CN107522876A - A kind of preparation method of plural gel and its in induction stem cell into the application in cartilage differentiation - Google Patents
A kind of preparation method of plural gel and its in induction stem cell into the application in cartilage differentiation Download PDFInfo
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Abstract
Comprise the following steps the invention discloses a kind of preparation method of plural gel and its in induction stem cell into the application in cartilage differentiation, methods described：(1) preparation of collagen, concentration is used to prepare the sterile ox-hide collagen dried frozen aquatic products acetum that concentration is 10 20mg/mL as solvent for 0.4 0.6M acetic acid；(2) preparation of Geniposide stoste；(3) take the collagen that certain volume is prepared by step (1) and add the Geniposide stoste prepared by step (2) and carbon point of certain volume, it is respectively the mixed liquor that 0.4 2vol% Geniposide and carbon are selected to prepare concentration, it is placed in after fully mixing under 37 DEG C of constant temperatures and stands 15 minutes, obtains Geniposide collagen carbon point plural gel.The present invention is by explanation after Geniposide crosslinking, the ROS releases of carbon point are restricted, the ROS that carbon point is discharged is controlled in low amounts state, play its cell proliferation and the facilitation of differentiation, and the timeliness of the effect of carbon point is kept, and the plural gel can be applied to differentiation of stem cells chondroblast.
Description
Technical field
The present invention relates to medical science and biomedical engineering field, more particularly to Geniposide-collagen-carbon point plural gel
Preparation method and its application in induction stem cell into cartilage differentiation.
Background technology
Articular cartilage defect is always the treatment problem of Orthopedic Clinical, and being primarily due to articular cartilage does not have blood supply, and soft
Osteocyte is embedded in the extracellular matrix of stiff, can not be moved to damage location and be participated in repairing.Traditional treatment method such as joint
Interior cleaning and lavation art etc. are difficult to obtain satisfied clinical effectiveness.
Tissue engineering technique meets timbering material by seed cell and builds organization engineered cartilage, is implanted to defect
And realize therapeutic purposes.In recent years, with the rise of nanometer technology, its conjunctive tissue engineering technology is combined applied to regeneration doctor
Field is forming a brand-new research direction.
Carbon point (CDots) is a kind of fluorescent nano material, has photoluminescence property, excellent optical stability and nontoxic
Property.Under laser action, carbon point can produce a certain amount of ROS, and the wherein ROS of low concentration with activating transcription factor and can promote thin
Born of the same parents' propagation, differentiation；And the ROS of middle and high concentration even results in meronecrosis by cellular stress inducing cell apoptosis.
The content of the invention
In order to solve the above-mentioned technical problem, an object of the present invention is to provide a kind of Geniposide-collagen-carbon point compound
The preparation method of gel, facilitates medication.
To achieve the above object, the technical scheme is that：A kind of preparation method of plural gel, including following step
Suddenly：
(1) preparation of collagen, concentration is used to add a certain amount of sterile ox-hide collagen as solvent for 0.4-0.6M acetic acid
Freeze-drying tablet prepares sterile ox-hide collagen dried frozen aquatic products-acetum that concentration is 10-20mg/mL, treats sterile ox-hide collagen dried frozen aquatic products
Fully after swelling, collagen is set fully to dissolve in being stirred continuously on ice；
(2) preparation of Geniposide stoste, take a certain amount of Geniposide to be completely dissolved in absolute ethyl alcohol, and add PBS constant volumes,
Prepare the Geniposide stoste that concentration is 60-200mg/mL
(3) preparation of plural gel, take collagen that certain volume is prepared by step (1) and add certain volume by step
(2) the Geniposide stoste and carbon point prepared, it is respectively the mixed liquor that 0.4-2vol% Geniposide and carbon are selected to prepare concentration, fully
It is placed in after mixing under 37 DEG C of constant temperatures and stands 15 minutes, obtains Geniposide-collagen-carbon point plural gel.
The beneficial effect of above-mentioned technical proposal is：The biological material for being compounded with carbon point is produced jointly using Geniposide and collagen
Material, facilitates carbon point as external used medicine, and its ROS release ratios are single after the collagen load carbon quantum dot after Geniposide is crosslinked
Pure carbon quantum dot is reduced with the mixed amount of collagen, illustrates after Geniposide crosslinking, and the ROS releases of carbon point are limited
System, reduce excessive ROS and produce to cell or body generation toxicity, but control the ROS of carbon point release to be in low amounts state, send out
Its cell proliferation and the facilitation of differentiation are waved, and keeps the timeliness of the effect of carbon point.
Further, in above-mentioned technical proposal, specifically, comprising the following steps：
(1) preparation of collagen, concentration is used to add a certain amount of sterile animal glue as solvent for 0.45-0.55M acetic acid
Former freeze-drying tablet prepares sterile ox-hide collagen dried frozen aquatic products-acetum that concentration is 12-18mg/mL, treats that sterile ox-hide collagen freezes
After product are fully swelled, collagen is set fully to dissolve in being stirred continuously on ice；
(2) preparation of Geniposide stoste, take a certain amount of Geniposide to be completely dissolved in absolute ethyl alcohol, and add PBS constant volumes,
Prepare the Geniposide stoste that concentration is 100-160mg/mL；
(3) preparation of plural gel, take collagen that certain volume is prepared by step (1) and add certain volume by step
(2) the Geniposide stoste and carbon point prepared, it is respectively the mixed liquor that 0.7-1.3vol% Geniposide and carbon are selected to prepare concentration, is filled
Divide after mixing to be placed under 37 DEG C of constant temperatures and stand 15 minutes, obtain Geniposide-collagen-carbon point plural gel.
The beneficial effect of above-mentioned technical proposal is：The proportioning of adjustment Geniposide, collagen and carbon point further improves it
Curative effect.
Further, in above-mentioned technical proposal, specifically, comprising the following steps：
(1) preparation of collagen, use concentration to add a certain amount of sterile ox-hide collagen as solvent for 0.5M acetic acid and freeze
Piece prepares sterile ox-hide collagen dried frozen aquatic products-acetum that concentration is 15mg/mL, treats that sterile ox-hide collagen dried frozen aquatic products is fully swelled
Afterwards, collagen is made fully to dissolve in being stirred continuously on ice；；
(2) preparation of Geniposide stoste, take a certain amount of Geniposide to be completely dissolved in absolute ethyl alcohol, and add PBS constant volumes,
Prepare the Geniposide stoste that concentration is 133mg/mL；
(3) preparation of plural gel, take collagen that certain volume is prepared by step (1) and add certain volume by step
(2) the Geniposide stoste and carbon point prepared, it is respectively the mixed liquor that 1vol% Geniposide and carbon are selected to prepare concentration, is fully mixed
After be placed under 37 DEG C of constant temperatures and stand 15 minutes, obtain Geniposide-collagen-carbon point plural gel.
The beneficial effect of above-mentioned technical proposal is：The proportioning of further adjustment Geniposide, collagen and carbon point is further
Raising its curative effect.
The amount ratio of Geniposide and absolute ethyl alcohol described in step described in above-mentioned technical proposal (2) is 1mg Geniposides pair
Answer 1-3 μ L absolute ethyl alcohol.
The beneficial effect of above-mentioned technical proposal is, is completely dissolved Geniposide using ethanol.
The second object of the present invention be to provide plural gel prepared by method a kind of as described above induction stem cell into
Application in cartilage differentiation.
The beneficial effect of above-mentioned technical proposal is：Carbon point is suppressed to ROS burst size by Geniposide and extends carbon point
Discharge ROS timeliness, it is ensured that ROS is chronically at a kind of low-level state, so as to play it for a long time to promoting cell propagation and dividing
The effect of change, it is cartilage cell to reach differentiation of stem cells.
Brief description of the drawings
Fig. 1 is with the infared spectrum after Geniposide-collagen cross-linking carbon point；
Fig. 2 is the XRD of Geniposide-collagen-carbon point plural gel；
Fig. 3 each components material and composite swelling rate；
Fig. 4 each components material and composite degradation rate
Fig. 5 each components material and composite fluorescence intensity；
Fig. 6 each components material and composite ROS releases；
The dynamic mechanical of Fig. 7 each components material and composite storage modulus；
The dynamic mechanical of Fig. 8 each components material and composite loss modulus；
Fig. 9 each component material cartilages are to ACAN factor expressions；
Figure 10 each component material cartilages are to COL2A1 factor expressions；
Figure 11 each component material cartilages are to SOX9 factor expressions；
Figure 12 each component material cartilages are to COL1A1 factor expressions；
The GAG contents of Figure 13 each component materials.
Embodiment
The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Embodiment 1
The preparation of plural gel, comprises the following steps：(1) preparation of collagen, the sterile ox-hide collagen dried frozen aquatic productses of 15mg are taken to add
Enter the acetic acid that 1mL concentration is 0.5M, after swelling, collagen is fully dissolved in being stirred continuously on ice；(2) system of Geniposide stoste
It is standby, weigh after 2mg Geniposides are dissolved in 2 μ L absolute ethyl alcohols and add PBS to be settled to 15 μ L；(3) preparation of plural gel, 100 μ L are taken
The collagen prepared by step (1), Geniposide stoste and each 1 μ L of carbon point prepared by step (2) are then added, is placed in after fully mixing
15 minutes are stood under 37 DEG C of constant temperatures, obtains Geniposide-collagen-carbon point plural gel A.
Embodiment 2
The preparation of plural gel：Comprise the following steps：(1) preparation of collagen, the sterile ox-hide collagen dried frozen aquatic productses of 10mg are taken to add
Enter the acetic acid that 1mL concentration is 0.4M, after swelling, collagen is fully dissolved in being stirred continuously on ice；(2) system of Geniposide stoste
It is standby, weigh after 2mg Geniposides are dissolved in 2 μ L absolute ethyl alcohols and add PBS to be settled to 15 μ L；(3) preparation of plural gel, 120 μ L are taken
The collagen prepared by step (1), Geniposide stoste and each 1.5 μ L of carbon point prepared by step (2) are then added, is fully mixed rearmounted
15 minutes are stood under 37 DEG C of constant temperatures, obtains Geniposide-collagen-carbon point plural gel B.
Embodiment 3
The preparation of plural gel：Comprise the following steps：(1) preparation of collagen, the sterile ox-hide collagen dried frozen aquatic productses of 20mg are taken to add
Enter the acetic acid that 1mL concentration is 0.6M, after swelling, collagen is fully dissolved in being stirred continuously on ice；(2) system of Geniposide stoste
It is standby, weigh after 2mg Geniposides are dissolved in 6 μ L absolute ethyl alcohols and add PBS to be settled to 15 μ L；(3) preparation of plural gel, take 80 μ L by
Collagen prepared by step (1), Geniposide stoste and each 0.5 μ L of carbon point prepared by step (2) are then added, is placed in after fully mixing
15 minutes are stood under 37 DEG C of constant temperatures, obtains Geniposide-collagen-carbon point plural gel C.
Embodiment 4
The preparation of plural gel：Comprise the following steps：(1) preparation of collagen, the sterile ox-hide collagen dried frozen aquatic productses of 12mg are taken to add
Enter the acetic acid that 1mL concentration is 0.45M, after swelling, collagen is fully dissolved in being stirred continuously on ice；(2) Geniposide stoste
Prepare, weigh after 2mg Geniposides are dissolved in 5 μ L absolute ethyl alcohols and add PBS to be settled to 15 μ L；(3) preparation of plural gel, 110 μ are taken
The collagen that L is prepared by step (1), Geniposide stoste and each 1.2 μ L of carbon point prepared by step (2) are then added, after fully mixing
It is placed under 37 DEG C of constant temperatures and stands 15 minutes, obtains Geniposide-collagen-carbon point plural gel D.
Embodiment 5
The preparation of plural gel：Comprise the following steps：(1) preparation of collagen, the sterile ox-hide collagen dried frozen aquatic productses of 18mg are taken to add
Enter the acetic acid that 1mL concentration is 0.55M, after swelling, collagen is fully dissolved in being stirred continuously on ice；(2) Geniposide stoste
Prepare, weigh after 2mg Geniposides are dissolved in 3 μ L absolute ethyl alcohols and add PBS to be settled to 15 μ L；(3) preparation of plural gel, 90 μ L are taken
The collagen prepared by step (1), Geniposide stoste and each 0.8 μ L of carbon point prepared by step (2) are then added, is fully mixed rearmounted
15 minutes are stood under 37 DEG C of constant temperatures, obtains Geniposide-collagen-carbon point plural gel E.
Embodiment 6
The physicochemical characterization of plural gel, as shown in figure 1, be the infared spectrum after Geniposide-collagen cross-linking carbon point,
3350cm-1NH2 absworption peak, 1631cm in the absorption peak position collagen at place-1The absworption peak at place is attributed to stretching for the C=O of amido link
Contracting vibration absorption peak, and in 2800cm-1-3000cm-1Absworption peak disappear, the appearance of these characteristic peaks or change explanation capital
After Buddhist nun divides son equally and is successfully connected to carbon quantum dot surface, and collagen is crosslinked, collagen accounts for mainly in system, infrared spectrum master
What is shown is the characteristic absorption of collagen.
As shown in Fig. 2 being the XRD of Geniposide-collagen-carbon point plural gel, 2 θ are that 18.75o weak diffraction occurs
Peak, it is the diffraction maximum of carbon quantum dot, 2 θ are that 31.6o strong and sharp diffraction maximum occurs, are the characteristic peak of collagen, illustrate crosslinking
Afterwards, collagen accounts for mainly, and its peak shape becomes apparent from, and the result with infrared spectrum is consistent.
Embodiment 7
BMSCs corresponding to embodiment 1 is wrapped in Geniposide-collagen-carbon azeotropic mixture, 15 points under the conditions of being placed in 37 DEG C
The bell gel into load cells, and corresponding blank control group i.e. collagen group (H groups), collagen-Geniposide group (HG are set respectively
Group), collagen-carbon point group (HC groups) and collagen-Geniposide-carbon point group (HGC groups), experimental group be collagen-Geniposide-carbon point-
Five groups of laser group (HGCL groups).
The swelling ratio and degradation rate of each component material and composite, as shown in Figure 3 and Figure 4, respectively collagen group (H
Group), collagen-Geniposide group (HG groups), collagen-carbon point group (HC groups), collagen-Geniposide-four groups of gels of carbon point group (HGC groups)
The performance of swelling and degraded is evaluated, and it is obvious to can be seen that more uncrosslinked group of after-poppet swelling ratio of crosslinking from swelling ratio figure line
Reduce.And the degradation rate of crosslinking group gradually increases over time, but more there is significant difference with uncrosslinked group.
Each component material and composite fluorescence and ROS releases
As it can be seen in figures 5 and 6, ROS measure is carried out to being crosslinked front and rear material, it can be found that the glue after Geniposide is crosslinked
Its ROS releases reduce than simple carbon quantum dot and the mixed amount of collagen after original load carbon quantum dot, illustrate to pass through Geniposide
After crosslinking, the ROS releases of material are restricted, and are reduced excessive ROS and are produced to cell or body generation toxicity.Further lead to
ROS production can also be illustrated to detect ROS consumption by crossing DPBF.
The dynamic mechanical of each component material and composite
As shown in FIG. 7 and 8, it is each component material and composite dynamic mechanical, at the same detecting to material, energy storage
Modulus reflects elastic properties of materials size, and dynamic mechanical detection finds the storage modulus highest of HGC groups, illustrates that nano material is handed over collagen
Collagen mechanical strength can be significantly improved after connection.Loss modulus reflects tack of materials size, and wherein HGC loss modulus is maximum,
Tack of materials increases after illustrating crosslinking.
PCR detection each component material softer bone photos close factor expression
Geniposide-collagen-carbon point plural gel induces effects of the BMSCs into cartilage differentiation
Collagen group (H groups), collagen-Geniposide group (HG groups), collagen-carbon point group (HC groups), collagen-Geniposide-carbon point group
(HGC groups), collagen-Geniposide-carbon point-laser group (HGCL groups), after cultivating 7,14,21 days respectively, wherein, HGCL groups are used every other day
808nm laser irradiates (optical density 1J/cm2, 3min) once, the cartilage-specific genes of above-mentioned five groups of experiments are detected respectively
SOX9, ACAN, COL2A1, cartilage differentiation type gene C OL1A1 and cartilage cell epimatrix GAP-associated protein GAP GAG expression, are commented
Valency Geniposide-collagen-carbon point plural gel induces effects of the BMSCs into cartilage differentiation.As described and depicted in figs. 9-13, the results showed that capital Buddhist nun
Flat-collagen-carbon point plural gel coordinates laser to be greatly facilitated GAG synthesis and cartilage-specific genes SOX9, ACAN and COL2A1
Expression, and it is relative suppress cartilage differentiation type gene C OL1A1, illustrate Geniposide-collagen-carbon point plural gel in laser action
It is lower to have the function that it is a kind of biology being potential very much for treating clinical joint cartilage defect into cartilage differentiation to promote BMSCs
Material.
PCR results show the gene expression dose of each group.With the extension of time, each material to ACAN, COL2A1,
The expression quantity of SOX9 genes has enhancing effect, and the wherein collagen after Geniposide crosslinking loads carbon quantum dot to gene expression amount
Enhancing effect is the most notable.But after laser irradiates, promoting composite to discharge more ROS, its corresponding gene contains
Amount significantly improves, and illustrates that a certain amount of ROS can promote Chondrocyte Differentiation.And COL1A1 expression quantity is over time
Gradually reduce, COL1A1 expression quantity is reduced and become apparent upon laser irradiation for material after crosslinking, illustrate it is compound after material
Material has into chondrocyte induction effect to mesenchymal stem cells MSCs (BMSCs), and makes to lure by producing a small amount of ROS after laser action
Effect is led to become apparent.
The GAG contents of each component material
GAG content is represented into cartilage differentiation ability.Over time, each group material is to albumen for it can be seen from the figure that
Polysaccharide secretory volume has the effect necessarily strengthened.But after laser irradiation, the collagen load carbon quantum dot group material after Geniposide crosslinking
The GAG secretory volumes of material significantly improve, consistent with Fig. 9-12 result, further illustrate that laser action carbon quantum dot produces on a small quantity
ROS becomes apparent inducing effect.
The operation as corresponding to embodiment 7 is carried out respectively by implementing the plural gel corresponding to 5 to embodiment 2-, as a result
Confirm after being irradiated by laser, carbon point can discharge a small amount of ROS to play its inducing action.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (5)
1. a kind of preparation method of plural gel, it is characterised in that comprise the following steps：
(1) preparation of collagen, use concentration to add a certain amount of sterile ox-hide collagen as solvent for 0.4-0.6M acetic acid and freeze
Piece prepares sterile ox-hide collagen dried frozen aquatic products-acetum that concentration is 10-20mg/mL, treats that sterile ox-hide collagen dried frozen aquatic products is abundant
After swelling, collagen is set fully to dissolve in being stirred continuously on ice；
(2) preparation of Geniposide stoste, take a certain amount of Geniposide to be completely dissolved in absolute ethyl alcohol, and add PBS constant volumes, prepare
Concentration is 60-200mg/mL Geniposide stoste；
(3) preparation of plural gel, take collagen that certain volume is prepared by step (1) and add certain volume by step (2)
Geniposide stoste and the carbon point of preparation, it is respectively the mixed liquor that 0.4-2vol% Geniposide and carbon are selected to prepare concentration, fully mixed
It is placed in after even under 37 DEG C of constant temperatures and stands 15 minutes, obtains Geniposide-collagen-carbon point plural gel.
2. the preparation method of plural gel according to claim 1, it is characterised in that comprise the following steps：
(1) preparation of collagen, use concentration to add a certain amount of sterile ox-hide collagen as solvent for 0.45-0.55M acetic acid and freeze
Dry plate prepares sterile ox-hide collagen dried frozen aquatic products-acetum that concentration is 12-18mg/mL, treats that sterile ox-hide collagen dried frozen aquatic products fills
After dividing swelling, collagen is set fully to dissolve in being stirred continuously on ice；
(2) preparation of Geniposide stoste, take a certain amount of Geniposide to be completely dissolved in absolute ethyl alcohol, and add PBS constant volumes, prepare
Concentration is 100-160mg/mL Geniposide stoste；
(3) preparation of plural gel, take collagen that certain volume is prepared by step (1) and add certain volume by step (2)
Geniposide stoste and the carbon point of preparation, it is respectively the mixed liquor that 0.7-1.3vol% Geniposide and carbon are selected to prepare concentration, fully
It is placed in after mixing under 37 DEG C of constant temperatures and stands 15 minutes, obtains Geniposide-collagen-carbon point plural gel.
3. the preparation method of plural gel according to claim 1 or 2, it is characterised in that comprise the following steps：
(1) preparation of collagen, concentration is used to add a certain amount of sterile ox-hide collagen freeze-drying tablet system as solvent for 0.5M acetic acid
Sterile ox-hide collagen dried frozen aquatic products-acetum that standby concentration is 15mg/mL, after sterile ox-hide collagen dried frozen aquatic products is fully swelled,
Collagen is set fully to dissolve in being stirred continuously on ice；；
(2) preparation of Geniposide stoste, take a certain amount of Geniposide to be completely dissolved in absolute ethyl alcohol, and add PBS constant volumes, prepare
Concentration is 133mg/mL Geniposide stoste；
(3) preparation of plural gel, take collagen that certain volume is prepared by step (1) and add certain volume by step (2)
Geniposide stoste and the carbon point of preparation, it is respectively the mixed liquor that 1vol% Geniposide and carbon are selected to prepare concentration, after fully mixing
It is placed under 37 DEG C of constant temperatures and stands 15 minutes, obtains Geniposide-collagen-carbon point plural gel.
4. the preparation method of the plural gel according to any one of claim 1-3, it is characterised in that the step (2)
Described in Geniposide and absolute ethyl alcohol amount ratio for the corresponding 1-3 μ L of 1mg Geniposides absolute ethyl alcohol.
5. plural gel prepared by a kind of method as any one of claim 1-4 is in induction stem cell into cartilage differentiation
Application.
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| US20150004138A1 (en) * | 2010-12-02 | 2015-01-01 | Icahn School Of Medicine At Mount Sinai | Method of repairing a tissue defect using genipin cross-linked fibrin gels |
| CN106620873A (en) * | 2016-11-17 | 2017-05-10 | 太原理工大学 | Composite hydrogel cartilage repair material and preparation method thereof |
| CN106620839A (en) * | 2017-01-17 | 2017-05-10 | 上海交通大学医学院附属第九人民医院 | Support material with function of promoting differentiation of stem cells as well as preparation method and application of support material |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20150004138A1 (en) * | 2010-12-02 | 2015-01-01 | Icahn School Of Medicine At Mount Sinai | Method of repairing a tissue defect using genipin cross-linked fibrin gels |
| CN106620873A (en) * | 2016-11-17 | 2017-05-10 | 太原理工大学 | Composite hydrogel cartilage repair material and preparation method thereof |
| CN106620839A (en) * | 2017-01-17 | 2017-05-10 | 上海交通大学医学院附属第九人民医院 | Support material with function of promoting differentiation of stem cells as well as preparation method and application of support material |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112972767A (en) * | 2021-02-05 | 2021-06-18 | 浙江大学 | Magnetic response tissue engineering material with osteogenesis promoting effect and preparation method and application thereof |
| CN112972767B (en) * | 2021-02-05 | 2022-03-11 | 浙江大学 | Magnetic response tissue engineering material with osteogenesis promoting effect and preparation method and application thereof |
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