CN105214128B - Collagen sponge with calcium ions enriched on surface and preparation method thereof - Google Patents
Collagen sponge with calcium ions enriched on surface and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a collagen sponge with calcium ions enriched on the surface and a preparation method thereof, wherein the preparation method comprises the following steps of S1, dissolving collagen in an aqueous solution of acetic acid, carrying out freeze drying to obtain the collagen sponge, S2, carrying out cross-linking on the collagen sponge by using glutaraldehyde, S3, soaking the collagen sponge in a calcium chloride solution of 0.01-0.1 mol/L for 1-12 hours, taking out, placing in a chromatographic column, washing for 2-4 hours, and carrying out freeze drying to obtain the collagen sponge with calcium ions enriched on the surface.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a collagen sponge with hemostatic and anti-adhesion effects and enriched calcium ions on the surface and a preparation method thereof.
Background
Intraoperative hemorrhage is a common symptom in surgical treatment. Blood loss can reduce the liquid in blood vessels to cause hypotension, can cause the oxygen supply of organism tissues to reduce and injure the organism, and a large amount of blood loss even can cause serious consequences such as hemorrhagic shock, and the like, thereby directly endangering the life safety of people. Therefore, the bleeding site must be subjected to a hemostatic operation at the first time during the operation.
Postoperative adhesion is also one of the important problems to be solved clinically at present. It has been reported that 50-100% of abdominal and pelvic surgeries can lead to postoperative adhesions of different degrees, which can lead to severe complications such as intestinal obstruction, infertility, chronic pelvic pain, etc.; post-craniotomy adhesions may cause post-operative epilepsy; adhesion after thyroid surgery may lead to secondary recurrent laryngeal nerve injury and the like. The symptoms caused by postoperative adhesion need to be treated by a conservative method of diet prohibition, intravenous infusion, taking traditional Chinese medicine and the like, even by secondary operation, so that the pain, economic burden and treatment risk of patients are increased.
Because the bleeding and postoperative adhesion sites are relatively close to each other during surgery, there is a need for an absorbable implantation device with hemostatic and anti-adhesion functions, which can effectively stop bleeding at the bleeding site during surgery and prevent postoperative adhesion of the viscera at the operation site. Meanwhile, the implantation instrument needs to be degradable in vivo, so that the implantation instrument is gradually metabolized and absorbed by a human body along with the recovery of surgical wounds, and the structure and physiological functions of normal tissues are not influenced after the patient recovers.
Collagen (collagen) is a main component of many tissues such as human bones, skin, tendons, cartilage, etc., and at present, a collagen sponge material has been developed for intraoperative hemostasis. The collagen sponge has good biocompatibility, can be degraded and absorbed in vivo, and can play a certain role in the application of hemostasis in operation. However, the existing collagen sponge products only rely on the porous structure and the hydrophilic property to absorb the water in the blood, and can only be used for tamponade hemostasis, and the effect is very limited.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a collagen sponge with rich calcium ions on the surface aiming at the defect of limited hemostatic effect of the existing collagen sponge product. The invention also provides the collagen sponge with the surface rich in calcium ions, which is prepared by the preparation method. The collagen molecules on the surface of the collagen sponge are combined with calcium ions with a certain concentration on a specific site, so that the blood coagulation effect can be effectively exerted; and the blood coagulation factor I, II, III and the like are cooperated to promote blood coagulation. The collagen sponge also has the effect of preventing postoperative adhesion.
In a first aspect of the present invention, there is provided a method for preparing a collagen sponge enriched in calcium ions on the surface, comprising:
step S1, dissolving collagen in an acetic acid water solution, and performing freeze drying to obtain collagen sponge;
step S2, crosslinking the collagen sponge by glutaraldehyde;
step S3, binding calcium ions on the surface of the collagen sponge, specifically comprising:
s3-1, preparing 0.01-0.1 mol/L of calcium chloride aqueous solution;
step S3-2, soaking the collagen sponge obtained in the step S2 in the calcium chloride solution prepared in the step S3-1 for 1-12 hours;
step S3-3, taking the collagen sponge obtained in the step S3-2 out of the calcium chloride solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge for 2-4 hours by using flowing pure water to remove the residual calcium chloride solution;
and S3-4, carrying out freeze drying on the collagen sponge obtained in the S3-3 to obtain the collagen sponge with the surface enriched with calcium ions.
In the method for preparing a collagen sponge with a surface enriched with calcium ions according to the present invention, the step S1 specifically includes:
s1-1, dissolving collagen in an aqueous solution of acetic acid to prepare a collagen solution, wherein the concentration of the collagen is 0.01-0.03 g/m L;
and S1-2, filling the collagen solution obtained in the step S1-1 into a mould, and then carrying out freeze drying to obtain the collagen sponge.
In the method for preparing a collagen sponge with a surface rich in calcium ions according to the present invention, the collagen solution is filled in the mold in the thickness of no more than 2cm in step S1-2, so as to prevent incomplete solvent removal from affecting the freeze-drying effect.
In the method for preparing a collagen sponge with a surface enriched with calcium ions according to the present invention, the step S2 specifically includes:
s2-1, preparing 0.005-0.25 wt% of ethanol solution of glutaraldehyde as a cross-linking agent solution;
step S2-2, soaking the collagen sponge obtained in the step S1 in the cross-linking agent solution prepared in the step S2-1 for 24-48 hours, and carrying out cross-linking;
and S2-3, taking the collagen sponge obtained in the step S2-2 out of the cross-linking agent solution, putting the collagen sponge into a chromatographic column, and washing for 48-72 hours to remove the residual cross-linking agent.
In the method for preparing a collagen sponge with a surface rich in calcium ions according to the present invention, the washing in step S2-3 is water washing, ethanol washing, or a combination of water washing and ethanol washing.
In a second aspect of the present invention, there is also provided a collagen sponge with a surface enriched with calcium ions, which is prepared by the above method for preparing a collagen sponge with a surface enriched with calcium ions.
In the collagen sponge with the surface enriched with calcium ions, the content of the calcium ions bound to the surface of the collagen sponge with the surface enriched with calcium ions is 1.5 × 10 per cubic centimeter of surface binding of the collagen sponge-6～3.0×10- 6And (5) mol of calcium ions.
In the collagen sponge surface-enriched with calcium ions according to the present invention, the collagen sponge surface-enriched with calcium ions has a high porosity of 90% to 99%.
The collagen sponge with the surface enriched with calcium ions and the preparation method thereof have the following beneficial effects: the collagen sponge with the surface rich in calcium ions prepared by the invention has high porosity, can quickly absorb water in blood to stop bleeding, can effectively play a blood coagulation role by the calcium ions combined on the surface, and can promote blood coagulation by the synergistic effect of the calcium ions and blood coagulation factors I, II, III and the like; in addition, the collagen sponge can be attached to the surface of a tissue organ to play a role in preventing postoperative adhesion, so that the collagen sponge with the surface rich in calcium ions has a good postoperative blood stopping effect and a postoperative anti-adhesion effect.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a flow chart of the preparation method of the collagen sponge with calcium ion enriched surface according to the present invention;
FIG. 2 is a Fourier transform infrared spectrum of a collagen sponge with calcium ions enriched on the surface according to the present invention;
FIG. 3 is a scanning electron microscope photograph of the collagen sponge with calcium ions enriched on the surface.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.
The invention provides a collagen sponge with calcium ions enriched on the surface and a preparation method thereof. Calcium ion (Ca)2+) The blood coagulation factor is the IV-type blood coagulation factor of human body, and is also the accessory factor of other various blood coagulation factors, and plays an important role in the blood coagulation process. It has been shown that there are many Ca on collagen molecules2+The binding sites, such as carbonyl, carboxyl, etc., can be enriched with Ca on the surface of the collagen material by a certain method2+. Therefore, if Ca is added2+Effectively combined on the surface of the collagen sponge, and activates the relevant blood coagulation factors of the human body when the collagen sponge is filled for hemostasis, thereby greatly improving the hemostatic effect of the collagen sponge material.
Please refer to fig. 1, which is a flowchart illustrating a method for preparing a collagen sponge with a surface enriched with calcium ions according to the present invention. As shown in fig. 1, the preparation method of the collagen sponge with the surface enriched with calcium ions provided by the invention mainly comprises the following operation steps:
step S1, preparing a collagen sponge, specifically comprising:
s1-1, dissolving collagen in an aqueous solution of acetic acid to prepare a collagen solution, wherein the concentration of the collagen is 0.01-0.03 g/m L;
and S1-2, filling the collagen solution obtained in the step S1-1 into a mold with a certain shape, and then carrying out freeze drying to obtain the collagen sponge.
Step S2, crosslinking the collagen sponge, specifically comprising:
s2-1, preparing 0.005-0.25 wt% of ethanol solution of glutaraldehyde as a cross-linking agent;
s2-2, soaking the collagen sponge obtained in the S1-2 in the cross-linking agent solution prepared in the S2-1 for 24-48 hours for cross-linking;
and S2-3, taking the collagen sponge obtained in the S2-2 out of the cross-linking agent solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge for 48-72 hours by using flowing pure water to remove the residual cross-linking agent.
Step S3, binding calcium ions on the surface of the collagen sponge, specifically comprising:
s3-1, preparing 0.01-0.1 mol/L of calcium chloride aqueous solution;
step S3-2, soaking the collagen sponge washed in the step S2-3 in the calcium chloride solution prepared in the step S3-1 for 1-12 hours;
step S3-3, taking the collagen sponge obtained in the step S3-2 out of the calcium chloride solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge for 2-4 hours by using flowing pure water to remove the residual calcium chloride solution;
and S3-4, carrying out freeze drying on the collagen sponge obtained in the S3-3 to obtain the collagen sponge with the surface enriched with calcium ions.
In step S1-2, the collagen solution is filled in the mold to a thickness of no more than 2cm, so as to prevent incomplete solvent removal from affecting the freeze-drying effect.
In addition to water washing, the cross-linking agent removing process of step S2-3 may also use ethanol washing, or a combination of water washing and ethanol washing.
The collagen sponge with the surface enriched with calcium ions is prepared by the method. The collagen sponge with the surface enriched with calcium ions comprises the outer surface of the collagen sponge and the inner surface of a pore structure, has high porosity of 90-99%, and is communicated with each other. Microcosmically, Ca is combined on the carbonyl and carboxyl equipotential points of collagen molecules on the surface of the collagen sponge2+. Ca bound to the surface of the collagen sponge2+In an amount of 1.5 × 10 per cubic centimeter of surface bonding of the collagen sponge-6～3.0×10-6molCa2+. And the collagen sponge with the surface rich in calcium ions is subjected to cross-linking treatment in the production process, and has certain toughness and collapsibility resistance. The collagen sponge with the surface rich in calcium ions can be cut and molded freely.
Example 1
S1-1, dissolving 1g of collagen in an aqueous solution of 100m L acetic acid to prepare a collagen solution;
step S1-2, filling the collagen solution obtained in the step S1-1 into a square mould with the bottom side length of 8cm, and then carrying out freeze drying to obtain collagen sponge;
s2-1, preparing 0.01 wt% ethanol solution of glutaraldehyde as a cross-linking agent;
step S2-2, soaking the collagen sponge obtained in the step S1-2 in the cross-linking agent solution prepared in the step S2-1 for 24 hours for cross-linking;
step S2-3, taking the collagen sponge obtained in the step S2-2 out of the cross-linking agent solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge with flowing pure water for 60 hours to remove the residual cross-linking agent;
s3-1, preparing 0.02 mol/L of calcium chloride aqueous solution;
step S3-2, soaking the collagen sponge washed in the step S2-3 in the calcium chloride solution prepared in the step S3-1 for 3 hours;
step S3-3, taking the collagen sponge obtained in the step S3-2 out of the calcium chloride solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge with flowing pure water for 2 hours to remove the residual calcium chloride solution;
and S3-4, carrying out freeze drying on the collagen sponge obtained in the S3-3 to obtain the collagen sponge with the surface enriched with calcium ions.
Ca on collagen sponge with enriched calcium ion surface of this example was detected by inductively coupled plasma mass spectrometry (ICP-MS)2+In an amount of 1.7 × 10 per cubic centimeter of surface bonding of the collagen sponge-6molCa2+。
Example 2
S1-1, dissolving 1.5g of collagen in an aqueous solution of 100m L acetic acid to prepare a collagen solution;
step S1-2, filling the collagen solution obtained in the step S1-1 into a square mould with the bottom side length of 8cm, and then carrying out freeze drying to obtain collagen sponge;
s2-1, preparing 0.05 wt% ethanol solution of glutaraldehyde as a cross-linking agent;
step S2-2, soaking the collagen sponge obtained in the step S1-2 in the cross-linking agent solution prepared in the step S2-1 for 36 hours for cross-linking;
step S2-3, the collagen sponge obtained in step S2-2 is taken out from the cross-linking agent solution, placed in a chromatographic column, washed with running absolute ethyl alcohol for 36 hours, and further washed with pure water for 24 hours to remove the residual cross-linking agent.
Step S3, binding calcium ions on the surface of the collagen sponge, specifically comprising:
s3-1, preparing 0.05 mol/L of calcium chloride aqueous solution;
step S3-2, soaking the collagen sponge washed in the step S2-3 in the calcium chloride solution prepared in the step S3-1 for 4 hours;
step S3-3, taking the collagen sponge obtained in the step S3-2 out of the calcium chloride solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge with flowing pure water for 3 hours to remove the residual calcium chloride solution;
and S3-4, carrying out freeze drying on the collagen sponge obtained in the S3-3 to obtain the collagen sponge with the surface enriched with calcium ions.
Ca on the collagen sponge enriched in calcium ions on the surface thereof according to ICP-MS detection2+In an amount of 2.2 × 10 per cubic centimeter of surface bonding of the collagen sponge-6mol Ca2+。
Fig. 2 shows a Fourier Transform Infrared (FTIR) spectrum of a collagen sponge with a surface enriched with calcium ions according to the present invention. Wherein a is unbound Ca2+B is the collagen sponge prepared in example 1, c is the collagen sponge prepared in example 2, and I, II and III are infrared absorption peaks of amide I, amide II and amide III in collagen molecules respectively. From FTIR spectrum, it is known that Ca is accompanied2+The intensity of the absorption peak of the amide bond in the collagen molecule is reduced, and the infrared absorption peak of the amide I bond shifts to a low wave number. It was confirmed that in the collagen molecules of examples 1 and 2, Ca was reacted at the site of amide bond2+Thereby affecting the infrared absorption properties of the amide bond, resulting in a change in the intensity and position of the absorption peak on the FTIR spectrum.
Example 3
S1-1, dissolving 2.5g of collagen in an aqueous solution of 100m L acetic acid to prepare a collagen solution;
step S1-2, filling the collagen solution obtained in the step S1-1 into a rectangular mould with the bottom side length of 6 × 9cm, and then carrying out freeze drying to obtain collagen sponge;
s2-1, preparing 0.1 wt% ethanol solution of glutaraldehyde as a cross-linking agent;
step S2-2, soaking the collagen sponge obtained in the step S1-2 in the cross-linking agent solution prepared in the step S2-1 for 36 hours for cross-linking;
step S2-3, taking the collagen sponge obtained in the step S2-2 out of the cross-linking agent solution, placing the collagen sponge in a chromatographic column, washing the collagen sponge with flowing absolute ethyl alcohol for 48 hours, and then washing the collagen sponge with pure water for 24 hours to remove the residual cross-linking agent;
s3-1, preparing 0.08 mol/L of calcium chloride aqueous solution;
step S3-2, soaking the collagen sponge washed in the step S2-3 in the calcium chloride solution prepared in the step S3-1 for 6 hours;
step S3-3, taking the collagen sponge obtained in the step S3-2 out of the calcium chloride solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge with flowing pure water for 4 hours to remove the residual calcium chloride solution;
and S3-4, carrying out freeze drying on the collagen sponge obtained in the S3-3 to obtain the collagen sponge with the surface enriched with calcium ions.
Ca on the collagen sponge enriched in calcium ions on the surface thereof according to ICP-MS detection2+In an amount of 2.8 × 10 per cubic centimeter of surface bonding of the collagen sponge-6mol Ca2+。
The hemostatic effect of the collagen sponge material prepared in the above examples was evaluated by adding Ca-free material2+The collagen sponge and the common medical gauze are used for hemostasis evaluation for comparison, and the specific operation steps are as follows:
1. establishing a rabbit femoral artery blood loss model: anaesthetizing rabbits, depilating, disinfecting, separating femoral artery, and then cutting off the femoral artery;
2. immediately using No. 1-3 collagen sponge without Ca2+Collagen sponge and common medical gauze to femoral arteryHemostasis was performed and the time to hemostasis and the amount of blood lost were recorded.
The hemostatic results of the collagen sponge with calcium ion enriched surface of the invention are shown in table 1:
table 1
| Product numbering | Hemostasis time (second) | Blood loss (gram) |
| Example 1 | 117.37±10.29 | 0.80±0.24 |
| Example 2 | 104.09±12.28 | 0.69±0.14 |
| Example 3 | 120.44±13.50 | 0.82±0.13 |
| Contains no Ca2+Collagen sponge of | 145.37±7.79 | 1.09±0.27 |
| Common gauze | 233.63±21.27 | 3.90±1.21 |
According to the hemostasis experimental dataIt can be seen that the wound is treated with a collagen sponge enriched in calcium ions on the surface, and the hemostatic time is shorter than that of Ca-free collagen sponge2+The collagen sponge is shortened by 17.15-28.40 percent, and is shortened by about 50 percent compared with the wound treated by common gauze. In terms of blood loss, the collagen sponge with enriched calcium ions on the surface is less Ca-free2+The collagen sponge is reduced by 24.77-36.70%, and the blood loss is reduced by about 80% compared with the common gauze for treating the wound, so that the collagen sponge has a good hemostatic effect.
The collagen sponge material prepared in the above examples was evaluated for anti-adhesion effect, and Ca-free collagen sponge material was used2+The collagen sponge and the control group without any anti-adhesion membrane were compared for evaluation of hemostasis, and the following specific procedures were carried out:
1. establishing a rabbit achilles tendon injury model: anaesthetizing rabbits, depilating, disinfecting, separating the achilles tendon, and scraping on the achilles tendon by using a scalpel to establish a 2 cm-long injury model;
2. immediately using collagen sponge No. 1-3 and containing no Ca2+The collagen sponge of (a) was wrapped around the achilles tendon injury and adhesion was observed and scored after 5 days.
The anti-adhesion experimental result of the collagen sponge with enriched calcium ions on the surface is shown in the table 2:
table 2
| Product numbering | Tendon injury adhesion length | Tendon injury adhesion score |
| Example 1 | 0.5±0.1 | 1 |
| Example 2 | 0 | 0 |
| Example 3 | 0.6±0.1 | 1 |
| Contains no Ca2+Collagen sponge of | 1.5±0.2 | 2 |
| Blank control | 2.6±0.3 | 3 |
According to the anti-adhesion experimental data, the collagen sponge with the surface rich in calcium ions is used for treating the injury of the achilles tendon, so that the achilles tendon can be effectively prevented from being adhered to autologous tissues in the repairing process after operation, and the anti-adhesion effect is good.
In conclusion, the present invention obtains a collagen sponge with a surface rich in calcium ions, the collagen sponge has a high porosity, and collagen molecules on the surface of the collagen sponge are combined with a certain concentration of calcium ions at specific sites. When used for intraoperative hemostasis, the collagen sponge has Ca bound to its surface, except for hemostasis by rapidly absorbing water in blood2+Can also effectively play a blood coagulation role, and can be synergistically acted with blood coagulation factors I, II, III and the like to promote blood coagulation. The collagen sponge can be attached to the surface of tissue and organs, thereby playing a role in preventing postoperative adhesion. In conclusion, the collagen sponge with the surface rich in calcium ions provided by the invention has good blood stopping effect and postoperative anti-adhesion effect, and has wide application prospect.
While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (6)
1. A method for preparing a collagen sponge with calcium ions enriched on the surface, which comprises the following steps:
step S1, dissolving collagen in an acetic acid water solution, and performing freeze drying to obtain collagen sponge;
step S2, crosslinking the collagen sponge by glutaraldehyde, which specifically comprises:
s2-1, preparing 0.005-0.25 wt% of ethanol solution of glutaraldehyde as a cross-linking agent solution;
step S2-2, soaking the collagen sponge obtained in the step S1 in the cross-linking agent solution prepared in the step S2-1 for 24-48 hours, and carrying out cross-linking;
s2-3, taking the collagen sponge obtained in the S2-2 out of the cross-linking agent solution, placing the collagen sponge in a chromatographic column, and washing for 48-72 hours to remove residual cross-linking agents;
step S3, binding calcium ions on the surface of the collagen sponge, specifically comprising:
s3-1, preparing 0.01-0.1 mol/L of calcium chloride aqueous solution;
step S3-2, soaking the collagen sponge obtained in the step S2 in the calcium chloride solution prepared in the step S3-1 for 1-12 hours;
step S3-3, taking the collagen sponge obtained in the step S3-2 out of the calcium chloride solution, placing the collagen sponge in a chromatographic column, and washing the collagen sponge for 2-4 hours by using flowing pure water to remove the residual calcium chloride solution;
step S3-4, carrying out freeze drying on the collagen sponge obtained in the step S3-3 to obtain the collagen sponge with the surface enriched with calcium ions;
the content of calcium ions bound to the surface of the collagen sponge with the enriched calcium ions on the surface is 1.5 × 10 per cubic centimeter of surface binding of the collagen sponge-6～3.0×10-6mol calcium ion。
2. The method for preparing a collagen sponge with calcium ion enriched surface as claimed in claim 1, wherein the step S1 specifically comprises:
s1-1, dissolving collagen in an aqueous solution of acetic acid to prepare a collagen solution, wherein the concentration of the collagen is 0.01-0.03 g/m L;
and S1-2, filling the collagen solution obtained in the step S1-1 into a mould, and then carrying out freeze drying to obtain the collagen sponge.
3. The method for preparing a collagen sponge with calcium ion enriched surface according to claim 2, wherein the collagen solution is filled in the mold to a thickness of not more than 2cm in step S1-2.
4. The method for preparing a collagen sponge with calcium ions enriched on the surface according to claim 1, wherein the washing in step S2-3 is water washing, ethanol washing, or a combination of water washing and ethanol washing.
5. A collagen sponge with calcium ions enriched on the surface, which is prepared by the method for preparing the collagen sponge with calcium ions enriched on the surface as claimed in any one of claims 1 to 4.
6. The collagen sponge with calcium ions enriched on the surface according to claim 5, wherein the porosity of the collagen sponge with calcium ions enriched on the surface is 90-99%.
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Effective date of registration: 20200603 Address after: 102600 north side of floor 1, floor 2, floor 3, floor 4, building 2, No. 26, Yongwang West Road, Daxing biomedical industry base, Zhongguancun Science and Technology Park, Daxing District, Beijing Applicant after: Beijing Jing Jing Medical Instrument Co.,Ltd. Address before: 7 liberty Ridge Road, basigingrich, NJ Applicant before: Cui Han |
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