CN109701073A - A kind of injectable cartilage repair hydrogel and preparation method thereof - Google Patents
A kind of injectable cartilage repair hydrogel and preparation method thereof Download PDFInfo
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- CN109701073A CN109701073A CN201910069720.6A CN201910069720A CN109701073A CN 109701073 A CN109701073 A CN 109701073A CN 201910069720 A CN201910069720 A CN 201910069720A CN 109701073 A CN109701073 A CN 109701073A
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- hydrogel
- cartilage repair
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Abstract
The present invention relates to a kind of injectable cartilage repair hydrogels and preparation method thereof, belong to technical field of biological materials.Injectable cartilage repair hydrogel of the invention includes modified hyaluronic acid, modified gelatin, platelet rich plasma and ultraviolet initiator.Repair of cartilage hydrogel material of the invention has good biocompatibility and syringeability, platelet rich plasma is loaded, the sustained release of a variety of growth factors, such as vascular endothelial growth factor, transforming growth factor can be achieved, can be applied in tissue engineering bone/cartilage reparation.
Description
Technical field
The present invention relates to a kind of injectable cartilage repair hydrogels and preparation method thereof, belong to technical field of biological materials.
Background technique
Cartilaginous tissue is made of cartilage cell and extracellular matrix, different from the huge regeneration potential of bone, cartilage due to a lack of
Blood fortune is supported and repair ability is limited.Cartilage cell can maintain natural phenotype in the three-dimensional rack for being similar to natural surroundings, and
The basis of extracellular matrix is generated, and then promotes regenerative agent of cartilaginous tissue.Hydrogel can keep flowing shape under certain condition
State, and the bulk material with certain shapes intensity is formed under outside stimulus, it can use this intelligence to prepare injection
Type bracket, play its repair complex-shaped defect and in terms of advantage.Therefore, research and develop it is novel can
Injection cartilage repair material is clinically of great significance.
Hyaluronic acid (HA) by β -1,4 glycosidic bond and β -1,3- glycosidic bond alternate links D-Glucose aldehydic acid and D-N- second
Acyl gucosamine is constituted, and is both the important glycosaminoglycan of cartilage cell epimatrix and the important component of synovia, and multinomial research is logical
Cross the ability that HA hydrogel has verified that HA reparation cartilage defect for preparing.
Gelatin (Gel) is the denaturation derivative obtained by collagen partial hydrolysis, therefore remains part collagen
Amino acid sequence, compared to collagen, gelatin has lower antigenicity.However, gelatin own mechanical intensity is low, it is seldom single
It is private to make cartilage repair material.
Platelet rich plasma (PRP) is the platelet concentrate that self whole blood obtains after being centrifuged, wherein containing a large amount of
Growth factor is difficult due to the problem of organizing interior diffusion and growth factor half-life short after these active small molecular injecting bodies are interior
To form optimum effective concentration in destination region and persistently play a role, it is therefore desirable to construct suitable material and load PRP realization
The sustained release of a variety of growth factors.
In recent years, scientific research personnel has been devoted to be modified natural polysaccharide, the excellent hydrogel material of processability
Meet various demands, it is field of tissue engineering technology that spy is standby.On the other hand, suitable material is found, PRP treatment advantages are given full play to,
It is also the hot spot of research.
Summary of the invention
A kind of injectable cartilage reparation water is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
Gel and preparation method thereof, hydrogel material prepared by the present invention have good biocompatibility and syringeability, load
Platelet rich plasma, it can be achieved that a variety of growth factors sustained release, can be applied in tissue engineering bone/cartilage reparation.
To achieve the above object, the technical scheme adopted by the invention is as follows: a kind of injectable cartilage repair hydrogel, the water
Gel includes modified hyaluronic acid, modified gelatin, platelet rich plasma and ultraviolet initiator.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the hydrogel includes following matter
Measure percentage component: modified hyaluronic acid 1~2.5%, modified gelatin 2.5~12.5%, platelet rich plasma 0.1~2%,
Ultraviolet initiator 0.1~2%, surplus are water.
In injectable cartilage repair hydrogel of the invention, gel time can be accelerated by improving ultraviolet initiator content, be examined
Consider as injectable gel, gel time should be depending on practical situations；And modified gelatin and modified hyaluronic acid contain
The raising of amount so that hydrogel intensity improve, on the contrary it is then be softness, can also be depending on practical application.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the matter of the platelet rich plasma
Measuring percentage is 1%.Platelet rich plasma can provide enough growth factors when mass percent is 1%；Rich platelet blood
Starch use of now withdrawing deposit as far as possible.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the ultraviolet initiator is
Irgacure2959。
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the system of the modified hyaluronic acid
Preparation Method are as follows: weigh hyaluronic acid, deionized water is added, stirring is added methacrylic anhydride, uses sodium hydroxide to being completely dissolved
Solution adjusts pH, and ice-water bath is stirred to react overnight, is then charged into bag filter and dialyses, and then freeze-drying is transparent to get modification
Matter acid.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the system of the modified hyaluronic acid
Preparation Method are as follows: weigh hyaluronic acid, deionized water is added, stirred under room temperature to being completely dissolved, methacrylic anhydride is added, used
The NaOH of 1mol/L adjusts pH to 8, and ice-water bath is stirred to react overnight, is then charged into the bag filter of 12~14KDa, pure water dialysis
A couple of days, then freeze-drying obtains modified hyaluronic acid, is stored at room temperature spare.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the system of the modified hyaluronic acid
In Preparation Method, the mass concentration of hyaluronic acid solution is 0.5~5%, and the dosage of methacrylic anhydride is 1~10 times of hyaluronic acid
Quality.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the preparation side of the modified gelatin
Method are as follows: weigh gelatin, deionized water is added, stirring is added methacrylic anhydride, continues to be stirred to react, then to being completely dissolved
It is fitted into bag filter and dialyses, then freeze-drying is to get modified gelatin.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the preparation side of the modified gelatin
Method are as follows: weigh gelatin, deionized water is added, stirred under 50 DEG C of water-baths to being completely dissolved, be added methacrylic anhydride, at 50 DEG C
Continue to be stirred to react a few hours, be then charged into the bag filter of 12~14KDa, pure water is dialysed a couple of days, and then freeze-drying obtains
Modified gelatin is stored at room temperature spare.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the preparation side of the modified gelatin
In method, the mass concentration of gelatin solution is 10~30%, and the dosage of methacrylic anhydride is 0.5~2 times of gelatin quality.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the system of the platelet rich plasma
Preparation Method are as follows: using centrifugal process twice, blood is centrifuged, draws under supernatant layer, middle layer and red blood cell 1~2 millimeter, is moved into another
In branch centrifuge tube；It is centrifuged again, discards supernatant liquid, remaining serum and blood platelet are uniformly mixed to get rich platelet blood
Slurry.
As the preferred embodiment of injectable cartilage repair hydrogel of the present invention, the system of the platelet rich plasma
Preparation Method are as follows: use traditional centrifugal process twice, 2mL blood is centrifuged 5~15 minutes under 200~300g revolving speed, draw supernatant
1~2 millimeter under layer, middle layer and red blood cell, move into another centrifuge tube, again with the speed of 1000~1500g again from
The heart 5~15 minutes, supernatant liquid is discarded, it is platelet rich plasma that remaining serum and blood platelet, which are uniformly mixed,.
The present invention also provides the preparation methods of above-mentioned injectable cartilage repair hydrogel, comprising the following steps:
(1) modified hyaluronic acid and modified gelatin are each configured to aqueous solution, are then uniformly mixed；
(2) platelet rich plasma is added, ultraviolet initiator is then added, obtains hydrogel precursor liquid；
(3) the resulting hydrogel precursor liquid of step (2) is injected into mold, mold is put and is irradiated in the UV lamp to get can
Inject repair of cartilage hydrogel.
The preferred embodiment of preparation method as injectable cartilage repair hydrogel of the present invention, the step
(1) in, the mass fraction of modified hyaluronic acid aqueous solution is 2~5%, and the mass fraction of modified gelatin aqueous solution is 5~25%,
The volume ratio of the aqueous solution of modified hyaluronic acid and modified gelatin mixing is 1:1.
Compared with prior art, the invention has the benefit that repair of cartilage hydrogel material of the invention has well
Biocompatibility and syringeability, loaded platelet rich plasma, it can be achieved that a variety of growth factors sustained release, such as blood vessel endothelium
Growth factor, transforming growth factor etc. can be applied in tissue engineering bone/cartilage reparation.
Detailed description of the invention
Fig. 1 is the external elution profiles figure of VEGF in embodiment 1.
Fig. 2 is the external elution profiles figure of TGF-β 1 in embodiment 1.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
Test method used in the embodiment of the present invention is conventional method unless otherwise specified；The embodiment of the present invention
Used in material, reagent etc., be commercially available unless otherwise specified.
Embodiment 1
A kind of injectable cartilage repair hydrogel, the component comprising following mass percent: modified hyaluronic acid 1% changes
Property gelatin 10%, platelet rich plasma 1%, ultraviolet initiator 1%, surplus be water.
The preparation method of the present embodiment hydrogel material includes the following steps:
(1) preparation of modified hyaluronic acid
5g hyaluronic acid is weighed, 100mL ionized water is added, is stirred under room temperature to being completely dissolved, 25g methacrylic acid is added
Acid anhydride adjusts pH to 8 with the NaOH of 1mol/L, and ice-water bath is stirred to react overnight, is then charged into the bag filter of 12~14KDa, pure
Water is dialysed 3 days, and then freeze-drying obtains modified hyaluronic acid, is stored at room temperature spare；
(2) preparation of modified gelatin
20g gelatin is weighed, 100mL deionized water is added, the first that 12g is added to being completely dissolved is stirred under 50 DEG C of water-baths
Base acrylic anhydride continues to be stirred to react a few hours at 50 DEG C, be then charged into the bag filter of 12~14KDa, and pure water is dialysed 3 days,
Then freeze-drying obtains modified gelatin, is stored at room temperature spare；
(3) preparation of platelet rich plasma (PRP)
Using traditional centrifugal process twice, 2mL blood is centrifuged 10 minutes under 200g revolving speed, draws supernatant layer, middle layer
And 1~2 millimeter under red blood cell, moves into another centrifuge tube, be centrifuged 10 minutes, discarded again with the speed of 1000g again
It is PRP that layer liquid, remaining serum and blood platelet, which are uniformly mixed,；
(4) preparation of injectable soft hydrogel
Respectively by step (1) and step (2) prepares resulting modified hyaluronic acid and modified gelatin is configured to 2% and 20%
Solution, be mixed evenly in equal volume, 1% step (3) resulting PRP be then added, finally adds 1% ultraviolet initiator
Irgacure2959 obtains hydrogel precursor liquid, and precursor liquid is injected mold, then mold is put and irradiates in the UV lamp
10min can arrive hydrogel material.
The present embodiment gel that volume is 1mL is prepared, totally three Duplicate Samples, are placed in centrifuge tube, are separately added into 2mL pbs
Buffer solution is sustained in the shaking table being placed in 37 DEG C of insulating boxs, takes out sustained-release liquid protect in -20 DEG C of refrigerators daily
It deposits, rejoins buffer, repeat 14 days.Finally the slow of all collections is tested using Enzyme-linked Immunosorbent Assay (ELISA) kit
Release the content of VEGF and TGF-β 1 in liquid.The external elution profiles figure of VEGF as shown in Figure 1, TGF-β 1 external elution profiles figure
As shown in Figure 2.
As shown in Figure 1, in two weeks, VEGF sustained release is up to 90% or so；As shown in Figure 2, sustained release connects in 1 liang of week of TGF-β
Nearly 85%, illustrate that the sustained release of both growth factors may be implemented in the hydrogel material of the present embodiment load platelet rich plasma.
Embodiment 2
A kind of injectable cartilage repair hydrogel, the component comprising following mass percent: modified hyaluronic acid 1% changes
Property gelatin 10%, platelet rich plasma 1%, ultraviolet initiator 2%, surplus be water.
The preparation method is the same as that of Example 1 for the present embodiment hydrogel material, wherein in step (4), modified hyaluronic acid and changes
Property gelatin is each configured to 2% and 20% solution, is then mixed evenly in equal volume；It is then respectively adding rich platelet blood
Starch 1%, ultraviolet initiator 2%.
In two weeks, the VEGF sustained release of the present embodiment gel rubber material is up to 90% or so；Sustained release is close in 1 liang of week of TGF-β
85%, illustrate that the sustained release of both growth factors may be implemented in the hydrogel material of the present embodiment load platelet rich plasma.
Embodiment 3
A kind of injectable cartilage repair hydrogel, the component comprising following mass percent: modified hyaluronic acid 1% changes
Property gelatin 7.5%, platelet rich plasma 1%, ultraviolet initiator 1%, surplus be water.
The preparation method is the same as that of Example 1 for the present embodiment hydrogel material, wherein in step (4), modified hyaluronic acid and changes
Property gelatin is each configured to 2% and 15% solution, is then mixed evenly in equal volume；It is then respectively adding rich platelet blood
Starch 1%, ultraviolet initiator 1%.
In two weeks, the VEGF sustained release of the present embodiment gel rubber material is up to 90% or so；Sustained release is close in 1 liang of week of TGF-β
85%, illustrate that the sustained release of both growth factors may be implemented in the hydrogel material of the present embodiment load platelet rich plasma.
Embodiment 4
A kind of injectable cartilage repair hydrogel, the component comprising following mass percent: modified hyaluronic acid 1% changes
Property gelatin 12.5%, platelet rich plasma 0.1%, ultraviolet initiator 2%, surplus be water.
The preparation method is the same as that of Example 1 for the present embodiment hydrogel material, wherein in step (4), modified hyaluronic acid and changes
Property gelatin is each configured to 2% and 25% solution, is then mixed evenly in equal volume；It is then respectively adding rich platelet blood
Starch 0.1%, ultraviolet initiator 2%.
In two weeks, the VEGF sustained release of the present embodiment gel rubber material is up to 90% or so；Sustained release is close in 1 liang of week of TGF-β
85%, illustrate that the sustained release of both growth factors may be implemented in the hydrogel material of the present embodiment load platelet rich plasma.
Embodiment 5
A kind of injectable cartilage repair hydrogel, the component comprising following mass percent: modified hyaluronic acid 2.5%,
Modified gelatin 2.5%, platelet rich plasma 2%, ultraviolet initiator 0.1%, surplus are water.
The preparation method is the same as that of Example 1 for the present embodiment hydrogel material, wherein in step (4), modified hyaluronic acid and changes
Property gelatin is each configured to 5% and 5% solution, is then mixed evenly in equal volume；It is then respectively adding rich platelet blood
Starch 2%, ultraviolet initiator 0.1%.
In two weeks, the VEGF sustained release of the present embodiment gel rubber material is up to 90% or so；Sustained release is close in 1 liang of week of TGF-β
85%, illustrate that the sustained release of both growth factors may be implemented in the hydrogel material of the present embodiment load platelet rich plasma.
Effect example 1
The gelation time of 1~3 hydrogel material of the embodiment of the present invention is analyzed, the results are shown in Table 1.
Table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Gelation time (min) | 15±2 | 10±1 | 15±2 |
As shown in Table 1, the percentage composition of modified gelatin and modified hyaluronic acid influences less, to improve light and draw on gelation time
Hair agent content can substantially speed up gelation time, subsequent that the constituent content meet demand can be adjusted according to practical application.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (9)
1. a kind of injectable cartilage repair hydrogel, which is characterized in that the hydrogel is bright comprising modified hyaluronic acid, modification
Glue, platelet rich plasma and ultraviolet initiator.
2. injectable cartilage repair hydrogel as described in claim 1, which is characterized in that the hydrogel includes following quality
The component of percentage: modified hyaluronic acid 1~2.5%, modified gelatin 2.5~12.5%, platelet rich plasma 0.1~2%, purple
Outer photoinitiator 0.1~2%, surplus are water.
3. injectable cartilage repair hydrogel as claimed in claim 2, which is characterized in that the quality of the platelet rich plasma
Percentage is 1%.
4. injectable cartilage repair hydrogel as described in claim 1, which is characterized in that the ultraviolet initiator is
Irgacure2959。
5. such as the described in any item injectable cartilage repair hydrogels of Claims 1 to 4, which is characterized in that the modification is transparent
Matter acid the preparation method comprises the following steps: weigh hyaluronic acid, deionized water is added, stirring is added methacrylic anhydride to being completely dissolved, uses
Sodium hydroxide solution adjust pH, ice-water bath be stirred to react overnight, be then charged into bag filter and dialyse, then freeze-drying to get
Modified hyaluronic acid.
6. such as the described in any item injectable cartilage repair hydrogels of Claims 1 to 4, which is characterized in that the modified gelatin
The preparation method comprises the following steps: weigh gelatin, deionized water is added, stirring is added methacrylic anhydride to being completely dissolved, it is anti-to continue stirring
It answers, is then charged into bag filter and dialyses, then freeze-drying is to get modified gelatin.
7. such as the described in any item injectable cartilage repair hydrogels of Claims 1 to 4, which is characterized in that the rich platelet
Blood plasma the preparation method comprises the following steps: using centrifugal process twice, blood is centrifuged, draws under supernatant layer, middle layer and red blood cell 1~2 millimeter,
It moves into another centrifuge tube；It is centrifuged again, discards supernatant liquid, remaining serum and blood platelet are uniformly mixed to get richness
Thrombocyte plasma.
8. the preparation method of injectable cartilage repair hydrogel as described in any one of claims 1 to 7, which is characterized in that packet
Include following steps:
(1) modified hyaluronic acid and modified gelatin are each configured to aqueous solution, are then uniformly mixed；
(2) platelet rich plasma is added, ultraviolet initiator is then added, obtains hydrogel precursor liquid；
(3) the resulting hydrogel precursor liquid of step (2) is injected into mold, mold is put and is irradiated in the UV lamp to get injectable
Repair of cartilage hydrogel.
9. the preparation method of injectable cartilage repair hydrogel as claimed in claim 8, which is characterized in that the step (1)
In, the mass fraction of modified hyaluronic acid aqueous solution is 2~5%, and the mass fraction of modified gelatin aqueous solution is 5~25%, is changed
Property hyaluronic acid and modified gelatin aqueous solution mixing volume ratio be 1:1.
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Cited By (8)
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|---|---|---|---|---|
| CN110237302A (en) * | 2019-07-22 | 2019-09-17 | 南京鼓楼医院 | A kind of preparation method of articular cartilage repair materials-autologous platelet rich plasma combination hyaluronic acid gel |
| CN110607076A (en) * | 2019-09-26 | 2019-12-24 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method of 3D printing material based on silk fibroin and hyaluronic acid |
| CN111184910A (en) * | 2020-01-08 | 2020-05-22 | 广州贝奥吉因生物科技股份有限公司 | Injectable cartilage repair hydrogel and preparation method thereof |
| CN112245395A (en) * | 2020-11-20 | 2021-01-22 | 佳木斯大学 | Medical cartilage repairing agent and preparation method thereof |
| CN112274702A (en) * | 2020-10-30 | 2021-01-29 | 南开大学 | Multifunctional injectable hydrogel and preparation method thereof |
| CN112870452A (en) * | 2020-03-12 | 2021-06-01 | 深圳市第二人民医院(深圳市转化医学研究院) | Manufacturing method of 3D printing gelatin-hydroxyapatite composite hydrogel scaffold |
| CN115531622A (en) * | 2022-09-28 | 2022-12-30 | 四川师范大学 | PRP composite injectable hydrogel and preparation method and application thereof |
| EP4147730A1 (en) | 2021-09-09 | 2023-03-15 | ETH Zurich | Method of preparing a functional hydrogel material that can be injected |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110237302A (en) * | 2019-07-22 | 2019-09-17 | 南京鼓楼医院 | A kind of preparation method of articular cartilage repair materials-autologous platelet rich plasma combination hyaluronic acid gel |
| CN110607076A (en) * | 2019-09-26 | 2019-12-24 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method of 3D printing material based on silk fibroin and hyaluronic acid |
| CN111184910A (en) * | 2020-01-08 | 2020-05-22 | 广州贝奥吉因生物科技股份有限公司 | Injectable cartilage repair hydrogel and preparation method thereof |
| CN112870452A (en) * | 2020-03-12 | 2021-06-01 | 深圳市第二人民医院(深圳市转化医学研究院) | Manufacturing method of 3D printing gelatin-hydroxyapatite composite hydrogel scaffold |
| CN112274702A (en) * | 2020-10-30 | 2021-01-29 | 南开大学 | Multifunctional injectable hydrogel and preparation method thereof |
| CN112245395A (en) * | 2020-11-20 | 2021-01-22 | 佳木斯大学 | Medical cartilage repairing agent and preparation method thereof |
| EP4147730A1 (en) | 2021-09-09 | 2023-03-15 | ETH Zurich | Method of preparing a functional hydrogel material that can be injected |
| WO2023036922A1 (en) | 2021-09-09 | 2023-03-16 | Eth Zurich | Method of preparing a functional hydrogel material that can be injected |
| CN115531622A (en) * | 2022-09-28 | 2022-12-30 | 四川师范大学 | PRP composite injectable hydrogel and preparation method and application thereof |
| CN115531622B (en) * | 2022-09-28 | 2024-04-19 | 四川师范大学 | PRP composite injectable hydrogel and preparation method and application thereof |
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Application publication date: 20190503 |