CN115747993A - Collagen fiber with high biocompatibility and high stability, and preparation method and application thereof - Google Patents
Collagen fiber with high biocompatibility and high stability, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedical materials, and particularly relates to a collagen fiber with high biocompatibility and high stability, a preparation method and an application thereof, wherein the preparation method comprises the following steps: (1) constructing collagen fibers; (2) crosslinking the collagen fibers by using a crosslinking agent; (3) dialyzing, homogenizing, centrifuging and collecting precipitates; the prepared cross-linked collagen fibers are closely stacked and highly cross-linked, and have good thermal stability and enzymolysis resistance; the cross-linked collagen fiber has high biocompatibility and promotes the proliferation of fibroblasts; has high biological safety, and the inflammatory reaction caused by the implantation of the implant in the body is obviously slight and is quickly eliminated; after the crosslinked collagen fibers are implanted, calcification nodes do not appear, and the crosslinked collagen fibers have excellent anti-calcification effect; the cross-linked collagen fiber implant provided by the invention can obviously promote the generation of new collagen, can be applied to the fields of implants, artificial skin, hemostatic sponges, stent materials, medical devices and the like, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a collagen fiber with high biocompatibility and high stability, a preparation method and application thereof.
Background
Collagen is a main structural component of dermis, and the content of collagen in skin is remarkably reduced with the increase of age, so that various aging phenomena such as skin wrinkles and the like are caused. Therefore, the preparation of injectable collagen implants using collagen fibers is widely used in the field of dermatology. However, the uncrosslinked collagen implant has poor stability after being implanted into a human body, and can be rapidly degraded by collagenase in the body, thereby seriously affecting the use effect of the collagen implant.
Chemical crosslinking is currently the predominant method used to crosslink collagen. Chinese patent CN113384748A discloses a method for preparing a collagen implant by crosslinking collagen hydrogel with glutaraldehyde; chinese patent CN109385682A discloses a method for preparing collagen fiber with good tensile strength by EDC/NHS crosslinking. Chinese patent CN101234216B discloses a method for preparing collagen gel with good thermal stability by adopting hydroformylation polysaccharide crosslinking. However, in these patents, the residue of a crosslinking agent such as glutaraldehyde causes health risks, and the stability of the prepared crosslinked collagen is yet to be further improved.
In view of the above technical problems, the inventors have unexpectedly found a method for preparing collagen fibers with high biocompatibility and high stability. The cross-linked collagen fibers prepared by the method are closely stacked and highly cross-linked, and have good thermal stability and enzymolysis resistance; the cross-linked collagen fiber has high biocompatibility and can remarkably promote the proliferation of fibroblasts; after the crosslinked collagen fibers are implanted, calcification nodes do not appear, and the crosslinked collagen fibers have excellent anti-calcification effect; the cross-linked collagen fiber implant provided by the invention can obviously promote collagen regeneration, can be applied to the fields of implants, artificial skin, hemostatic sponges, stent materials, medical devices and the like, and has wide application prospect.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide a collagen fiber preparation method, which comprises the following steps: (1) constructing collagen fibers; (2) crosslinking the collagen fibers by using a crosslinking agent; (3) dialyzing, homogenizing and collecting precipitates;
the method for constructing the collagen fiber in the step (1) comprises the following steps:
preparing collagen solution with concentration of 0.1-5mg/mL, adding 10mM-100mM disodium hydrogen phosphate/sodium dihydrogen phosphate solution (pH 6-8), centrifuging, and collecting precipitate; dispersing the obtained precipitate uniformly by using 20mM disodium hydrogen phosphate/sodium dihydrogen phosphate solution (pH is 6-9) to make the collagen concentration be 3mg/mL, and incubating at 17-25 deg.C for 8-19h; homogenizing the incubated collagen at 4 ℃ and 10000rpm for 20min; centrifuging to obtain collagen fiber.
Preferably, the crosslinking agent in step (2) is one or more selected from carbodiimide, epichlorohydrin, sodium metaphosphate, 1, 4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, procyanidins, hesperidin, tannic acid, genipin, riboflavin, naringenin, quercetin, epigallocatechin gallate, oleuropein, L-threose (LT), phytic acid, isocyanate, acyl azide and diphenyl phosphate, and bis (N-hydroxysuccinimide) suberate (NHS-SA).
Preferably, the crosslinking agent in the step (2) is bis (N-hydroxysuccinimide) suberate.
Preferably, the concentration of the cross-linking agent in the step (2) is 0.01-5mg/mL.
Preferably, the addition amount of the cross-linking agent in the step (2) is 4-5mg/mL.
Preferably, the reaction temperature of the cross-linking agent in the step (2) is 4-25 ℃, and the reaction time is 12-24h.
Preferably, the reaction temperature of the crosslinking agent in the step (2) is 8-25 ℃.
Preferably, the reaction temperature of the crosslinking agent in the step (2) is 8 ℃.
Preferably, the reaction time of the crosslinking agent in the step (2) is 24h.
Preferably, the collagen in step (1) is animal collagen, including type I, type II, and type III.
Preferably, the collagen of step (1) is type I collagen.
Preferably, the dialysis in step (3) is carried out by adding glycine after crosslinking, dialyzing for 48-72h in a buffer at 4-25 ℃, centrifuging and collecting the precipitate after 20-30min at 4-25 ℃ and 10000 rpm.
The second purpose of the invention is to provide the collagen fiber prepared by the preparation method.
The third purpose of the invention is to provide the application of the collagen fiber in the preparation of implants, artificial skin, hemostatic sponges, stent materials and medical devices.
The fourth purpose of the invention is to provide a collagen fiber implant, which is prepared by the collagen fiber.
The fifth purpose of the invention is to provide the application of bis (N-hydroxysuccinimide) suberate as a collagen crosslinking agent.
The beneficial effects of the invention are: (1) The invention provides a preparation method of collagen fibers, which is simple and convenient, and the prepared crosslinked collagen fibers are closely stacked and highly crosslinked and have good thermal stability and enzymolysis resistance; (2) The cross-linked collagen fiber has high biocompatibility and can obviously promote the proliferation of fibroblasts; the cross-linked collagen fiber has high biological safety, and the inflammatory reaction caused by the cross-linked collagen fiber after being implanted into a body is obviously slight and is quickly eliminated; (3) After the crosslinked collagen fibers are implanted, calcification nodes do not appear, and the crosslinked collagen fibers have excellent anti-calcification effect; (4) The crosslinked collagen fiber implant can remarkably promote the regeneration of collagen, can be applied to the fields of implants, artificial skin, hemostatic sponge, bracket materials, medical instruments and the like, and has wide application prospect; (5) The invention also provides a new application of the bis (N-hydroxysuccinimide) suberate as a collagen crosslinking agent, and collagen fibers obtained by crosslinking the bis (N-hydroxysuccinimide) suberate.
Drawings
FIG. 1 Effect of different concentrations of NHS-SA on crosslinking collagen fibers
FIG. 2 scanning electron microscope images of NHS-SA crosslinked collagen fibers at different temperatures
FIG. 3 the enzymatic resistance of NHS-SA crosslinked collagen fibers
FIG. 4 denaturation assay for NHS-SA Cross-Linked collagen fibers
FIG. 5 thermal stability of NHS-SA Cross-Linked collagen fibers
FIG. 6 cellular Activity of NHS-SA Cross-Linked collagen fibers
FIG. 7 viable/dead cell staining pattern of NHS-SA crosslinked collagen fibers
FIG. 8 photograph of mouse skin implanted with NHS-SA crosslinked collagen fibers
FIG. 9 HE staining of tissue from implantation of NHS-SA Cross-Linked collagen fibers into mouse skin
FIG. 10 Masson staining of tissue of NHS-SA Cross-Linked collagen fibers implanted into mouse skin
FIG. 11 tissue alizarin Red staining of NHS-SA Cross-Linked collagen fibers implanted into mouse skin
Detailed Description
The present invention is described in detail by the following specific embodiments, and any technical solutions that can be conceived by a person skilled in the art based on the present invention and the common general knowledge in the field are all within the protection scope of the present invention.
Refer to Chinese patent CN 112778412A to prepare medical grade yak collagen, the steps are as follows: cleaning yak tendon, removing foreign matter, and pulverizing into small pieces; degreasing the crushed small blocks with 10% n-butanol, taking the precipitate, and washing the precipitate to be neutral; decalcifying the precipitate with 0.5M hydrochloric acid, collecting precipitate, and washing with water to neutrality; soaking the tissue precipitate in 0.1M sodium hydroxide solution, stirring at 25 deg.C for 4 hr, collecting precipitate, and washing with water to neutrality; extracting collagen with 0.5M acetic acid solution containing 1g/L pepsin to obtain crude collagen extractive solution; adjusting the pH value to be neutral, and performing enzyme inactivation; dialyzing with 8-14kDa dialysis bag, and freeze-drying after dialysis to obtain collagen with low endotoxin content.
EXAMPLE I preparation and characterization of Cross-Linked collagen fibers with different concentrations of NHS-SA
1.1 preparation of collagen fibers
(1) Preparing a collagen solution: dissolving collagen powder in water, adding 0.5M acetic acid and/or hydrochloric acid and/or phosphoric acid solution, adjusting pH to 3.0-5.0 to obtain clear collagen solution with collagen concentration of 0.1-5mg/mL;
(2) inducing self-assembly: adding 10mM-100mM disodium hydrogen phosphate/sodium dihydrogen phosphate solution (pH 6-8) into the prepared collagen acidic solution, standing for 1.5 hr, centrifuging, and collecting collagen precipitate;
(3) redissolving, namely uniformly dispersing the obtained collagen precipitate by adopting a 20mM disodium hydrogen phosphate/sodium dihydrogen phosphate solution (the pH is 6-9), wherein the concentration of the collagen is 3mg/mL;
(4) and (3) incubation: incubating the re-dissolved collagen solution at a constant temperature of 17-25 ℃ for 8-19h;
(5) homogenizing, namely homogenizing the incubated collagen for 20min at the temperature of 4 ℃ and the rotating speed of 10000 rpm;
(6) sieving: sieving the homogenized collagen by using a 40-mesh sieve;
(7) centrifuging: and collecting the sieved collagen solution, and centrifuging to obtain the collagen fiber.
1.2 preparation of NHS-SA Cross-Linked collagen fibers at different concentrations
Taking the prepared collagen fiber, preparing the collagen fiber with the concentration of 3mg/mL, respectively adding NHS-SA solutions of 0.25, 1.25, 2.5, 4 and 5mg/mL, crosslinking at 8 ℃ and 220rpm for 24h, adding glycine to remove unreacted NHS-SA, dialyzing with 0.02M PBS (pH 7.4) for 72h, homogenizing at 10000rpm for 20min, centrifuging at 10000rpm and 30min, and collecting the obtained precipitate, namely the crosslinked collagen fiber.
1.3 characterization of the degree of crosslinking
The crosslinking degree of crosslinked collagen was measured by the TNBS method, the collagen before and after crosslinking was freeze-dried, 11mg of the freeze-dried sample before and after crosslinking was taken and placed in 4% sodium bicarbonate, 0.5% TNBS was added thereto, after reaction at 40 ℃ for 4 hours, 6mol/L HCl was added thereto, after reaction at 60 ℃ for 1.5 hours, after dilution was carried out, anhydrous ether was added to remove unreacted TNBS, and then the ultraviolet absorbance at 345nm was measured to calculate the crosslinking degree.
Calculation of the degree of crosslinking: α = (W) 1 -W 0 )-(W 2 -W 0 )/(W 1 -W 0 )×100％
Wherein a: a degree of crosslinking; wo: absorbance of the background; w is a group of 1 : absorbance of uncrosslinked collagen; w 2 : absorbance of collagen after crosslinking.
The results of the UV detection are shown in FIG. 1, and the degree of crosslinking is calculated. With increasing concentrations of the cross-linking agent (0.25, 1.25, 2.5, 4, 5 mg/mL), the degree of cross-linking of NHS-SA cross-linked collagen fibers gradually increased from 67% to 85%, 96%, 100%, and 100%. The results show that NHS-SA crosslinked collagen fiber has high crosslinking degree, and NHS-SA is one kind of efficient collagen crosslinking agent.
EXAMPLE two preparation and characterization of Cross-Linked collagen fibers at different temperatures
2.1 preparation of collagen fibers
The preparation method refers to the first embodiment.
2.2 preparation of Cross-Linked collagen fibers at different temperatures
Taking the prepared long-acting collagen fiber, preparing the concentration of 3mg/mL, adding 4mg/mL NHS-SA solution into the long-acting collagen fiber, respectively crosslinking at 8 ℃ and 25 ℃ after 24h of crosslinking, adding glycine to remove unreacted NHS-SA, dialyzing for 72h, homogenizing for 20min at the rotation speed of 10000rpm, centrifuging (10000 rpm and 30min) after the homogenization is finished, and collecting the obtained precipitate, namely the crosslinked collagen fiber.
2.3 characterization of the degree of crosslinking
Samples of NHS-SA crosslinked collagen fibers at different temperatures are subjected to freeze drying, 11mg of NHS-SA crosslinked collagen fibers and 11mg of freeze-dried samples of uncrosslinked collagen fibers are weighed respectively and placed in 4% of sodium bicarbonate, 0.5% of TNBS is added into the samples, the mixture is reacted for 4 hours at 40 ℃, 6mol/L of HCl is added into the mixture, the mixture is reacted for 1.5 hours at 60 ℃, absolute ethyl ether is added into the mixture after dilution to remove the unreacted TNBS, and then the ultraviolet absorbance at 345nm is measured to calculate the crosslinking degree.
Calculation of the degree of crosslinking: α = (W) 1 -W 0 )-(W 2 -W 0 )/(W 1 -W 0 )×100％
Wherein α: a degree of crosslinking; wo: absorbance of the background; w is a group of 1 : absorbance of uncrosslinked collagen; w 2 : absorbance of collagen after crosslinking.
The cross-linking degree was calculated and the results are shown in table 1, where the cross-linking degree of NHS-SA cross-linked collagen fibers at 25 ℃ at 8 ℃ was 100%, indicating that NHS-SA can also achieve highly efficient cross-linking of collagen fibers at low temperature.
TABLE 1 Cross-linking degree results for cross-linking collagen fibers at different temperatures
| Temperature of | Degree of crosslinking |
| 8℃ | 100％ |
| 25℃ | 100％ |
2.4 characterization by scanning Electron microscope
The freeze-dried collagen sample obtained in example 2.2 after cross-linking was sliced and sampled, and subjected to SEM characterization after gold spraying.
The results of the experiment are shown in fig. 2, the NHS-SA crosslinked collagen fibers are closely arranged to form a uniform fiber network structure.
Comparative example preparation and characterization of glutaraldehyde crosslinked collagen fibers
1. Preparation of glutaraldehyde crosslinked collagen fiber
Adding glutaraldehyde into the collagen fiber solution with the concentration of 3mg/mL, stirring the mixture for 24 hours at the temperature of 37 ℃ and pH7.4, and centrifuging the collected collagen fibers after the crosslinking reaction is finished.
2. Characterization of the degree of crosslinking of glutaraldehyde crosslinked collagen fibers
Untreated collagen, 11mg of samples lyophilized for glutaraldehyde crosslinked collagen fibers of comparative example and NHS-SA crosslinked collagen fibers prepared in example III were weighed, respectively, placed in 4% sodium bicarbonate, 0.5% TNBS was added thereto, reacted at 40 ℃ for 4 hours, then 6mol/L HCl was added thereto, reacted at 60 ℃ for 1.5 hours, diluted, and then anhydrous ether was added to remove unreacted TNBS, and then the ultraviolet absorbance at 345nm was measured to calculate the degree of crosslinking.
Calculation of the degree of crosslinking: α = (W) 1 -W 0 )-(W 2 -W 0 )/(W 1 -W 0 ) X100%, wherein α: a degree of crosslinking; wo: absorbance of the background; w 1 : absorbance of uncrosslinked collagen; w is a group of 2 : absorbance of collagen after crosslinking.
The experimental results are shown in Table 2, the crosslinking degree of the crosslinked collagen fibers is 85% when the glutaraldehyde concentration is 5mg/mL, and the crosslinking degree of the crosslinked collagen fibers can reach 100% when the NHS-SA concentration is 4mg/mL, which indicates that NHS-SA is a more efficient collagen crosslinking agent.
TABLE 2 comparison of Cross-linking of glutaraldehyde cross-linked collagen fibers with NHS-SA cross-linked collagen fibers
| Type (B) | Degree of crosslinking |
| Glutaraldehyde crosslinking | 85％ |
| Crosslinking with NHS- |
100％ |
The collagen fibers prepared in this comparative example were used in the control groups in the following examples.
EXAMPLE III preparation and characterization of NHS-SA Cross-Linked collagen fibers
Preparation of NHS-SA Cross-Linked collagen fibers
Taking the prepared long-acting collagen fiber, preparing the concentration of the long-acting collagen fiber to be 3mg/mL, adding 4mg/mL NHS-SA solution into the long-acting collagen fiber, crosslinking the long-acting collagen fiber at 8 ℃ for 24 hours, adding glycine to remove unreacted NHS-SA, dialyzing the long-acting collagen fiber for 72 hours, homogenizing the long-acting collagen fiber at the rotation speed of 10000rpm for 20 minutes, centrifuging the long-acting collagen fiber (10000rpm and 30min) after the homogenization is finished, and collecting the obtained precipitate to obtain the crosslinked collagen fiber.
The collagen fibers used in the examples of the subsequent experiments were prepared by the method described above in this example.
2. Enzymolysis test
10mg of untreated collagen fibers were weighed out as blanks, glutaraldehyde-crosslinked collagen fibers prepared for the comparative example, and NHS-SA-crosslinked collagen fibers prepared for example three, respectively, and the initial dry weight (about 10 mg) was recorded. Formulated glueProenzyme was dissolved in buffer solution (TES, 1mM CaCl) 2 pH 7.4), collagenase concentration 5U/mL,37 ℃, and carrying out enzymolysis experiments at 220rpm, washing the sample after enzymolysis for the same time, centrifuging, freeze-drying the centrifuged sample, weighing, and calculating the enzymolysis rate.
AW％＝(Wo-W)/W×100％
Wherein AW%: the enzymolysis rate; wo: initial weight of sample before enzymolysis; w: weight of sample after enzymolysis.
The experimental results are shown in fig. 3, and the enzymolysis rates of the blank group (untreated collagen fibers) on days 1, 2, 3 and 7 are respectively 25%, 94%, 98% and 100%; the enzymatic hydrolysis rates of comparative examples (glutaraldehyde-crosslinked collagen fibers prepared) on days 1, 2, 3 and 7 were 38%, 41%, 45% and 47%, respectively; the enzymatic hydrolysis rates of the experimental group (NHS-SA crosslinked collagen fibers prepared in example three) on days 1, 2, 3 and 7 were 4%, 11%, 14% and 23%, respectively. The results show that glutaraldehyde crosslinking can improve the enzymolysis resistance of the collagen fibers, but the enzymolysis resistance of the NHS-SA crosslinked collagen fibers is more remarkable.
3. Denaturation detection
Taking the non-crosslinked collagen fiber leaching solution with the concentration of 0.01mg/mL and the NHS-SA crosslinked collagen fiber leaching solution prepared in the third embodiment with the concentration of 0.01 mg/mL; 0.01mg/mL of boiling denatured type I collagen and 0.01mg/mL of gelatin were prepared, 100. Mu.L of each sample was put in a 96-well plate in parallel 5 times, and after 12 hours at 4 ℃ in the dark, the supernatant was aspirated. Targeting denatured collagen at a concentration of 20. Mu.M to fluorescent probe FAM- (GPO) 10 After heating at 90 ℃ for 20min, ice water was added to the reaction mixture immediately after cooling, 100. Mu.L of each of the solutions was added to each sample well, and after washing with 200. Mu.L of 10mM PBS (pH 7.4) for 3 times at 4 ℃ in a dark environment, the fluorescence intensity was measured with a microplate reader.
As shown in FIG. 4, the boiling denatured type I collagen and gelatin were combined with the denatured collagen targeting fluorescent probe FAM- (GPO) 10 After binding, very strong fluorescence intensity was exhibited (42754 and 35839); while the uncrosslinked collagen fiber with complete triple-helical structure and the collagen fiber crosslinked by NHS-SA are combined with the denatured collagen targeting fluorescent probe FAM- (GPO) 10 After binding, a similar, very weak fluorescence signal is present. The results show that the NHS-SA crosslinked collagen fiber is prepared under the conditions of low temperature and mild conditions, the complete triple-helix structure is maintained, and the possible denaturation risk in the collagen crosslinking process is completely avoided.
4. DSC characterization
5mg of the samples of the NHS-SA crosslinked collagen fiber and the uncrosslinked collagen fiber prepared in the third embodiment after freeze-drying are respectively weighed, and then the samples are placed in a crucible under the protection of nitrogen, and the thermal stability of the samples within the range of 25-120 ℃ is measured under the condition that the temperature rise rate is 5 ℃/min.
The experimental results are shown in fig. 5, and compared with the uncrosslinked collagen fibers, the heat-altered temperature of the NHS-SA crosslinked collagen fibers was significantly increased from 96 ℃ to 104 ℃, indicating that the NHS-SA crosslinked collagen fibers have better thermal stability.
Example four bioactivity and biocompatibility of NHS-SA Cross-Linked collagen fibers
1. Cytotoxicity and proliferation
To investigate the cytotoxicity and proliferation of crosslinked and uncrosslinked samples, HFF-1 human fibroblasts were treated at 37 ℃ and 5% CO 2 Cultured under the conditions with high-glucose DMEM medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin. Placing the NHS-SA crosslinked collagen fiber prepared in the third example, the glutaraldehyde crosslinked collagen fiber prepared in the comparative example and the freeze-dried sample of the uncrosslinked collagen fiber in a DMEM high-glucose culture solution, soaking for 72h at 37 ℃, and preparing a leaching solution according to a standard cytotoxicity test ISO 10993-5. Digesting the cells with 0.25% (w/w) trypsin, seeding in 96-well plates at a density of 7000 per well, at 37 ℃ and 5% 2 After 24h incubation, 100 uL/well extract of different samples was used instead of medium for 24h, 48h, 72h incubation, respectively, 10 uL Cell Counting Kit-8 (Dojindo Molecular Technologies, japan) was added, and incubation was performed at 37 ℃ for 1-3h. Basal medium was a blank (n = 6) and OD at 450nm was read. Cell viability was calculated as follows:
cell viability (%) = (A-C)/(B-C) x100%
Wherein A: OD values of different samples; b: OD value of a positive control group; c: blank OD value.
The experimental results are shown in fig. 6, wherein the cell survival rates of 24h, 48h and 72h of the uncrosslinked collagen fibers are 105%,108% and 110%, respectively, and the cell survival rates of the NHS-SA crosslinked collagen fibers are 104%,109% and 114%, respectively, while the cell survival rates of the glutaraldehyde crosslinked collagen fibers are greatly reduced to 90%,61% and 59%, respectively. The result shows that the glutaraldehyde crosslinking collagen fiber has obvious cytotoxicity, while the NHS-SA crosslinking collagen fiber has no cytotoxicity completely and has good biocompatibility.
2. Live/dead cell staining
To investigate the cell survival of the crosslinked and uncrosslinked samples, HFF-1 human fibroblasts were treated at 37 ℃ and 5% CO 2 Cultured under the conditions with high-glucose DMEM medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin. The NHS-SA crosslinked collagen fibers prepared in example three, the glutaraldehyde crosslinked collagen fibers prepared in comparative example, and the lyophilized samples of the uncrosslinked collagen fibers were placed in a DMEM high glucose medium, soaked at 37 ℃ for 72 hours, and a leaching solution was prepared according to standard cytotoxicity test ISO 10993-5. Digesting the cells with 0.25% (w/w) trypsin, inoculating in a laser confocal culture dish at a density of 40000 per well, at 37 ℃ and 5% 2 And (4) performing middle incubation for 24h, then respectively incubating for 24h and 72h by using 100 mu L/hole leaching liquor of different samples to replace a culture medium, then dyeing by using CA/PI, and then photographing by using olympus laser confocal technology.
The results are shown in FIG. 7, and the number of viable cells of 24h and 72h non-crosslinked collagen fibers is 173/mm 2 (ii) a 225 pieces/mm 2 (ii) a The number of viable cells of the 24h NHS-SA crosslinked collagen fiber and the 72h NHS-SA crosslinked collagen fiber is 184/mm 2 (ii) a 206 pieces/mm 2 (ii) a The number of viable cells of the glutaraldehyde crosslinked collagen fiber is obviously reduced at 24h and 72h, and the number of viable cells is 113/mm respectively 2 (ii) a 95 pieces/mm 2 . The results show that NHS-SA crosslinked collagen fibers have good biocompatibility and can provide beneficial conditions for cell survival.
EXAMPLE V animal experiments with NHS-SA Cross-Linked collagen fiber implants
The mouse subcutaneous implantation model is a common method for evaluating the biocompatibility of materials in vivo. Male Kunming mice (30-40 g) were used for in vivo evaluation in this study. Samples before and after crosslinking were soaked overnight in 75% (v/v) ethanol and rinsed thoroughly with sterile physiological saline. Mice without implant were blanked, and the NHS-SA crosslinked collagen fiber prepared in example three and the glutaraldehyde crosslinked collagen fiber prepared in comparative example were injected with the crosslinked collagen fiber to the subcutaneous tissue of the mice, respectively, and 5 mice were injected in parallel per group.
1. Implant degradation
Mice were anesthetized with 1mL of 10% chloral hydrate at weeks 2, 4, 6, and 8, and then shaved, sacrificed after shaving, and the skin of the implantation sites was harvested as shown in fig. 8. The NHS-SA crosslinked collagen fibers were not significantly degraded within 8 weeks, while both the glutaraldehyde crosslinked collagen fibers and the uncrosslinked collagen fibers were significantly degraded. The results show that NHS-SA crosslinked collagen fibers still maintain significant resistance to degradation in vivo.
HE staining
Anesthetizing the mice with 1mL of 10% chloral hydrate in weeks 2, 4, 6 and 8 respectively, shaving the mice, killing the mice after shaving, taking materials from the skin of the implantation part, slicing the skin of the mice with paraffin after taking the materials, and carrying out HE staining.
The experimental result is shown in fig. 9, when the NHS-SA crosslinked collagen fibers are implanted into subcutaneous tissues of mice, a mild inflammatory response of the implant subcutaneous at week 2, which is a normal foreign body response at the initial stage of implantation, can be observed; inflammatory cells were significantly reduced at week 4; there were few inflammatory cells at weeks 6 and 8. Glutaraldehyde cross-linked collagen fibers are implanted into subcutaneous tissue, and severe inflammatory reaction is generated at week 2; inflammatory cells decreased at week 4; obvious inflammatory cells still exist at weeks 6 and 8. The results show that the biological safety of the NHS-SA crosslinked collagen fiber is obviously better than that of the glutaraldehyde crosslinked collagen fiber.
Masson staining
The mice were anesthetized with 1mL of 10% chloral hydrate at weeks 2, 4, 6, and 8, shaved, sacrificed, and the skin of the implanted sites was harvested, paraffin-sectioned and Masson-stained.
The experimental results are shown in fig. 10, the NHS-SA crosslinked collagen fibers were implanted into the subcutaneous tissue of the mice, and fibroblasts were generated at the 2 nd implantation site; new collagen fibril production was observed at week 4; the newly grown collagen fibers increased greatly at weeks 6 and 8. The glutaraldehyde cross-linked collagen fibers are implanted into subcutaneous tissues of mice, and obvious fibroblasts can be observed only in 4 weeks; a small amount of nascent collagen fibers was observed at week 8. The result shows that the NHS-SA crosslinked collagen fiber can obviously promote the regeneration of collagen, and the effect is obviously superior to that of glutaraldehyde crosslinked collagen fiber.
4. Alizarin red staining
The mouse subcutaneous implantation model is a common method for evaluating the biocompatibility of materials in vivo. Male Kunming mice (30-40 g) were used for in vivo evaluation in this study. Samples before and after crosslinking were soaked overnight in 75% (v/v) ethanol and rinsed thoroughly with sterile physiological saline. The mice without implant were blanked, and the third example and the comparative example were injected with crosslinked collagen fibers into the subcutaneous tissue of the mice, respectively, and 5 mice were injected in parallel per group.
Anesthetizing the mice with 1mL of 10% chloral hydrate in weeks 2, 4, 6 and 8 respectively, shaving after using depilatory cream, killing the mice after shaving, taking materials from the skin of the implanted parts of the mice, slicing the skin of the mice with paraffin after taking the materials, and staining the mice with alizarin red.
The experimental results are shown in fig. 11, the mouse subcutaneous tissue of the collagen fiber cross-linked by glutaraldehyde has orange-red nodes in week 2; at weeks 4-8, calcification was more evident and nodes of calcification increased significantly. While subcutaneous tissue of mice implanted with NHS-SA crosslinked collagen fibers showed substantially no calcification nodes during 8 weeks. The results show that NHS-SA crosslinked collagen fibers have excellent anti-calcification ability.
In conclusion, the invention provides the collagen fiber with high biocompatibility and high stability, the preparation method and the application, the preparation method is simple and convenient, and the prepared crosslinked collagen fiber is closely stacked and highly crosslinked and has good thermal stability and enzymolysis resistance; the cross-linked collagen fiber has high biocompatibility and can remarkably promote the proliferation of fibroblasts; has high biological safety, and the inflammatory reaction caused by the implantation of the implant in the body is obviously slight and is quickly eliminated; after the crosslinked collagen fibers are implanted, calcification nodes do not appear, and the crosslinked collagen fibers have excellent anti-calcification effect; the cross-linked collagen fiber implant provided by the invention can obviously promote the generation of new collagen, can be applied to the fields of implants, artificial skin, hemostatic sponges, stent materials, medical appliances and the like, and has wide application prospect; the invention also provides a new application of the bis (N-hydroxysuccinimide) suberate as a collagen crosslinking agent, and collagen fibers obtained by crosslinking the bis (N-hydroxysuccinimide) suberate.
Claims (10)
1. A preparation method of collagen fibers with high biocompatibility and high stability is characterized by comprising the following steps: (1) constructing collagen fibers; (2) crosslinking the collagen fibers by using a crosslinking agent; (3) dialyzing, homogenizing, centrifuging and collecting precipitates; the method for constructing the collagen fiber in the step (1) comprises the following steps:
preparing collagen solution with concentration of 0.1-5mg/mL, adding 10mM-100mM disodium hydrogen phosphate/sodium dihydrogen phosphate solution, centrifuging, and collecting precipitate; dispersing the obtained precipitate uniformly by using 20mM disodium hydrogen phosphate/sodium dihydrogen phosphate solution to ensure that the collagen concentration is 3mg/mL, and incubating at 17-25 ℃ for 8-19hrs; homogenizing the incubated collagen at 4 ℃ and 10000rpm for 20min; centrifuging to obtain collagen fiber.
2. The method of claim 1, wherein the cross-linking agent in step (2) is one or more selected from the group consisting of carbodiimide, epichlorohydrin, sodium metaphosphate, 1, 4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, procyanidins, hesperidin, tannic acid, genipin, riboflavin, naringenin, quercetin, epigallocatechin gallate, oleuropein, L-threose (LT), phytic acid, isocyanate, acyl azide, diphenyl phosphate, and bis (N-hydroxysuccinimide) suberate (NHS-SA).
3. The method of claim 2, wherein the crosslinking agent of step (2) is bis (N-hydroxysuccinimide) suberate.
4. The method according to claim 3, wherein the concentration of the crosslinking agent in the step (2) is 0.01 to 5mg/mL; the reaction temperature of the cross-linking agent is 4-25 ℃, and the reaction time is 12-24h.
5. The method according to claim 1, wherein the collagen of step (1) is animal collagen, including one or more of type I, type II, and type III.
6. The method of claim 1, wherein the dialysis in step (3) is performed by adding glycine after the crosslinking, dialyzing in a buffer at 4 ℃ to 25 ℃ for 48 to 72 hours, and centrifuging at 4 ℃ to 25 ℃ for 20 to 30 minutes at 10000rpm to collect the precipitate.
7. A collagen fiber produced by the production method according to any one of claims 1 to 6.
8. Use of the collagen protein fiber according to claim 7 for the preparation of implants, artificial skin, hemostatic sponges, scaffold materials, medical devices.
9. An implant of collagen fibers, wherein the implant is prepared from the collagen fibers according to claim 7.
10. Use of bis (N-hydroxysuccinimide) suberate as a collagen cross-linking agent.
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