CN118001452A - Recombinant collagen sponge and manufacturing method thereof - Google Patents
Recombinant collagen sponge and manufacturing method thereof Download PDFInfo
- Publication number
- CN118001452A CN118001452A CN202410156816.7A CN202410156816A CN118001452A CN 118001452 A CN118001452 A CN 118001452A CN 202410156816 A CN202410156816 A CN 202410156816A CN 118001452 A CN118001452 A CN 118001452A
- Authority
- CN
- China
- Prior art keywords
- recombinant collagen
- collagen sponge
- recombinant
- aqueous solution
- sponge
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000515 collagen sponge Substances 0.000 title claims abstract description 54
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- 102000008186 Collagen Human genes 0.000 claims abstract description 38
- 108010035532 Collagen Proteins 0.000 claims abstract description 38
- 229920001436 collagen Polymers 0.000 claims abstract description 38
- 238000004132 cross linking Methods 0.000 claims abstract description 14
- 230000002439 hemostatic effect Effects 0.000 claims abstract description 14
- 239000007864 aqueous solution Substances 0.000 claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 238000001816 cooling Methods 0.000 claims abstract description 7
- 238000004108 freeze drying Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 10
- 206010052428 Wound Diseases 0.000 claims description 8
- 208000027418 Wounds and injury Diseases 0.000 claims description 8
- 230000023597 hemostasis Effects 0.000 claims description 5
- 230000005855 radiation Effects 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- 238000010894 electron beam technology Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 abstract description 13
- 239000007788 liquid Substances 0.000 abstract description 10
- 238000002360 preparation method Methods 0.000 abstract description 4
- 239000011148 porous material Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 239000011343 solid material Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000001338 self-assembly Methods 0.000 description 4
- 238000003917 TEM image Methods 0.000 description 3
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 3
- 229910052753 mercury Inorganic materials 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000012620 biological material Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 238000009736 wetting Methods 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241001484259 Lacuna Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100058594 Mus musculus Hlcs gene Proteins 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 102000034240 fibrous proteins Human genes 0.000 description 1
- 108091005899 fibrous proteins Proteins 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000002459 porosimetry Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/102—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0036—Porous materials, e.g. foams or sponges
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
Landscapes
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Surgery (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
The patent application discloses a recombinant collagen sponge and a manufacturing method thereof. The preparation method of the recombinant collagen sponge comprises the steps of pre-cooling the recombinant collagen aqueous solution to enable the temperature to be 2-5 ℃ and keeping the temperature for more than 2 hours; immediately carrying out irradiation crosslinking on the precooled recombinant collagen aqueous solution; freeze-drying the recombinant collagen aqueous solution subjected to irradiation crosslinking to obtain the recombinant collagen sponge; the thickness of the recombinant collagen sponge is greater than 1mm; wherein the amino acid sequence of the recombinant collagen is obtained by repeating the short amino acid sequence shown in SEQ ID NO. 152 times. Under the same pore-forming condition, the recombinant collagen sponge can obtain higher porosity and has better liquid absorption performance (hemostatic performance).
Description
Technical Field
The application belongs to the field of biological materials, and particularly relates to a recombinant collagen sponge capable of being absorbed by a body and a manufacturing method thereof.
Background
Collagen is a family of fibrous proteins that are most widely distributed in animals, are most abundant in content, and are of a wide variety, with collagen present in various organ tissues accounting for more than about 30% of the total protein in humans. Collagen is considered as the most useful biomaterial as the major component of the extracellular matrix. Collagen has low antigenicity, biodegradability, easy absorption, no toxicity, good biocompatibility, hemostatic effect and important role in tissue repair. For example, the use of collagen sponges for artificial skin grafts at wounds can promote cell proliferation and increase tissue healing rates by virtue of their porous structure for cell growth and the role of collagen itself in tissue repair.
The collagen sponge is mainly used for hemostasis in the blood seepage area of the wound surface in vivo, emergency hemostasis and operation hemostasis, or is used for filling the lacuna and the wound surface. In order to fully exert the hemostatic effect of the collagen sponge, a certain volume of the collagen sponge is required to absorb and gather more liquid at the wound. The hemostatic performance of the collagen sponge is closely related to indexes such as porosity, water absorption and the like, and the greater the porosity and the water absorption, the better the hemostatic effect of the product.
In general, the pores in the solid material include open pores and closed pores, wherein an open pore is a pore having at least one end attached to the outer surface of the solid material that contributes to the liquid absorbing properties of the solid material; closed cells are cells that are not attached to the outer surface of the solid material and do not contribute to the liquid absorbing properties of the solid material.
Therefore, how to increase the number of open pores and the proportion thereof in the total pores is a technical problem that the skilled person always focuses on for solid materials.
Disclosure of Invention
In view of the above-mentioned technical problems in the prior art, an object of the present invention is to provide a new recombinant collagen sponge and a method for manufacturing the same, wherein the recombinant collagen sponge can obtain higher porosity and better liquid absorption performance (hemostatic performance) under the same pore-forming condition.
The inventors have conducted intensive studies to solve the above-mentioned technical problems, and as a result, found that: the type I recombinant collagen obtained by repeating the short amino acid sequence shown in SEQ ID NO. 1 (G A P G A P G SQ G AP G L Q) 52 times can self-assemble at a low temperature (2-5 ℃) to form a polymer, and the polymer is formed to ensure that the connectivity among the pores of the collagen sponge manufactured by the type I recombinant collagen is better, more open pores can be formed, and the porosity and the liquid absorption performance (hemostatic performance) of the type I recombinant collagen are improved.
Namely, the present invention includes:
1. A method of manufacturing a recombinant collagen sponge, comprising:
Step A: pre-cooling the recombinant collagen aqueous solution to 2-5 ℃ and keeping the temperature for more than 2 hours;
And (B) step (B): immediately carrying out irradiation crosslinking on the precooled recombinant collagen aqueous solution;
step C: freeze-drying the recombinant collagen aqueous solution subjected to irradiation crosslinking to obtain the recombinant collagen sponge; the thickness of the recombinant collagen sponge is greater than 1mm;
wherein the amino acid sequence of the recombinant collagen is obtained by repeating the short amino acid sequence shown in SEQ ID NO. 1 (G A P G A P G SQ G A P G L Q) 52 times.
2. The production method according to item 1, wherein the aqueous solution of recombinant collagen contains 0.5 to 10wt%, preferably 1 to 8 wt%, more preferably 1 to 4 wt% of the recombinant collagen.
3. The production method according to item 1, wherein the irradiation crosslinking temperature in step B is 2 to 8 ℃.
4. The method according to claim 1, wherein the irradiation crosslinking agent amount in step B is 10 to 100kGy, preferably 20 to 80kGy, more preferably 40 to 60kGy.
5. The production method according to item 1, wherein the radiation source for radiation crosslinking is selected from ultraviolet rays, gamma rays, X rays, neutron beams, particle beams, or electron beams.
6. The production method according to item 1, wherein in the step A, the recombinant collagen aqueous solution is subjected to a pre-cooling treatment at a temperature of 2 to 5℃for 4 hours or longer.
7. The production method according to item 1, wherein the thickness of the recombinant collagen sponge is 20mm or less, preferably 10mm or less, and more preferably 2 to 5mm.
8. The production method according to item 1, further comprising a step of cutting the recombinant collagen sponge obtained after freeze-drying into a predetermined size and sterilizing the same.
9. The recombinant collagen sponge produced by the production method according to any one of claims 1 to 8.
10. Use of the recombinant collagen sponge produced by the production method according to any one of claims 1 to 8 in the production of hemostatic products. The hemostatic product is preferably suitable for hemostasis of wounds, burns and operation wounds and filling of residual cavities.
Based on the invention, the recombinant collagen can be obtained through genetic engineering expression, which effectively reduces the potential immunogenicity and the virus infection risk of the product; under the same pore-forming condition, the recombinant collagen sponge can obtain higher porosity and has better liquid absorption performance (hemostatic performance).
Drawings
Fig. 1 is a transmission electron micrograph taken immediately after precooling of the collagen solution in example 2.
Fig. 2 is a transmission electron micrograph taken immediately after precooling of the collagen solution in comparative example 2.
Detailed Description
The application will be further illustrated with reference to the following examples, which are to be understood as merely further illustrating and explaining the application and are not to be construed as limiting the application.
Type I recombinant collagen (i.e., HLC protein, referred to by the above-mentioned chinese patent application publication) was prepared according to example 2 of chinese patent application publication CN115947828 a.
EXAMPLE 1 preparation of recombinant collagen sponge 1
1G of the type I recombinant collagen is precisely weighed, 99g of water for injection is fully dissolved, and 1 weight percent of recombinant collagen solution is obtained.
The recombinant collagen solution was filtered with a 0.22 μm filter to remove insoluble material.
Filling the filtered recombinant collagen solution into a specific mold, and pre-cooling for 6 hours at 4 ℃.
Setting the irradiation dose to be 20kgy, and immediately completing the cross-linking of the precooled recombinant collagen solution by using an electron accelerator at the temperature of 5+/-3 ℃.
Setting freeze-drying parameters, and completing freeze-drying of the recombinant collagen solution after crosslinking by using a vacuum freeze dryer.
And cutting the freeze-dried recombinant collagen sponge by using related equipment to obtain recombinant collagen sponges with different specifications and sizes, wherein the thickness of the recombinant collagen sponge is 1.5mm, and the recombinant collagen sponge is 1.
EXAMPLE 2 preparation of recombinant collagen sponge 2
A recombinant collagen sponge was prepared as in example 1, except that: the concentration of the type I recombinant collagen was 4% by weight, the pre-cooling temperature was 4 ℃, the pre-cooling time was 4 hours, and the irradiation crosslinking amount was 40kgy, to prepare a recombinant collagen sponge, which was recombinant collagen sponge 2 of the example.
Comparative example 1
A recombinant collagen sponge was prepared as in example 2, except that: precooling time was 0.5h, and a recombinant collagen sponge, which was recombinant collagen sponge 1 of comparative example, was prepared.
Comparative example 2
A recombinant collagen sponge was prepared as in example 2, except that: the precooling temperature was 8 ℃, and a recombinant collagen sponge, which is recombinant collagen sponge 2 of comparative example, was prepared.
Using the recombinant collagen sponges obtained in examples 1-2 and comparative examples 1-2, the pore size distribution and the first part of porosity of the solid material were determined using a high-performance fully automatic mercury porosimeter with reference to GB/T21650.1-2008 mercury porosimetry and gas adsorption: mercury intrusion method, the porosity of the recombinant collagen sponge was determined. The porosity measured by this method represents the ratio of the total volume of the pores in the recombinant collagen sponge to the apparent volume thereof.
In addition, the liquid absorbency of the recombinant collagen sponge was determined using the following method: about 20mg of the product was taken, precisely weighed, recorded as m1, immersed in a beaker containing water at 20+ -1deg.C, lightly kneaded with fingers until completely soaked, and all air was removed, care was taken not to break, after sufficient water was absorbed, one corner was gently clamped with small forceps, taken out of the water, and after draining on the water surface with light forceps for 1 minute, again weighed, recorded as m2, and calculated as follows. Samples were randomly drawn 5 times together and the average water absorption capacity was calculated.
A＝(m2-m1)/m1
Wherein:
A- -absorption capacity of sample
M1- -mass of the sample before wetting in grams (g);
m2- -mass of the sample after wetting in grams (g);
The main experimental conditions and the results of the porosity and the water absorption multiple test of the prepared recombinant collagen sponge of all the above examples and comparative examples are summarized in table 1.
TABLE 1 recombinant collagen sponge preparation conditions and related indicators
From the test results in table 1, it is known that, in the case where the solution of HLC protein was previously kept at 2 to 5 ℃ for 2 hours or more, higher porosity and water absorption multiple can be achieved under the same pore-forming conditions, thereby enabling the recombinant collagen sponge to have better liquid absorption properties (hemostatic properties). After the inventors studied this phenomenon, it was considered that the reason for this was probably that the HLC was able to self-assemble to some extent at 2 to 5 ℃ to form multimers, which were immobilized by irradiation crosslinking to be subsequently performed, and that depolymerization did not occur even when the temperature was returned to normal temperature. The presence of these polymers allows better connectivity between the pores of the collagen sponge, enabling more open pores to be formed, thus improving its porosity and liquid absorption properties (hemostatic properties).
EXAMPLE 5 Low temperature self-Assembly Performance study of HLC
Example 3 of chinese patent application publication CN115947828a demonstrates that the HLC does not self-assemble at normal temperature. Further studies by the inventors have shown that the HLCs can undergo a degree of self-assembly at low temperatures (see chinese patent application 202410092488.9). Fig. 1 and 2 (i.e., fig. 9 and 10 of chinese patent application 202410092488.9) are transmission electron micrographs taken immediately after precooling of the collagen solution in example 2 and comparative example 2, respectively, and it is known that the HLC undergoes some degree of self-assembly under the experimental conditions of the examples. Note that, in fig. 1, the arrows indicate protein fibers formed by self-assembly of HLC.
Claims (10)
1. A method of manufacturing a recombinant collagen sponge, comprising:
Step A: pre-cooling the recombinant collagen aqueous solution to 2-5 ℃ and keeping the temperature for more than 2 hours;
And (B) step (B): immediately carrying out irradiation crosslinking on the precooled recombinant collagen aqueous solution;
step C: freeze-drying the recombinant collagen aqueous solution subjected to irradiation crosslinking to obtain the recombinant collagen sponge; the thickness of the recombinant collagen sponge is greater than 1mm;
wherein the amino acid sequence of the recombinant collagen is obtained by repeating the short amino acid sequence shown in SEQ ID NO. 1 (G A P G A P G SQ G A P G L Q) 52 times.
2. The method according to claim 1, wherein the aqueous solution of recombinant collagen contains 0.5 to 10 wt%, preferably 1 to 8 wt%, more preferably 1 to 4 wt% of the recombinant collagen.
3. The production method according to claim 1, wherein the irradiation crosslinking temperature in the step B is 2 to 8 ℃.
4. The method according to claim 1, wherein the irradiation crosslinking agent amount in step B is 10-100 kGy, preferably 20-80 kGy, more preferably 40-60 kGy.
5. The method of manufacturing according to claim 1, wherein the radiation source for radiation crosslinking is selected from the group consisting of ultraviolet, gamma, X-ray, neutron, particle or electron beam.
6. The method according to claim 1, wherein in the step A, the aqueous solution of the recombinant collagen is pre-cooled to a temperature of 2 to 5℃for 4 hours or longer.
7. The method according to claim 1, wherein the thickness of the recombinant collagen sponge is 20mm or less, preferably 10mm or less, more preferably 2 to 5mm.
8. The method according to claim 1, further comprising a step of cutting the recombinant collagen sponge obtained after freeze-drying into a predetermined size and sterilizing the same.
9. A recombinant collagen sponge produced by the production method according to any one of claims 1 to 8.
10. Use of the recombinant collagen sponge produced by the production method according to any one of claims 1 to 8 in the production of a hemostatic product. The hemostatic product is preferably suitable for hemostasis of wounds, burns and operation wounds and filling of residual cavities.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410156816.7A CN118001452A (en) | 2024-02-02 | 2024-02-02 | Recombinant collagen sponge and manufacturing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410156816.7A CN118001452A (en) | 2024-02-02 | 2024-02-02 | Recombinant collagen sponge and manufacturing method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118001452A true CN118001452A (en) | 2024-05-10 |
Family
ID=90957409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410156816.7A Pending CN118001452A (en) | 2024-02-02 | 2024-02-02 | Recombinant collagen sponge and manufacturing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118001452A (en) |
-
2024
- 2024-02-02 CN CN202410156816.7A patent/CN118001452A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101574539B (en) | Gelatin sponge and preparation method thereof | |
| CN107551312B (en) | Flocculent collagen hemostatic fiber and preparation method thereof | |
| CN103463668A (en) | Preparation method of porous dressing of silk fibroin-calcium alginate biological wound | |
| CN110917386A (en) | Stem cell sponge scaffold, preparation method and application | |
| JP6663012B2 (en) | Method for producing scaffold for cell growth having structural memory properties | |
| CN110507842B (en) | Bacterial cellulose/hyaluronic acid/epsilon-polylysine functional dressing and preparation method thereof | |
| Bao et al. | Agar/collagen membrane as skin dressing for wounds | |
| WO2016141892A1 (en) | Sheet-like cross-linked hyaluronate hydrogel and preparation method thereof | |
| KR20060134346A (en) | Microporous dressing material of chitosan complex and production method | |
| KR100440239B1 (en) | Method for the preparation of hydrogels for wound dressings | |
| JP2007197649A (en) | Sponge comprised of polysaccharide material | |
| Nam et al. | Evaluations of poly (vinyl alcohol)/alginate hydrogels cross-linked by γ-ray irradiation technique | |
| JP5076141B2 (en) | Composition for tissue regeneration and scaffold using the same | |
| KR20130111849A (en) | Bilayer hydrogel moist wound dressing containig amniotic membranes-loaded and method for preparing the same | |
| CN118001452A (en) | Recombinant collagen sponge and manufacturing method thereof | |
| KR100608192B1 (en) | A Method for Producing a Neutralized Chitosan Sponge for Wound Dressing and Scaffold, and the Chitosan Sponge produced by the same | |
| CN107261189B (en) | Preparation method of sisal fiber environment-friendly medical gauze | |
| KR100459494B1 (en) | Method for the preparation of hydrogels for wound dressings | |
| CN111481731A (en) | Collagen-chitosan electrostatic spinning membrane composite alginate anti-seawater soaking application and preparation method thereof | |
| EP3838302B1 (en) | Sandwich and composite collagen sponge for controlled release of active substances, and method of preparation thereof | |
| CN112121228B (en) | Bone defect cavity filling implant | |
| KR20180076063A (en) | Wound dressing materials based on dextran and manufacturing method thereof | |
| CN113842492A (en) | Hydrogel/non-woven material composite dressing loaded with centella asiatica extract | |
| CN111450315A (en) | Inflammation-inhibiting hydrogel-coated polypropylene patch and preparation method thereof | |
| KR101080380B1 (en) | Film for adhesion of hydrogel having enhanced adhesive strength and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |