CN113318273A - ECM gradient microfiber tube and preparation method thereof - Google Patents
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Abstract
The invention relates to the technical field of medical treatment and health, and provides an ECM gradient microfiber tube and a preparation method thereof, wherein the preparation method comprises the following steps: step one, preparing ECM-sodium alginate aqueous solution with final concentration of 5-10 mg/ml-1% and 1-3 mg/ml-1%, and preparing calcium chloride with mass concentration of 2-5% and polyvinyl alcohol with mass concentration of 10-15%; secondly, respectively sucking ECM-sodium alginate aqueous solution and polyvinyl alcohol with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; fixing the microfluidic device on the collecting tank by using a clamp; thirdly, setting the flow rate of the injection pump; and fourthly, simultaneously opening a polyvinyl alcohol micro-injection pump, a high-concentration ECM micro-injection pump and a low-concentration ECM micro-injection pump, closing a high-concentration ECM external phase channel after a mixing tube appears in the collection pool, only opening a low-concentration ECM channel, closing the low-concentration ECM channel and opening the high-concentration ECM channel after the low-concentration ECM becomes stable, thereby obtaining the gradient-concentration ECM microfiber tube.
Description
Technical Field
The invention relates to the technical field of medical treatment and health, in particular to an ECM gradient microfiber tube and a preparation method thereof.
Background
Extracellular matrix (ECM) is a complex structure that surrounds and supports cells found in mammalian or human tissue. ECM of mammalian or human tissue includes structural proteins (collagen and elastin), specific (particulate) proteins (fibrillar proteins, fibronectin and laminin), and proteoglycans. The major fibrous structural proteins collagen and elastin are responsible for tissue strength and elasticity and play a significant role in promoting cell growth and differentiation. Thus, ECM plays an important role in human tissue. Methods and devices for synthesizing ECM fiber catheters and other vascular prostheses, tissues have been developed in the prior art.
CN102899249A discloses a method of producing blood vessels by injecting extracellular matrix ECM and blood vessel-forming cells into the blood vessel-forming channel of a blood vessel producing device. The method provides a device and method for creating a blood vessel in vitro. A device for creating a blood vessel, comprising: a) a sump channel in fluid communication with the sump inlet; b) a source channel in fluid communication with the source inlet, substantially parallel to the sink channel; c) a blood vessel-forming channel in fluid communication with the blood vessel-forming channel inlet, disposed between and in contact with one side of the sink channel and the source channel, and substantially parallel to the source channel and the sink channel; d) a first culture channel in fluid communication with the first culture channel inlet, disposed in contact with the other side of the sink channel, and substantially parallel to the sink channel; and e) a second culture channel in fluid communication with the second culture channel inlet, in contact with the other side of the source channel, and substantially parallel to the source channel, wherein the plurality of microstructures are configured to allow interaction between the biochemical substances contained in each channel, and are arranged at set intervals on the interface of each adjacent channel.
CN102137926A discloses a method for promoting the production of ECM from fibroblasts and/or for promoting the migration of fibroblasts in a biological system, and the present invention relates to a method for promoting the production of one or more components of extracellular matrix from one or more fibroblasts in a biological system and/or for promoting the migration of one or more fibroblasts in a biological system. The method comprises exposing one or more fibroblast cells in a biological system to an effective amount of an agent with peroxidase activity.
CN110201223A relates to a degradable synthetic polymer and natural extracellular matrix composite material, an artificial blood vessel and a preparation method thereof, wherein the degradable synthetic polymer component in the preparation process can be selected from one or more material proportions, and the scaffold material with different fiber diameters, different fiber arrangements, different pore diameters and different pore structures can be prepared by various technologies such as electrostatic spinning, wet spinning, melt spinning, 3D printing, pouring, phase separation, particle leaching and the like. The natural extracellular matrix components are wide in source, can be selected from vascular tissues of different animal sources (such as pig and cow arteries, veins and the like) or vascular tissues of human donors (such as umbilical cords and the like), and can be flexibly adjusted in components and content according to requirements. The composite material prepared by the preparation technology and the artificial blood vessel have good mechanical property, controllable spatial structure and proper degradation speed, and also have excellent biocompatibility and biological induction activity. The preparation method disclosed by the invention is simple in preparation process, high in controllability, mild in condition and suitable for large-scale industrial production.
However, although some methods for preparing artificial catheters exist in the prior art, most of the artificial blood vessels and catheters used for preparing the peripheral nerve defects are uniform in concentration, the tropism of chemical factors to cell migration is not considered, most of the catheters carrying concentration gradients are grafted with proteins or medicines by a chemical method, the preparation is difficult and the process is complicated, and the prepared catheters are poor in biocompatibility. There is therefore a need to develop a microfibrous tube with ECM gradient and a method for preparing the same.
Disclosure of Invention
The invention aims to provide an ECM gradient microfiber tube and a preparation method thereof, which construct a decellularized scaffold concentration gradient tube for promoting and guiding nerve growth, and are used for repairing long-section defects of peripheral nerves so as to solve at least one of the technical problems in the background art.
In order to achieve the purpose, the invention adopts the technical scheme that: a method of making an ECM gradient microfiber tube comprising the steps of:
firstly, mixing 100mg/ml ECM gel with a sodium alginate solution with the mass concentration of 2-4% to prepare an ECM-sodium alginate aqueous solution with the final concentration of 5-10 mg/ml-1% and 1-3 mg/ml-1%, and preparing calcium chloride with the mass concentration of 2-5% and polyvinyl alcohol with the mass concentration of 10-15%;
secondly, respectively sucking ECM-sodium alginate aqueous solution and polyvinyl alcohol with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; after the connection is finished, fixing the microfluidic device on a collection pool filled with calcium chloride with the mass concentration of 2-5% by using a clamp, and ensuring that the lower edge of the microfluidic device is immersed in the collection pool;
step three, after the connection is finished, setting the flow rate of the injection pump: the flow rate of the inner phase PVA solution injection pump is 0.5-1.5ml/h, and the flow rate of the outer phase injection pump is 2-5 ml/h;
and fourthly, the micro-injection pump comprises a polyvinyl alcohol micro-injection pump, a high-concentration ECM micro-injection pump and a low-concentration ECM micro-injection pump, the polyvinyl alcohol micro-injection pump, the high-concentration ECM micro-injection pump and the low-concentration ECM micro-injection pump are opened at the same time to enable the micro-fluidic device to be filled with liquid, the high-concentration ECM external phase channel is closed after a mixing tube appears in the collection pool, only the low-concentration ECM channel is opened, and the low-concentration ECM channel is closed and the high-concentration ECM channel is opened after the low-concentration ECM tube is stable to obtain the gradient-concentration ECM microfiber tube.
Preferably, the microfluidic device comprises a square capillary tube fixed at the center of the slide by epoxy resin; the square capillary tube is provided with an injection pool capillary tube and a collection pool capillary tube, and the injection pool capillary tube and the collection pool capillary tube are coaxially arranged and fixed by resin; 5-hole capillaries are inserted into the injection pool capillaries, and the 5-hole capillaries are fixed in the injection pool capillaries by resin; a capillary tube is inserted into each hole of the 5-hole capillary tube to serve as an external connection tube, and the external connection tubes are fixed in the holes of the 5-hole capillary tube by resin; the micro-fluidic device is fixed on a collecting pool filled with 2-5% of calcium chloride by a clamp, and a collecting pool capillary tube 5 at the lower edge of the micro-fluidic device is immersed in the collecting pool filled with 2-5% of calcium chloride.
The assembly process of the microfluidic device comprises: fixing a square capillary tube in the center of a glass slide by using epoxy resin, loading a collection pool capillary tube and an injection pool capillary tube in the square tube, and adjusting the positions to ensure that the collection pool capillary tube and the injection pool capillary tube are coaxially arranged and are fixed by using resin; inserting a 5-hole capillary into the loaded injection pool capillary to enable the tip of the 5-hole capillary to be in contact with the tube wall of the injection pool capillary; after the 5-hole capillary tube is fixed by resin, inserting a capillary tube into each hole to serve as an external connecting tube, and fixing the external connecting tube by resin; the microfluidic device is soaked in ethanol and dried for later use after being loaded.
In some embodiments, the extension tubes inserted into each well of the 5-well capillary tube preferably include a first high-concentration ECM extension tube, a first low-concentration ECM extension tube, a polyvinyl alcohol extension tube, a second high-concentration ECM extension tube, and a second low-concentration ECM extension tube.
In some embodiments, preferably, the sterile syringes include a polyvinyl alcohol sterile syringe, a high concentration ECM sterile syringe, and a low concentration ECM sterile syringe.
In some embodiments, preferably, the internal phase PVA solution syringe pump is a polyvinyl alcohol micro syringe pump; the polyvinyl alcohol external connecting pipe is connected to the outlet of the polyvinyl alcohol sterile injector through a medical micro-pipe; the polyvinyl alcohol sterile injector is fixed on a polyvinyl alcohol micro-injection pump, and polyvinyl alcohol with the mass concentration of 10-15% is sucked in the polyvinyl alcohol sterile injector.
In some embodiments, preferably, the external phase syringe pump comprises a high concentration ECM microinjection pump and a low concentration ECM microinjection pump; the first high-concentration ECM external connecting pipe and the second high-concentration ECM external connecting pipe are connected to an outlet of the high-concentration ECM sterile syringe through a medical microtubule; the high-concentration ECM sterile injector is fixed on a high-concentration ECM micro-injection pump; the first low-concentration ECM extension tube and the second low-concentration ECM extension tube are connected to an outlet of the low-concentration ECM sterile syringe through a medical microtubule; the low-concentration ECM sterile injector is fixed on a low-concentration ECM micro-injection pump.
In some embodiments, preferably, the high concentration ECM sterile syringe draws an aqueous solution of 5-10 mg/ml-1% ECM-sodium alginate.
In some embodiments, preferably, the low-concentration ECM sterile syringe draws 1-3 mg/ml-1% ECM-sodium alginate in water.
Meanwhile, the invention also provides an ECM gradient microfiber tube, which is prepared by adopting the preparation method.
The beneficial effects of the invention include:
the ECM gradient microfiber tube and the preparation method thereof adopt a novel microfluidic technology, and can artificially design and assemble a microfluidic device to obtain an ideal material configuration. The ECM preparation method has controllable required concentration and gradient and simple and convenient process; the ECM gradient microfiber tube obtained by the method has the advantages of ECM concentration gradient, good strength, flexibility and elasticity, high tissue activity and high biocompatibility, can meet the requirement of serving as a tissue engineering blood vessel, and can realize the concentration gradient of a decellularized scaffold on one fiber tube.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed for the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
Fig. 1 is a schematic structural view of a device for manufacturing an ECM gradient microfiber tube according to the present invention.
Fig. 2 is an assembly view of the apparatus for manufacturing the ECM gradient microfiber tube according to the present invention.
FIG. 3 is a NF-200 immunofluorescence staining pattern of neurofilaments in experiments in which ECM gradient microfiber tubes prepared in example 1 of the present invention were bridged across sciatic nerve defects of rats of 1 cm.
FIG. 4 is a MBP immunofluorescence staining pattern of myelin sheath in a 1cm sciatic nerve defect experiment in ECM gradient microfiber tube-bridged rats prepared in example 1 of the present invention.
Fig. 5 is a concentration gradient test of ECM gradient microfiber tubes prepared in example 1 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly or indirectly secured to the other element. When an element is referred to as being "connected to" another element, it can be directly or indirectly connected to the other element. The terms "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like indicate orientations or positions based on the orientations or positions shown in the drawings, and are for convenience of description only and not to be construed as limiting the technical solution. The terms "first", "second" and "first" are used merely for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features. The meaning of "plurality" is two or more unless specifically limited otherwise.
The preparation method of the ECM gradient microfiber tube comprises the following steps:
firstly, mixing 100mg/ml ECM gel with a sodium alginate solution with the mass concentration of 2-4% to prepare an ECM-sodium alginate aqueous solution with the final concentration of 5-10 mg/ml-1% and 1-3 mg/ml-1%, and preparing calcium chloride with the mass concentration of 2-5% and polyvinyl alcohol (PVA) with the mass concentration of 10-15%;
secondly, respectively sucking ECM-sodium alginate solution and PVA with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; after the connection is finished, fixing the microfluidic device on a collection pool filled with calcium chloride with the mass concentration of 2-5% by using a clamp, and ensuring that the lower edge of the microfluidic device is immersed in the collection pool;
step three, after the device connection is finished, setting the flow rate of the injection pump: the flow rate of the internal phase PVA solution syringe pump (i.e., polyvinyl alcohol microinjection pump 16) is 0.5-1.5ml/h, and the flow rate of the external phase syringe pump (i.e., high concentration ECM microinjection pump 18 and low concentration ECM microinjection pump 20) is 2-5 ml/h.
And fourthly, firstly, simultaneously opening three injection pumps (namely a polyvinyl alcohol micro-injection pump 16, a high-concentration ECM micro-injection pump 18 and a low-concentration ECM micro-injection pump 20) to fill the liquid into the device, closing a high-concentration ECM external phase channel (namely the high-concentration ECM micro-injection pump 18) after a mixing pipe appears in the collection pool, only opening a low-concentration ECM channel (namely the low-concentration ECM micro-injection pump 20), and closing the low-concentration ECM channel and opening the high-concentration ECM channel after the low-concentration ECM11 is stably formed into a pipe to obtain the gradient-concentration ECM microfiber pipe. It can be seen in fig. 2 that the low concentration ECM11 and the high concentration ECM12 resulted in a gradient concentration ECM microfiber tube condition after tubing.
In particular, the ECM of the invention is an extracellular matrix, and in the examples the ECM is derived from healthy adult porcine sciatic nerve. Preferably, the method of processing the ECM sample comprises: is prepared from fresh sciatic nerve of pig by cutting into small segments with surgical scissors, soaking in 1% TrionX-100, and shaking at 120rpm and 4 deg.C for 12 hr. After shaking and washing for 3 times with Phosphate Buffered Saline (PBS), the membrane is soaked in 1 percent Sodium Dodecyl Sulfate (SDS) solution and further shaken for 48 hours. The cell-free samples were then freeze-dried after washing with PBS for 12h to remove residual SDS. Pulverizing the lyophilized cell-removed sample into coarse powder, digesting with 1mg/ml pepsin at normal temperature for 48h, adjusting pH to 7.4, and packaging at-80 deg.C.
Specifically, as shown in fig. 1 and 2, the apparatus for preparing the ECM gradient microfibre tube of the present invention comprises a microfluidic device including a square capillary tube 1, and a collection chamber 3 filled with calcium chloride at a mass concentration of 2-5%, the square capillary tube 1 being fixed to the center of a slide 2 with epoxy resin; the square capillary tube 1 is provided with an injection pool capillary tube 4 and a collection pool capillary tube 5, and the injection pool capillary tube 4 and the collection pool capillary tube 5 are coaxially arranged and fixed by resin; 5-hole capillaries are inserted into the injection pool capillaries, and the 5-hole capillaries are fixed in the injection pool capillaries by resin; a capillary tube is inserted into each hole of the 5-hole capillary tube to serve as an external connection tube, and the external connection tubes are fixed in the holes of the 5-hole capillary tube by resin; the micro-fluidic device is fixed on a collecting pool filled with 2-5% of calcium chloride by a clamp, and a collecting pool capillary tube 5 at the lower edge of the micro-fluidic device is immersed in the collecting pool filled with 2-5% of calcium chloride.
The tip 14 of the 5-hole capillary is in contact with the tube wall 13 of the injection pool capillary.
The external connecting pipes inserted into each hole of the 5-hole capillary comprise a first high-concentration ECM external connecting pipe 6, a first low-concentration ECM external connecting pipe 7, a polyvinyl alcohol external connecting pipe 8, a second high-concentration ECM external connecting pipe 9 and a second low-concentration ECM external connecting pipe 10.
The first high-concentration ECM extension pipe 6 and the second high-concentration ECM extension pipe 9 are connected to the outlet of a high-concentration ECM sterile syringe 17 through medical microtubules.
The high-concentration ECM sterile injector 17 is fixed on a high-concentration ECM micro-injection pump 18, and 5-10 mg/ml-1% ECM-sodium alginate aqueous solution (namely 5-10mg/ml 1% sodium alginate aqueous solution for removing the cell scaffold) is absorbed in the high-concentration ECM sterile injector 17.
The first low-concentration ECM extension pipe 7 and the second low-concentration ECM extension pipe 10 are connected to the outlet of a low-concentration ECM sterile syringe 19 through a medical microtubule.
The low-concentration ECM sterile injector 19 is fixed on a low-concentration ECM micro-injection pump 20, and 1-3 mg/ml-1% ECM-sodium alginate aqueous solution (namely 1-3mg/ml 1% sodium alginate aqueous solution of the acellular scaffold) is sucked into the low-concentration ECM sterile injector 19.
The polyvinyl alcohol external connecting pipe 8 is connected to the outlet of the polyvinyl alcohol sterile injector 15 through a medical micro-pipe.
The polyvinyl alcohol sterile injector 15 is fixed on a polyvinyl alcohol micro-injection pump 16, and polyvinyl alcohol with the mass concentration of 10-15% is sucked in the polyvinyl alcohol sterile injector 15.
The assembly process of the microfluidic device described in the present invention comprises:
the square capillary tube was fixed to the center of the slide with epoxy, the collection reservoir capillary tube and the injection reservoir capillary tube were loaded in the square tube, the positions were adjusted to ensure their coaxial arrangement and they were fixed with resin. And inserting the 5-hole capillary into the loaded injection pool capillary to enable the tip of the capillary to touch the wall of the injection pool capillary, fixing the capillary by using resin, inserting one capillary into each hole to serve as an external connecting pipe, and fixing the external connecting pipe by using resin. After the micro-fluidic device is loaded, the micro-fluidic device is soaked in ethanol and dried for standby.
The invention also provides an ECM gradient microfiber tube, which is prepared by adopting the preparation method.
Example 1
A method of making an ECM gradient microfiber tube comprising the steps of:
firstly, mixing 100mg/ml ECM gel with a sodium alginate solution with the mass concentration of 4% to prepare an ECM-sodium alginate aqueous solution with the final concentration of 5 mg/ml-1% and 1 mg/ml-1%, and preparing calcium chloride with the mass concentration of 2% and polyvinyl alcohol with the mass concentration of 10%;
secondly, respectively sucking ECM-sodium alginate aqueous solution and polyvinyl alcohol with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; after the connection is finished, the microfluidic device is fixed on a collection pool filled with calcium chloride with the mass concentration of 2% by using a clamp, and the lower edge of the microfluidic device is ensured to be immersed in the collection pool;
step three, after the connection is finished, setting the flow rate of the injection pump: the flow rate of an inner phase PVA solution injection pump is 0.5ml/h, and the flow rate of an outer phase injection pump is 2 ml/h;
and fourthly, the micro-injection pump comprises a polyvinyl alcohol micro-injection pump, a high-concentration ECM micro-injection pump and a low-concentration ECM micro-injection pump, the polyvinyl alcohol micro-injection pump, the high-concentration ECM micro-injection pump and the low-concentration ECM micro-injection pump are opened at the same time to enable the micro-fluidic device to be filled with liquid, the high-concentration ECM external phase channel is closed after a mixing tube appears in the collection pool, only the low-concentration ECM channel is opened, and the low-concentration ECM channel is closed and the high-concentration ECM channel is opened after the low-concentration ECM tube is stable to obtain the gradient-concentration ECM microfiber tube.
Example 2
A method of making an ECM gradient microfiber tube comprising the steps of:
firstly, mixing 100mg/ml ECM gel with a sodium alginate solution with the mass concentration of 4% to prepare ECM-sodium alginate aqueous solutions with the final concentrations of 8 mg/ml-1% and 1 mg/ml-1%, and preparing calcium chloride with the mass concentration of 3% and polyvinyl alcohol with the mass concentration of 12%;
secondly, respectively sucking ECM-sodium alginate aqueous solution and polyvinyl alcohol with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; after the connection is finished, the microfluidic device is fixed on a collecting pool filled with calcium chloride with the mass concentration of 3% by using a clamp, and the lower edge of the microfluidic device is ensured to be immersed in the collecting pool;
step three, after the connection is finished, setting the flow rate of the injection pump: the flow rate of an inner phase PVA solution injection pump is 0.8ml/h, and the flow rate of an outer phase injection pump is 5 ml/h;
and fourthly, the micro-injection pump comprises a polyvinyl alcohol micro-injection pump, a high-concentration ECM micro-injection pump and a low-concentration ECM micro-injection pump, the polyvinyl alcohol micro-injection pump, the high-concentration ECM micro-injection pump and the low-concentration ECM micro-injection pump are opened at the same time to enable the micro-fluidic device to be filled with liquid, the high-concentration ECM external phase channel is closed after a mixing tube appears in the collection pool, only the low-concentration ECM channel is opened, and the low-concentration ECM channel is closed and the high-concentration ECM channel is opened after the low-concentration ECM tube is stable to obtain the gradient-concentration ECM microfiber tube.
Example 3
A method of making an ECM gradient microfiber tube comprising the steps of:
firstly, mixing 100mg/ml ECM gel with a sodium alginate solution with the mass concentration of 4% to prepare ECM-sodium alginate aqueous solutions with the final concentrations of 5 mg/ml-1% and 2 mg/ml-1%, and preparing calcium chloride with the mass concentration of 2% and polyvinyl alcohol with the mass concentration of 10%;
secondly, respectively sucking ECM-sodium alginate aqueous solution and polyvinyl alcohol with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; after the connection is finished, the microfluidic device is fixed on a collection pool filled with calcium chloride with the mass concentration of 2% by using a clamp, and the lower edge of the microfluidic device is ensured to be immersed in the collection pool;
step three, after the connection is finished, setting the flow rate of the injection pump: the flow rate of an inner phase PVA solution injection pump is 0.5ml/h, and the flow rate of an outer phase injection pump is 3 ml/h;
and fourthly, the micro-injection pump comprises a polyvinyl alcohol micro-injection pump, a high-concentration ECM micro-injection pump and a low-concentration ECM micro-injection pump, the polyvinyl alcohol micro-injection pump, the high-concentration ECM micro-injection pump and the low-concentration ECM micro-injection pump are opened at the same time to enable the micro-fluidic device to be filled with liquid, the high-concentration ECM external phase channel is closed after a mixing tube appears in the collection pool, only the low-concentration ECM channel is opened, and the low-concentration ECM channel is closed and the high-concentration ECM channel is opened after the low-concentration ECM tube is stable to obtain the gradient-concentration ECM microfiber tube.
Physical properties and performance characterization of ECM gradient fiber tubes prepared for the examples are shown in table 1.
TABLE 1
Example 4
Experiment of bridging rat 1cm sciatic nerve defect by ECM gradient microfiber tube
SD rats of 8 weeks old are taken and randomly grouped, after adaptive feeding, the SD rats are subjected to pentobarbital sodium abdominal anesthesia, the lower femoral edge incision of the right lower limb is taken, and muscles are separated in a blunt manner to expose sciatic nerves. The sciatic nerve transection after nerve dissection was performed with a glass needle to form 1cm nerve defect. Group 1 was an end-to-end suture and nerve endings of silicone tubing loaded with ECM gradient microfiber tubing of example 1 with 9/0 national nylon thread; group 2 was an end-to-end suture and nerve endings of silicone tubing loaded with ECM gradient microfiber tubing of comparative example 1 with 9/0 national nylon thread; group 3 was an end-to-end suture and nerve endings of silicone tubing loaded with ECM gradient microfiber tubing of comparative example 2 with 9/0 national nylon thread. Penicillin was administered intramuscularly at 20 wu/d for 7 days after surgery. Among them, comparative example 1 is an ECM fiber tube prepared by injecting only ECM solution having a concentration of 5mg/ml to 1% (prepared in the same manner as in example 1); comparative example 2 was an ECM solution injected only at a concentration of 1 mg/ml-1% (prepared as in example 1).
And (4) performing behavioral step imprint detection after 2 weeks, 4 weeks and 8 weeks of operation, and calculating the sciatic nerve function index SFI according to a formula. The sciatic nerve function index is an important index for expressing motor function recovery after nerve injury by measuring and analyzing foot prints on an affected side and a healthy side of a rat when the rat walks and calculating by a formula. Smaller SFI results in poorer functional recovery and vice versa. And 8 weeks after the operation, taking out a transplant section specimen, fixing by paraformaldehyde, freezing and embedding, carrying out 5-micrometer-layer thick longitudinal section, carrying out immunofluorescence staining on the nerve filaments NF-200 and myelin MBP, and observing the nerve growth and docking conditions by using a laser confocal microscope.
And (4) evaluating the results:
the behavioral index SFI of each group at 2, 4 and 8 weeks post-surgery is shown in table 2.
| |
2 |
4 |
8 weeks |
| Example 1 | -76.7±4.0 | -63.2±2.5 | -57.6±1.1 |
| Comparative example 1 | -87.2±3.8 | -78.9±3.3 | -69.1±2.5 |
| Comparative example 2 | -79.4±4.2 | -75.8±2.7 | -71.6±1.4 |
As can be seen from the above table, the ECM gradient microfibrous tubes of the example 1 group promoted restoration of motor function after nerve injury. Immunofluorescent staining of the graft at 8 weeks post-surgery in FIGS. 3 and 4, it is seen that the gradient of ECM microfibrous tubes promotes nerve fiber growth in axial alignment and axonal remyelination.
Whereas the high concentration ECM fiber tube of comparative example 1 had poor recovery of motor function at an early stage, which was significantly improved after 4 weeks, the low concentration ECM fiber tube of comparative example 2 had good recovery of motor function at an early stage, and had poor improvement of motor function index with time.
Example 5
ECM gradient microfiber tube concentration gradient characterization
To verify the effectiveness of the products prepared by the preparation method of the present invention on ECM gradient concentration, the product of example 1 was used as a sample. For the ECM gradient microfiber tube obtained in example 1, the concentration gradient in the radial direction was tested. ECM fiber tubes were first obtained at a specific length of 1cm, followed by the fiber tube centerline (the centerline is taken to be the axis of the fiber tube and is perpendicular to the vessel cross-section), and then the concentration of the specific ECM was measured by determining 5 points, ABCDE, on the fiber tube at an equally spaced distance based on the determined centerline, as shown in fig. 5. The results are given in Table 2, using standard 5mg ECM as reference.
TABLE 2
| ECM | A | B | C | D | E |
| ％ | 25％ | 45％ | 65％ | 85％ | 95％ |
The ECM gradient microfiber tube prepared by the invention has obvious ECM concentration gradient, good strength, flexibility and elasticity, high tissue activity and high biocompatibility, and can meet the requirement of serving as a tissue engineering blood vessel.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method of making an ECM gradient microfiber tube, comprising: the preparation method comprises the following steps:
firstly, mixing 100mg/ml ECM gel with a sodium alginate solution with the mass concentration of 2-4% to prepare an ECM-sodium alginate aqueous solution with the final concentration of 5-10 mg/ml-1% and 1-3 mg/ml-1%, and preparing calcium chloride with the mass concentration of 2-5% and polyvinyl alcohol with the mass concentration of 10-15%;
secondly, respectively sucking ECM-sodium alginate aqueous solution and polyvinyl alcohol with different concentrations by using a 1ml sterile injector, fixing the sterile injector on a micro-injection pump and connecting the sterile injector on a micro-fluidic device by using a medical micro-tube in sequence; after the connection is finished, fixing the microfluidic device on a collection pool filled with calcium chloride with the mass concentration of 2-5% by using a clamp, and ensuring that the lower edge of the microfluidic device is immersed in the collection pool;
step three, after the connection is finished, setting the flow rate of the injection pump: the flow rate of the inner phase PVA solution injection pump is 0.5-1.5ml/h, and the flow rate of the outer phase injection pump is 2-5 ml/h;
and fourthly, the micro-injection pump comprises a polyvinyl alcohol micro-injection pump, a high-concentration ECM micro-injection pump and a low-concentration ECM micro-injection pump, the polyvinyl alcohol micro-injection pump, the high-concentration ECM micro-injection pump and the low-concentration ECM micro-injection pump are opened at the same time to enable the micro-fluidic device to be filled with liquid, the high-concentration ECM external phase channel is closed after a mixing tube appears in the collection pool, only the low-concentration ECM channel is opened, and the low-concentration ECM channel is closed and the high-concentration ECM channel is opened after the low-concentration ECM tube is stable to obtain the gradient-concentration ECM microfiber tube.
2. The method of preparing an ECM gradient microfibre tube of claim 1, wherein the microfluidic device comprises a square capillary tube fixed to the center of a glass slide with epoxy; the square capillary tube is provided with an injection pool capillary tube and a collection pool capillary tube, and the injection pool capillary tube and the collection pool capillary tube are coaxially arranged and fixed by resin; 5-hole capillaries are inserted into the injection pool capillaries, and the 5-hole capillaries are fixed in the injection pool capillaries by resin; a capillary tube is inserted into each hole of the 5-hole capillary tube to serve as an external connection tube, and the external connection tubes are fixed in the holes of the 5-hole capillary tube by resin; the micro-fluidic device is fixed on a collecting pool filled with 2-5% of calcium chloride by a clamp, and a collecting pool capillary tube 5 at the lower edge of the micro-fluidic device is immersed in the collecting pool filled with 2-5% of calcium chloride.
3. The method of making an ECM gradient microfiber tube of claim 2, wherein the microfluidic device assembly process comprises: fixing a square capillary tube in the center of a glass slide by using epoxy resin, loading a collection pool capillary tube and an injection pool capillary tube in the square tube, and adjusting the positions to ensure that the collection pool capillary tube and the injection pool capillary tube are coaxially arranged and are fixed by using resin; inserting a 5-hole capillary into the loaded injection pool capillary to enable the tip of the 5-hole capillary to be in contact with the tube wall of the injection pool capillary; after the 5-hole capillary tube is fixed by resin, inserting a capillary tube into each hole to serve as an external connecting tube, and fixing the external connecting tube by resin; the microfluidic device is soaked in ethanol and dried for later use after being loaded.
4. The method of claim 2, wherein the extension tubes inserted into each well of the 5-well capillary tube comprise a first high concentration ECM extension tube, a first low concentration ECM extension tube, a polyvinyl alcohol extension tube, a second high concentration ECM extension tube, and a second low concentration ECM extension tube.
5. The method of preparing an ECM gradient microfiber tube according to claim 4, wherein said sterile syringes comprise polyvinyl alcohol sterile syringes, high concentration ECM sterile syringes and low concentration ECM sterile syringes.
6. The method of making an ECM gradient microfiber tube of claim 5, wherein said internal phase PVA solution syringe pump is a polyvinyl alcohol micro syringe pump; the polyvinyl alcohol external connecting pipe is connected to the outlet of the polyvinyl alcohol sterile injector through a medical micro-pipe; the polyvinyl alcohol sterile injector is fixed on a polyvinyl alcohol micro-injection pump, and polyvinyl alcohol with the mass concentration of 10% is sucked in the polyvinyl alcohol sterile injector.
7. The method of making an ECM gradient microfiber tube of claim 5, wherein said external phase syringe pump comprises a high concentration ECM microinjection pump and a low concentration ECM microinjection pump; the first high-concentration ECM external connecting pipe and the second high-concentration ECM external connecting pipe are connected to an outlet of the high-concentration ECM sterile syringe through a medical microtubule; the high-concentration ECM sterile injector is fixed on a high-concentration ECM micro-injection pump; the first low-concentration ECM extension tube and the second low-concentration ECM extension tube are connected to an outlet of the low-concentration ECM sterile syringe through a medical microtubule; the low-concentration ECM sterile injector is fixed on a low-concentration ECM micro-injection pump.
8. The method for preparing an ECM gradient microfiber tube according to claim 7, wherein the ECM sterile syringe with high concentration sucks the ECM-sodium alginate aqueous solution with concentration of 5-10 mg/ml-1%, and the ECM sterile syringe with low concentration sucks the ECM-sodium alginate aqueous solution with concentration of 1-3 mg/ml-1%.
9. The method of claim 1, wherein the ECM gel is derived from the sciatic nerve of a healthy adult pig.
10. An ECM gradient microfiber tube prepared by the method of any one of claims 1 to 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115806411A (en) * | 2022-09-16 | 2023-03-17 | 中国航发北京航空材料研究院 | Polymer fiber geopolymer composite material and preparation method thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173871A (en) * | 2011-12-22 | 2013-06-26 | 中国科学院大连化学物理研究所 | Method for producing nano electrospining with concentration gradient based on microfluidics technology |
| JP2014136128A (en) * | 2013-01-18 | 2014-07-28 | Univ Of Tokyo | Nerve fascicle for implantation and method of manufacturing the same |
| CN104233479A (en) * | 2014-09-02 | 2014-12-24 | 北京理工大学 | Method for adjusting concentration of magnetic particles in magnetic calcium alginate microfiber preparing process |
| JP2017077473A (en) * | 2015-10-21 | 2017-04-27 | 国立大学法人 東京大学 | Microtube, method for producing microtube, and device for producing microtube |
| US20170130184A1 (en) * | 2014-05-20 | 2017-05-11 | The University Of Tokyo | Hollow microfiber |
| JP2018016901A (en) * | 2016-07-27 | 2018-02-01 | 国立大学法人宇都宮大学 | Gel fiber composite body and method for producing the same |
| CN109927282A (en) * | 2019-04-17 | 2019-06-25 | 中国科学院长春应用化学研究所 | A kind of Method of printing of 3D printing system and fiber |
| US20200002681A1 (en) * | 2017-03-09 | 2020-01-02 | Universite de Bordeaux | Hollow cellular microfibre and method for producing such a hollow cellular microfibre |
| CN112138732A (en) * | 2020-09-27 | 2020-12-29 | 陕西科技大学 | Simple micro-fluidic device, magnetic alginate microspheres based on simple micro-fluidic device, and preparation method and application of magnetic alginate microspheres |
-
2021
- 2021-06-25 CN CN202110712194.8A patent/CN113318273B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173871A (en) * | 2011-12-22 | 2013-06-26 | 中国科学院大连化学物理研究所 | Method for producing nano electrospining with concentration gradient based on microfluidics technology |
| JP2014136128A (en) * | 2013-01-18 | 2014-07-28 | Univ Of Tokyo | Nerve fascicle for implantation and method of manufacturing the same |
| US20170130184A1 (en) * | 2014-05-20 | 2017-05-11 | The University Of Tokyo | Hollow microfiber |
| CN104233479A (en) * | 2014-09-02 | 2014-12-24 | 北京理工大学 | Method for adjusting concentration of magnetic particles in magnetic calcium alginate microfiber preparing process |
| JP2017077473A (en) * | 2015-10-21 | 2017-04-27 | 国立大学法人 東京大学 | Microtube, method for producing microtube, and device for producing microtube |
| JP2018016901A (en) * | 2016-07-27 | 2018-02-01 | 国立大学法人宇都宮大学 | Gel fiber composite body and method for producing the same |
| US20200002681A1 (en) * | 2017-03-09 | 2020-01-02 | Universite de Bordeaux | Hollow cellular microfibre and method for producing such a hollow cellular microfibre |
| CN109927282A (en) * | 2019-04-17 | 2019-06-25 | 中国科学院长春应用化学研究所 | A kind of Method of printing of 3D printing system and fiber |
| CN112138732A (en) * | 2020-09-27 | 2020-12-29 | 陕西科技大学 | Simple micro-fluidic device, magnetic alginate microspheres based on simple micro-fluidic device, and preparation method and application of magnetic alginate microspheres |
Non-Patent Citations (2)
| Title |
|---|
| XIANG WANG ET AL: "concentration gradient generation methods based on microfluidic systems", 《RSC ADVANCES》 * |
| YAO CHENG ET AL: "controlled fabrication of bioactive microfibers for creating tissue constructs using microfluidic techniques", 《ACS APPLIED MATERIALS AND INTERFACES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115806411A (en) * | 2022-09-16 | 2023-03-17 | 中国航发北京航空材料研究院 | Polymer fiber geopolymer composite material and preparation method thereof |
| CN115806411B (en) * | 2022-09-16 | 2024-04-09 | 中国航发北京航空材料研究院 | Polymer fiber geopolymer composite material and preparation method thereof |
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