are shown in Table 6:
TABLE 6 gel TNF-alpha, IL-6 content of each formulation
| Group of | TNF-α(μg/mL) | IL-6(μg/mL) |
| Formulation 26 | 0.6682 | 0.7055 |
| Formulation 27 | 0.5417 | 0.6648 |
| Formulation 28 | 0.5386 | 0.6592 |
| Formulation 29 | 0.5928 | 0.7121 |
| Formulation 30 | 0.6612 | 0.7348 |
| Formulation 31 | 0.7054 | 0.7562 |
| Formulation 33 | 0.7346 | 0.7884 |
| LPS group | 0.7715 | 0.8113 |
| Dexamethasone group | 0.5917 | 0.6785 |
| Normal cell group | 0.5218 | 0.5702 |
From Table 6, it can be seen that the collagen gel has an obvious anti-inflammatory activity after a certain concentration of D-mannose is added, but the anti-inflammatory activity is reduced when the concentration exceeds a certain amount; wherein, when the percentage of D-mannose is 0.2% and 0.4%, the difference of anti-inflammatory activity is not obvious, and the formula 27 with low D-mannose content is preferable.
Example 5
In this example, the collagen gels of example 4 were subjected to a low-temperature test and an acceleration test to examine whether or not the gel had significantly changed in terms of its appearance, properties, and the like. The low temperature test comprises the steps of taking a proper amount of sample, subpackaging the sample in an ointment tube, placing the ointment tube in a refrigerator at 4 ℃, placing the ointment tube for 10 days, and sampling and detecting on days 5 and 10; the acceleration experiment is to take a proper amount of samples, split-charge the samples into ointment tubes, put the ointment tubes into a constant temperature and constant humidity incubator, place the ointment tubes for 6 months at 35+/-2 ℃ and take samples for detection on days 30, 60, 90 and 180. The results are shown in Table 7:
table 7 examination of gel stability for each formulation
The recombinant III type humanized collagen gel obtained from the formulas 26 to 33 in the table 7 can maintain gel state after low-temperature test and acceleration test, and has unchanged color, smell and other properties.
Example 6
This example is a study of the repair of skin lesions in rats promoted by recombinant type III humanized collagen gel of formulation 27 of example 4. 20 adult healthy female SD rats (220 g.+ -.20 g) were randomly divided into a model group, a blank gel group (no recombinant III type humanized collagen, other same formula 27), a collagen gel single use group (formula 11, no glycerol, tween 80, D-mannose, pH adjusted to 4.5-7.5) and a collagen gel combination group (formula 27), 4 total groups, 5 each. Each gel group was sterilized and used. The back was shaved with Mao Qiti hairs and then short Mao Chujin with depilatory cream, and then anesthetized with 3% pentobarbital sodium at 30mg/kg intraperitoneal injection. After skin is disinfected by iodophor, a puncher with the diameter of 6mm is taken to take a round wound surface at the corresponding position, and the whole layer of skin is cut off. After hemostasis, the blank dressing set and the collagen dressing set are fixed by medical gauze and adhesive tape after corresponding dressing is used for covering wounds, and the model set is directly fixed by the medical gauze and the adhesive tape. The postoperative rats were fed singly in single cages, fed and drunk freely, changed dressings daily and observed for wound healing. Calculating the wound healing rate at 2, 5, 7 and 14 days after operation; at the same time, each group of new skin was cut off and the VEGF content was measured. The results are detailed in tables 8 and 9.
Table 8 skin injury healing rate (n=5) for animals of each group 2, 5, 7, 14d after gel administration
Note that: healing rate = (original wound area-unhealed wound area)/original wound area×100%
Table 9 comparison of VEGF levels in neonatal skin at 14d injury after gel administration (n=5) for animals of each group
| Group of | VEGF(μg/mL) |
| Model group | 120.02±8.19 |
| Blank gel set | 132.46±12.52 |
| Formulation 11 | 155.25±9.64 |
| Formulation 27 | 183.22±9.79 |
Experiment result one: compared with the model group, the healing rate of the rat damaged skin of the collagen gel group in the formula 11 is increased, and the healing rate of the collagen gel group in the formula 27 is obviously improved. The healing rate of the gel of formulation 27 was also significantly enhanced compared to the gel of formulation 11, with a significant difference between the two at 2d and 5d after gel administration. It is shown that the glycerol, the tween 80 and the D-mannose are added to have synergistic effect on promoting skin injury repair of the recombinant type III humanized collagen.
Experimental results two: compared with the model group, the VEGF content in the skin repaired by the formula 11 and the formula 27 is obviously improved, and the VEGF content in the formula 11 of the formula 27 is obviously improved. It is presumed that the addition of glycerol, tween 80 and D-mannose can promote the generation of VEGF in cooperation with collagen, promote the migration, proliferation and angiogenesis of vascular endothelial cells, thereby promoting the repair of skin injury.
Example 7
This example demonstrates the irritation of the prepared synergistic repair-promoting thermostable recombinant type III humanized collagen gel to animal skin. 6 healthy, young female albino rabbits, weighing 3 kg.+ -. 0.5kg, were selected and randomly divided into 2 groups, blank and formula 27 gel groups, respectively. Blank groups were given 0.5mL of sterile water and formula 27 groups were given 0.5mL of the recombinant type iii humanized collagen gel described above. The animals were dehaired on both sides of the back spine (approximately 10X 15cm area) 12h prior to the test, taking care not to damage the skin during dehairing. The test article was applied to both sides of the back of the rabbit, the contact area was covered with a 2.5X2.5 cm absorbent gauze piece, and then fixed with a bandage for 6 hours. After the contact period, the gauze is taken off, the contact part is marked by using the durable ink, and the residual test material is removed by washing with warm water and is wiped dry. Part 10 of the biological evaluation of medical devices according to GB/T16886.10-2017: the scoring system (Table 10) given by the animal skin irritation test in the irritation and skin sensitization response test describes that each contact site was scored and scored for skin erythema and edema response after (1.+ -. 0.1) h, (24.+ -. 2) h, (48.+ -. 2) h, and (72.+ -. 2) h after the gauze was removed. The skin irritation test results are shown in table 11:
TABLE 10 skin reaction scoring system
TABLE 11 integral of stimulus response
As can be seen from the results of the animal skin irritation test in Table 11, the recombinant type III humanized collagen gel showed no significant irritation response in the skin irritation test in albino rabbits.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. The repair-promoting recombinant humanized collagen gel is characterized by comprising the following components in percentage by mass:
0.05% of recombinant III type humanized collagen;
0.1 to 1.0 percent of carbomer;
0-20% of glycerol;
tween 80 0.1% -0.5%;
1.0 to 1.5 percent of triethanolamine;
d-mannose 0.1-2.0%;
the balance being water.
2. A repair-promoting recombinant humanized collagen gel according to claim 1, wherein said recombinant type iii humanized collagen has a molecular weight of 55kDa.
3. A repair-promoting recombinant humanized collagen gel according to claim 1, wherein said carbomers comprise one or more of carbomer 940, carbomer 941, carbomer 980, carbomer 981, carbomer 934P, carbomer 971P, carbomer 974P, carbomer 71G, carbomer 5984 and carbomer 1342.
4. The repair-promoting recombinant humanized collagen gel according to claim 1, wherein the mass ratio of carbomer to triethanolamine is 1-10:10-15.
5. A repair-promoting recombinant humanized collagen gel according to claim 4, wherein the mass ratio of carbomer to triethanolamine is 5:12.
6. The repair-promoting recombinant humanized collagen gel according to claim 1, wherein the mass ratio of the glycerol, the tween 80 and the D-mannose is 0-200:1-5:1-20.
7. The repair-promoting recombinant humanized collagen gel according to claim 6, wherein the mass ratio of glycerol, tween 80 and D-mannose is 75:2:1.
8. The repair-promoting recombinant humanized collagen gel according to claim 1, wherein the gel comprises the following components in percentage by mass:
0.05% of recombinant III type humanized collagen;
carbomer 0.5%;
15% of glycerol;
tween 80.4%;
triethanolamine 1.2%;
d-mannose 0.2%;
the balance being water.
9. A method of preparing a repair-promoting recombinant humanized collagen gel according to any one of claims 1 to 8, comprising the steps of:
1) Uniformly mixing carbomer with water, and stirring for a certain time to obtain a first solution;
2) Uniformly mixing glycerol, tween 80, D-mannose and water, and stirring to obtain a second solution;
3) Adding the second solution into the first solution, and uniformly stirring to obtain a third solution;
4) Adding recombinant III type humanized collagen and triethanolamine into the third solution, regulating pH value, stirring for a certain time, discharging, and sterilizing with ethylene oxide after filling.
10. The method for preparing a repair-promoting recombinant humanized collagen gel according to claim 9, wherein in the step 1), the stirring speed is 400 r.min -1 Stirring time is 12h; in the step 2), the stirring speed is 300 r.min -1 Stirring for 30min; in the step 3), the stirring speed is 300 r.min -1 Stirring for 30min; in the step 4), the stirring speed is 600 r.min -1 Stirring for 1h.
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