CN115282338A - Multimode composite scaffold with immune mediation and effect of promoting healing of rotator cuff aponeurosis and preparation method thereof - Google Patents
Multimode composite scaffold with immune mediation and effect of promoting healing of rotator cuff aponeurosis and preparation method thereof Download PDFInfo
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- CN115282338A CN115282338A CN202111592474.6A CN202111592474A CN115282338A CN 115282338 A CN115282338 A CN 115282338A CN 202111592474 A CN202111592474 A CN 202111592474A CN 115282338 A CN115282338 A CN 115282338A
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Abstract
The invention provides a multimode composite scaffold with immune mediation and capability of promoting healing of rotator cuff aponeurosis and a preparation method thereof. The preparation method of the multimode composite stent comprises the following steps: preparing a cell membrane by adopting the adipose-derived stem cells; adopting a medicament or an active substance with the effect of promoting bone formation or cartilage formation to obtain a medicament-carrying polylactic acid slightly-soluble gel electrospun fiber scaffold; preparing an etanercept liposome solution by adopting a reverse evaporation method technology; and soaking the slightly-soluble gel electrospun fiber scaffold in a liposome solution, loading the liposome containing the etanercept through a Schiff base bond, and loading the adipose-derived stem cell membrane. The multimode composite scaffold provided by the invention can promote the regeneration of cartilage at the tendon-bone interface of the rotator cuff in an immune-mediated manner, improve the reconstruction of a multi-layer fibrocartilage structure instead of the formation of simple scar tissues, and increase the biomechanical strength of tendon-bone healing, thereby improving the success rate of rotator cuff repair and reducing postoperative injury.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a multimode composite stent with immune mediation and capability of promoting healing of rotator cuff aponeurosis and a preparation method thereof.
Background
Rotator cuff injuries are one of the common shoulder lesions, affecting shoulder joint function and causing daily life. Scar connection is often formed at the tendon-bone interface after rotator cuff repair, the biomechanical strength is reduced, and stress is concentrated, so that the rotator cuff repair is torn again after the operation. With the gradual understanding of the tendon structure and the development of new technology, various methods for promoting tendon-bone insertion healing are developed at home and abroad, including improvement of internal fixation implant materials, strategies based on various exogenous growth factors, tissue-engineered mesenchymal stem cells, low-intensity pulsed ultrasound and the like.
Although these measures at present improve the ultimate load of the repaired structure to different degrees, the local action time is short, and the effective concentration or strength can not be maintained for a long time, so the scarring at the tendon-bone interface is enhanced, the original multi-layer tissue structure is not reconstructed, and the strength and ultimate load of the tendon-bone dead point are not restored to the level before the injury.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a multimode composite scaffold with immune mediation and capability of promoting healing of rotator cuff tendon bones and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a preparation method of a multimode composite scaffold with immune mediation and capability of promoting healing of rotator cuff tendon bones, which comprises the following steps:
step one, preparing a cell membrane: preparing a cell membrane by adopting the adipose-derived stem cells;
step two, preparing the micro-sol electrospun fiber scaffold
(1) Completely dissolving Hyaluronic Acid (HA) in deionized water, adding medicine or active substance with effect of promoting osteogenesis or chondrogenesis, and mixing;
(2) Adding Span80 and DCM (dichloromethane), stirring at high speed to obtain uniform and stable water-oil emulsion;
(3) Adding aminated PLA (aminated polylactic acid) and DMF (N, N-dimethylformamide) into the water-oil emulsion to obtain a slightly soluble glue spinning solution, and then obtaining the slightly soluble glue electrospun fiber scaffold by a slightly soluble glue electrospinning technology;
step three, preparing liposome solution by adopting reverse evaporation method technology
(1) Dissolving lecithin, cholesterol and DSPE-PEG-CHO in trichloromethane, then adding octadecylamine dissolved in trichloroethane, and fully dissolving; dropwise adding an etanercept aqueous solution, performing water bath ultrasonic treatment after dropwise adding to obtain a stable emulsion, and performing water bath reduced pressure evaporation to remove an organic solution to obtain a colloidal product;
(2) Sequentially carrying out hydration treatment and ultrasonic treatment on the colloidal product, and filtering to obtain an etanercept liposome solution;
step four, preparing the multimode composite bracket: and soaking the slightly soluble glue electrospun fiber scaffold in a liposome solution for 20-30 hours, and finally washing the slightly soluble glue electrospun fiber scaffold with deionized water to load the cell membrane on the slightly soluble glue electrospun fiber scaffold.
Further, in step one, adipose-derived stem cells were continuously cultured in a temperature-sensitive culture dish for 2 weeks without passage, during which the medium was changed every 2 days and vitamin C was added to promote extracellular matrix secretion; after 2 weeks of culture, slices were formed and the incubation temperature was lowered to 15-22 ℃ and the bottom of the temperature sensitive dish was separated from the cells and the cell membrane was harvested.
Further, the above-mentioned drug or active substance having an osteogenesis or chondrogenesis promoting effect is selected from one or more of BMP-12 (bone morphogenetic factor 12), BMP-2 (bone morphogenetic factor 2), PDGF (platelet-derived growth factor) and phosphorus.
Further, the added mass of the drug or active substance having an osteogenesis or chondrogenesis promoting effect is 1 to 100 μ g.
Further, the above-mentioned drug or active substance having osteogenesis promoting or chondrogenesis promoting effect comprises BMP-12 and PDGF, and the addition mass ratio of the two is preferably 1:1 to 10.
Further, the adding mass ratio of the hyaluronic acid to the aminated PLA is 1:8 to 12.
Further, in the third step, the mass ratio of the lecithin, the cholesterol and the DSPE-PEG-CHO is 40:10:1; the addition mass ratio of DSPE-PEG-CHO to octadecylamine is 4:5.
further, the parameters of the ultrasonic treatment in step three are: the time is 3min, the power is 10 percent, and the work is stopped for 1s for 2 s.
Further, the filtration in the third step is a filtration sequentially through 0.45 μm and 0.22 μm microporous filtration membranes.
The second aspect of the invention is to provide the multimode composite stent prepared by the preparation method.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the drug-loaded polylactic acid electrospun fiber membrane is prepared by applying drugs or active substances with the effect of promoting osteogenesis or chondrogenesis, and liposome containing etanercept is loaded on the membrane through Schiff base and an adipose-derived stem cell membrane is loaded on the membrane. The multimode scaffold can ensure the long-term slow release of the drug or active substance with the effect of promoting bone formation or cartilage formation and the etanercept, and maintain effective concentration at local parts. Meanwhile, the polylactic acid electrospun fiber membrane has high histocompatibility as a stent main body, controllable degradation speed, and synergistic effect with the adipose-derived stem cell membrane, and is beneficial to cell growth and tissue regeneration.
The multimode composite scaffold provided by the invention can promote the regeneration of cartilage at the tendon-bone interface of the rotator cuff in an immune-mediated manner, improve the reconstruction of a multi-layer fibrocartilage structure instead of the formation of simple scar tissues, and increase the biomechanical strength of tendon-bone healing, thereby improving the success rate of rotator cuff repair and reducing postoperative injury.
Drawings
FIG. 1 is a schematic flow diagram of one embodiment of the present invention for making a multimodal composite scaffold;
FIG. 2 is a photograph showing a cell patch obtained by culturing adipose-derived stem cells according to an embodiment of the present invention;
FIG. 3 is a graph of the particle size distribution of liposomes prepared in one embodiment of the present invention;
FIG. 4 is a slow release profile of PDGF (panel A) and BMP-12 (panel B) from a sparingly soluble electrospun fibrous scaffold in an embodiment of the invention;
FIG. 5 illustrates the failure load that the prosthetic rotator cuff of the stent assembly can withstand in failure-load experiments in accordance with an embodiment of the present invention;
FIG. 6 shows the results of histological examination of the supraspinatus-bone complex in accordance with an embodiment of the present invention; wherein, panel a is a hematoxylin and eosin stained image showing morphological features of the healing interface; panel B is a toluidine blue stained image showing fibrocartilage formation.
Detailed Description
The invention provides a multimode composite scaffold with immune mediation and capability of promoting healing of rotator cuff tendon bones and a preparation method thereof.
The first aspect of the invention provides a preparation method of a multimode composite scaffold with immune mediation and capability of promoting healing of aponeurosis of a rotator cuff, which comprises the following steps:
step one, preparing a cell membrane: preparing a cell membrane by adopting adipose-derived stem cells;
step two, preparing the micro-sol electrospun fiber scaffold
(1) Completely dissolving hyaluronic acid in deionized water, adding medicine or active substance with osteogenesis or chondrogenesis promoting effect, and mixing;
(2) Adding Span80 and DCM, and stirring at high speed to obtain a uniform and stable water-oil emulsion;
(3) Adding aminated PLA and DMF into the water-oil emulsion to obtain a slightly soluble gel spinning solution, and then obtaining the slightly soluble gel electrospun fiber scaffold by a slightly soluble gel electrospinning technology;
step three, preparing liposome solution by adopting reverse evaporation method technology
(1) Dissolving lecithin, cholesterol and DSPE-PEG-CHO in trichloromethane, then adding octadecylamine dissolved in trichloroethane, and fully dissolving; dropwise adding an etanercept aqueous solution, carrying out water bath ultrasound to obtain a stable emulsion, and carrying out water bath reduced pressure evaporation to remove an organic solution to obtain a colloidal product;
(2) Sequentially carrying out hydration treatment and ultrasonic treatment on the colloidal product, and filtering to obtain an etanercept liposome solution;
step four, preparing the multimode composite bracket: and soaking the slightly soluble glue electrospun fiber scaffold in a liposome solution for 20-30 hours, and finally washing the slightly soluble glue electrospun fiber scaffold with deionized water to load the cell membrane on the slightly soluble glue electrospun fiber scaffold.
In a preferred embodiment of the present invention, in step one, adipose-derived stem cells are cultured in a temperature sensitive culture dish for 2 weeks without passage, during which the medium is replaced every 2 days and vitamin C is added to promote extracellular matrix secretion; after 2 weeks of culture, sheets were formed and the incubation temperature was lowered to 15-22 ℃ and the bottom of the temperature sensitive dish was separated from the cells and the cell membrane was harvested.
In a preferred embodiment of the present invention, the drug or active substance having an osteogenic or chondrogenic effect is selected from one or more of BMP-12, BMP-2, PDGF and phosphorus.
In a preferred embodiment of the present invention, the pharmaceutical or active substance having an osteogenic or chondrogenic activity is added in an amount of 1 to 100. Mu.g by mass.
In a preferred embodiment of the present invention, the drug or active substance having osteogenesis promoting or chondrogenesis promoting effect comprises BMP-12 and PDGF, and the ratio of the two added mass is preferably 1:1 to 10.
In a preferred embodiment of the present invention, the added mass ratio of HA to aminated PLA is 1:8 to 12. In a preferred embodiment of the invention, in step three, the mass ratio of lecithin, cholesterol and DSPE-PEG-CHO is 40:10:1; the addition mass ratio of DSPE-PEG-CHO to octadecylamine is 4:5.
in a preferred embodiment of the present invention, the parameters of the ultrasonic treatment in the third step are: the time is 3min, the power is 10 percent, and the work is stopped for 1s for 2 s.
In a preferred embodiment of the present invention, the filtration in step three is sequentially performed through 0.45 μm and 0.22 μm microporous filtration membranes.
The second aspect of the invention provides the multimode composite stent prepared by the preparation method.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, conventional methods were used unless otherwise specified, and reagents used were, for example, conventional commercially available reagents or reagents prepared by conventional methods.
Example 1
The embodiment provides a multimode composite scaffold with immune mediation and capability of promoting healing of rotator cuff tendon bones, and a preparation method thereof comprises the following steps (as shown in figure 1):
step one, preparing a cell membrane: continuously culturing the adipose-derived stem cells in a temperature sensitive culture dish for 2 weeks without passage, and changing the culture medium every 2 days and adding vitamin C to promote the secretion of extracellular matrix during the period; after 2 weeks of culture, thin slices are formed, the incubation temperature is reduced to 20 ℃, the bottom of the temperature sensitive culture dish is separated from the cells, and then cell membranes are obtained (shown in figure 2);
step two, preparing a micro-sol electrospun fiber scaffold:
(1) 1.5. Mu.g of BMP-12 and 1.5. Mu.g of PDGF were mixed with 50. Mu.l of HA solution by dissolving 0.1g of HA in 9.9g of deionized water and spinning at room temperature until complete dissolution;
(2) Then 0.01g of Span80 and 4g of DCM are added to the mixture and stirred at high speed for 30 minutes at room temperature to obtain a uniform and stable water-oil emulsion;
(3) Adding 0.5g of aminated PLA and 2g of DMF into the emulsion to finally obtain a micro-sol spinning solution;
(4) Active factors PDGF and BMP-12 are wrapped in polylactic acid electrospun fibers by a slightly soluble gel electrospinning technology to form a slightly soluble gel electrospun fiber scaffold;
step three, preparing liposome solution by adopting a reverse evaporation method technology:
(1) Weighing 160mg of lecithin, 40mg of cholesterol and 4mg of DSPE-PEG-CHO, dissolving in 5mL of trichloromethane, dissolving 5mg of octadecylamine in 1mL of trichloroethane, transferring the solution into a 250mL eggplant-shaped bottle after the two solutions are fully dissolved, dropwise adding an etanercept aqueous solution into the eggplant-shaped bottle, carrying out water bath ultrasound after the dropwise adding is finished to obtain stable emulsion, and carrying out water bath decompression evaporation at 10 ℃ to remove the organic solution to obtain a colloidal product;
(2) Adding a proper amount of deionized water into an eggplant-shaped bottle for hydration treatment, then carrying out ultrasonic treatment for 3min with the power of 10 percent for 2s and stopping for 1s, and after the ultrasonic treatment, sequentially passing through 0.45 mu m and 0.22 mu m microporous filter membranes to obtain an etanercept liposome solution; after the preparation, the particle size and uniformity were measured by Dynamic Light Scattering (DLS), and the results are shown in FIG. 3;
as shown in figure 3, the particle size of the etanercept liposome suspension is 162.8nm, the particle size distribution is good, and the PDI is 0.241;
step four, preparing the multimode composite scaffold: soaking the slightly soluble gel electrospun fiber scaffold in 5ml of liposome solution, placing the solution in a drying oven at 37 ℃ for at least 24 hours, finally washing the solution with deionized water for three times to obtain the required scaffold, and loading the adipose-derived stem cell membrane on the scaffold.
Verification of the embodiments
This example performed the following performance tests on the composite scaffold provided in example 1:
1. in vitro Release characteristics
In order to explore the in-vitro release characteristics of BMP-12 and PDGF drugs in the stent carrier, the prepared stent is immersed in a PBS buffer solution, and an in-vitro release experiment is carried out in a constant temperature shaking table with the temperature of 37 ℃ and the rotating speed of 100 rpm; all release media were replaced at predetermined time points of 1 day, 2 days, 1 week, etc., samples were tested by HPLC and BCA kits and in vitro release profiles were plotted, with the results shown in figure 4; in vitro release of PDGF and BMP-12 can be maintained for about 1 month and the desired local effective concentration achieved.
2. Biomechanical test (failure-load experiment)
Biomechanical testing of the supraspinatus-bone complex at 4 and 8 weeks was performed using a uniaxial testing system to analyze the ability of the composite scaffolds to promote tissue regeneration in a rotator cuff injury model. Supraspinatus tendon was covered with polyester cloth and braided with suture to prevent tissue slippage. In addition, the proximal humerus is rigidly fixed in a polyvinyl chloride tube with plaster. A preload of 0.1N was first applied, five rounds were performed for 5 minutes, and then a uniaxial tension of 1mm/min was applied to obtain a failure load (failure load). The results are shown in FIG. 5.
As can be seen in fig. 5, the repair rotator cuff of the stent group can bear Failure load in the snap-off test more than the control group (simple repair) at both 4 weeks and 8 weeks.
3. Histological examination
At 4 and 8 weeks, rats of each group were sacrificed for histological analysis. The removed supraspinatus-bone complex was fixed in 10% neutral buffered formalin for 3 days and decalcified with 10% ethylenediaminetetraacetic acid at 37 ℃ for 14 days. The supraspinatus-bone complex was then dehydrated and embedded in paraffin and multiple coronal sections of 5 μm were performed. The obtained tissue sections were stained with hematoxylin, eosin and toluidine blue to observe the morphological characteristics of the healing interface and the formation of fibrocartilage, and the results are shown in fig. 6.
As can be seen from fig. 6, the advantage of the scaffold group in healing at the interface of the tendon and bone is significantly better than the control group, and the scaffold group has more fibrocartilage formed at each time.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Equivalent modifications and substitutions of the present invention are within the scope of the present invention for any person skilled in the art. Accordingly, equivalent alterations and modifications are intended to be included within the scope of the invention, without departing from the spirit and scope of the invention.
Claims (10)
1. The preparation method of the multimode composite scaffold with the functions of immune mediation and promotion of rotator cuff tendon bone healing is characterized by comprising the following steps:
step one, preparing a cell membrane: preparing a cell membrane by adopting adipose-derived stem cells;
step two, preparing the micro-sol electrospun fiber scaffold
(1) Completely dissolving hyaluronic acid in deionized water, adding medicine or active substance with osteogenesis or chondrogenesis promoting effect, and mixing;
(2) Adding Span80 and DCM, and stirring at high speed to obtain uniform and stable water-oil emulsion;
(3) Adding aminated PLA and DMF into the water-oil emulsion to obtain a slightly soluble glue spinning solution, and then obtaining a slightly soluble glue electrospun fiber scaffold by a slightly soluble glue electrospinning technology;
step three, preparing liposome solution by adopting a reverse evaporation method technology
(1) Dissolving lecithin, cholesterol and DSPE-PEG-CHO in trichloromethane, then adding octadecylamine dissolved in trichloroethane, and fully dissolving; dropwise adding an etanercept aqueous solution, carrying out water bath ultrasound to obtain a stable emulsion, and carrying out water bath reduced pressure evaporation to remove an organic solution to obtain a colloidal product;
(2) Sequentially carrying out hydration treatment and ultrasonic treatment on the colloidal product, and filtering to obtain an etanercept liposome solution;
step four, preparing the multimode composite scaffold: and soaking the slightly soluble glue electrospun fiber scaffold in a liposome solution for 20-30 hours, and finally washing with deionized water to load the cell membrane on the slightly soluble glue electrospun fiber scaffold.
2. The preparation method according to claim 1, wherein in the first step, the adipose-derived stem cells are cultured in a temperature-sensitive culture dish for 2 weeks without passage, during which the medium is replaced every 2 days and vitamin C is added to promote extracellular matrix secretion; after 2 weeks of culture, sheets were formed and the incubation temperature was lowered to 15-22 ℃ and the bottom of the temperature sensitive dish was separated from the cells and the cell membrane was harvested.
3. The method for preparing a drug or an active substance having an osteogenesis or chondrogenesis effect according to claim 1, wherein the drug or the active substance is selected from one or more of BMP-12, BMP-2, PDGF, and Hexaphos.
4. The method according to claim 3, wherein the pharmaceutical or active substance having an osteogenic or chondrogenic effect is added in an amount of 1 to 100 μ g in mass.
5. The method for preparing a drug or an active substance having an osteogenesis or chondrogenesis effect according to claim 3, wherein the drug or the active substance includes BMP-12 and PDGF.
6. The method according to claim 1, wherein the mass ratio of hyaluronic acid to aminated PLA is 1:8 to 12.
7. The preparation method according to claim 1, wherein in step three, the mass ratio of lecithin, cholesterol and DSPE-PEG-CHO is 40:10:1; the addition mass ratio of DSPE-PEG-CHO to octadecylamine is 4:5.
8. the method of claim 1, wherein the parameters of the sonication in step three are: the time is 3min, the power is 10 percent, and the work is stopped for 1s for 2 s.
9. The method according to claim 1, wherein the filtration in the third step is sequentially performed through a 0.45 μm, 0.22 μm microporous membrane.
10. A multimodal composite scaffold prepared according to the method of any one of claims 1 to 9.
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| US20120189587A1 (en) * | 2011-01-26 | 2012-07-26 | The Chinese University Of Hong Kong | Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same |
| CN104841022A (en) * | 2014-02-14 | 2015-08-19 | 赵金忠 | Application of nanofiber membrane in preparation of rotator cuff injury treatment material |
| CN111494723A (en) * | 2020-04-22 | 2020-08-07 | 苏州大学附属第一医院 | Preparation method of micro-nano fiber for promoting nerve regeneration through micro-environment responsive immune regulation |
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| US20120189587A1 (en) * | 2011-01-26 | 2012-07-26 | The Chinese University Of Hong Kong | Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same |
| CN104841022A (en) * | 2014-02-14 | 2015-08-19 | 赵金忠 | Application of nanofiber membrane in preparation of rotator cuff injury treatment material |
| CN111494723A (en) * | 2020-04-22 | 2020-08-07 | 苏州大学附属第一医院 | Preparation method of micro-nano fiber for promoting nerve regeneration through micro-environment responsive immune regulation |
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