CN115721781A - Preparation process of artificial tendon with cell density and mechanical strength - Google Patents
Preparation process of artificial tendon with cell density and mechanical strength Download PDFInfo
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- CN115721781A CN115721781A CN202211114799.8A CN202211114799A CN115721781A CN 115721781 A CN115721781 A CN 115721781A CN 202211114799 A CN202211114799 A CN 202211114799A CN 115721781 A CN115721781 A CN 115721781A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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- 239000000661 sodium alginate Substances 0.000 claims abstract description 21
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 12
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Abstract
The invention provides a preparation process of an artificial tendon with cell density and mechanical strength, which is used for preparing a novel artificial tendon and is applied to the field of tendon tissue repair, and the process comprises the following steps: preparing a PCL grid by using an electrofluid jet printing technology; (2) rolling PCL into a tubular shape; (3) Forming a film on the periphery of the tubular PCL support by utilizing the cross-linking characteristic of sodium alginate and calcium ions; (4) injecting cells into the scaffold capsule; (5) After culturing for a certain time, dissolving the film outside the bracket capsule by utilizing the crosslinking removal characteristic of sodium alginate and sodium citrate; (6) After the culture was carried out for a certain period of time, cyclic stretching was started for a certain period of time. The cell concentration of the artificial tendon prepared by the technology can reach3×10 8 Individual cells/ml.
Description
Technical Field
The invention provides a preparation process of an artificial tendon with cell density and mechanical strength, which is used for preparing a novel artificial tendon and is applied to the field of tendon tissue repair.
Background
Many studies have been reported so far on the preparation of artificial tendons from biomaterials for tissue repair therapy, and various artificial tendons have been prepared. The prior art mainly has the following problems:
1. the preparation of the tendon tissue engineering fiber scaffold needs biological materials, and the popular materials at present comprise synthetic polymer materials such as PLA, PCL, PLGA and the like and natural materials such as silk, sodium alginate and the like, and although the materials have biocompatibility, the microstructure and the activity of the biological materials are greatly different from those of a natural tendon matrix.
2. Regardless of the structure of the prepared scaffold, most of the space of the scaffold is occupied by the biological material, so that the density of cells on the scaffold is low (such as 1X 10) 6 Individual cells/mL) cells have weak interactions that are detrimental to cell spreading, proliferation, 3D culture and ECM formation.
3. The structure manufactured based on the cell cluster technology only contains cells, although the cell density is high, the structure can have higher strength (the compression modulus is approximately equal to 1.1 MPa) after long-time culture due to lack of support of biological materials, but the structure still cannot meet the requirements of tendon tissue strength (tendon tissue strength is different in position and strength, the whole range is approximately 65-820MPa of Young modulus and 11-80MPa of ultimate strength), and cannot be applied to tendon tissue repair.
The prior art CN201610534817.6 discloses a method for preparing an artificial tendon, the cell concentration inoculated on the artificial tendon is 1 × 10 6 cells/mL, but the number of seeded cells has yet to be increased.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a preparation process of an artificial tendon with cell density and mechanical strength, which comprises the following steps:
(1) The PCL grid is prepared by utilizing an electrofluid jet printing technology, and the preferable technical scheme is as follows: setting printing parameters: printing needle 21G, printing voltage 3kv, injection speed: 1 microliter/min, and preparing a PCL grid with the size of 30 x 20mm;
(2) Rolling PCL into a tubular shape;
(3) The method utilizes the cross-linking characteristic of sodium alginate and calcium ions to form a film on the periphery of the tubular PCL stent, and the preferable technical scheme is as follows: soaking in 0.8% sodium alginate aqueous solution to ensure that the sodium alginate solution is quickly placed in 4% calcium chloride aqueous solution to form a film on the periphery of the PCL support after being uniformly attached;
(4) Injecting cells into the bracket capsule, wherein the preferable technical scheme is as follows: the method comprises the steps of utilizing a tendon differentiation culture medium prepared from 2.5mg of ascorbic acid, 5 mu g of TGF-beta 3 growth factor, 75ml of bovine serum albumin (corning), 5ml of penicillin-streptomycin and 500ml of DMEM culture medium (FBS), differentiating the human mesenchymal stem cell tendon for 7 days, digesting and centrifuging by trypsin to obtain a cell mass, sucking the cell mass by a micro-syringe, injecting the cell mass into a bracket capsule, clamping two ends by a medium-sized vascular clamp, and placing the cell mass into a culture dish (corning) of 60mm for starting culture, wherein liquid is changed every two days, and 6-8ml of liquid is needed each time;
(6) After culturing for a certain time, dissolving the film outside the bracket capsule by utilizing the de-crosslinking characteristics of sodium alginate and sodium citrate, and the preferable technical scheme is as follows: placing the mixture into an aqueous solution of sodium citrate with the mass concentration of 4%, shaking for 5 minutes by using a shaking table at the speed of 100rpm, and dissolving the film outside the bracket capsule by utilizing the crosslinking removal characteristic of sodium alginate and sodium citrate;
(6) After the culture is carried out for a certain time, the cyclic stretching is carried out for a certain time, and the preferable technical scheme is as follows: the stretching frequency was 0.1Hz, and the radial stretching amplitude was 3%.
Through a large number of studies, it was found that: the sodium alginate aqueous solution with the mass concentration of 0.8% is selected to ensure that the sodium alginate aqueous solution on the tubular PCL bracket can be evenly distributed at all parts of the bracket and has proper thickness. If the concentration of the solution is too high, the viscosity of the solution is too high, so that huge droplets are formed on the tubular PCL support, and the thickness of the prepared film is too thick.
And, through a large number of studies, found that: the calcium chloride aqueous solution with the mass concentration of 4% is selected to enable the crosslinking to be faster, so that the film is ensured to be formed quickly, and no extra deformation is generated; the sodium citrate aqueous solution with the mass concentration of 4% is selected for use, so that the crosslinking can be removed more quickly, the film can be dissolved more quickly and better, the time of the artificial tendon in the sodium citrate aqueous solution is shortened, the damage to cells is avoided, and in addition, the damage to the cells by the concentration is small.
In a preferred embodiment of the present invention, the culturing time in the step (5) is from the 7 th day of culturing.
In a preferred embodiment of the present invention, the culture time in the step (6) is from the 14 th day of culture, and the cycle stretching is performed for seven days.
The beneficial effects of the invention compared with the prior art comprise:
1. compared with the traditional tendon scaffold preparation technology, the technology has higher cell concentration and is close to natural human tissues. For example, CN201610534817.6, the cell concentration of the prepared artificial tendon is 1 × 10 6 cell/mL, and the cell concentration of the technology can reach 3X 10 8 Individual cells/ml. In addition, the mechanical strength of the artificial tendon prepared by the process can also meet the requirements of natural tendon tissues.
2. The process further adopts stretching culture and circular stretching culture, can bring new stimulation to cells, and can further promote the expression of tendon-related genes (Tnmd, scx) of the cells in the prepared artificial tendon, tendon-related proteins Col I and tenomodulin and tendon-related matrix genes (Col I, fmod, fn, tnC, and Thbs 4).
Drawings
FIG. 1 is a flow chart of the process for preparing an artificial tendon;
FIG. 2 is a flow chart of PCL grid preparation by electrofluid jet printing technology;
FIG. 3, a PCL grid schematic;
FIG. 4, schematically, rolling PCL into a tubular shape;
FIG. 5 is a schematic diagram of a film formed on the periphery of a tubular PCL stent by utilizing the cross-linking property of sodium alginate and calcium ions;
fig. 6 is a detailed schematic diagram of the structure of the stent capsule.
FIG. 7, schematic illustration of injection of cells into a scaffold capsule;
FIG. 8, schematic representation of the completion of the injection with both ends clamped with vascular clamps;
FIG. 9, schematic of cell aggregation over time in culture;
FIG. 10, which shows the dissolution of the membrane outside the scaffold capsule by the seventh day of culture using the de-crosslinking property of sodium alginate and sodium citrate;
FIG. 11 is a schematic view showing that the cyclic stretching culture was started for seven days from the culture on day 14.
Detailed Description
The invention will be further illustrated, but is not limited, by the following examples and the accompanying drawings.
Example 1 referring to fig. 1-11, a process for preparing an artificial tendon having cell density and mechanical strength comprises the following steps:
1) With the electrofluid ejection printing technique (printing parameter setting: printing needle 21G, printing voltage 3kv, injection speed: 1 microliter/min), and PCL meshes (0.5 mm in transverse mesh gap and 1mm in longitudinal mesh gap, and formed by stacking 1 layer of coarse fiber mesh with a diameter of about 100 micrometers and 3 layers of fine fiber mesh with a diameter of about 20 micrometers) with a size of 30 × 20mm are prepared.
2) It was rolled into a tube using a 0.8mm diameter 304 stainless steel rod.
3) The steel bar and the PCL bracket on the steel bar are put into an aqueous solution of sodium alginate with the mass concentration of 0.8% to be soaked together, the sodium alginate solution is ensured to be quickly put into an aqueous solution of calcium chloride with the mass concentration of 4% after being uniformly attached, and a film (called bracket capsule) is formed at the periphery of the tubular PCL bracket by utilizing the characteristic of cross-linking of sodium alginate and calcium ions.
4) Differentiating human mesenchymal stem cell tendon by using a tendon differentiation culture medium prepared from 2.5mg of ascorbic acid, 5 mu g of TGF-beta 3 growth factor, 75ml of bovine serum albumin (corning), 5ml of penicillin-streptomycin and 500ml of DMEM (FBS) culture medium for 7 days, digesting by trypsin, centrifuging (2000rpm, 5 min) to obtain a cell mass, sucking the cell mass by using a micro-injector, injecting the cell mass into a bracket capsule, clamping two ends by using vascular clamps, and enabling the cell concentration to reach 3 multiplied by 10 8 The cells per ml were placed in a 60mm cultureThe dish (Corning) is started to culture, wherein, the liquid is changed every two days, and 6-8ml of the liquid is needed each time.
5) After culturing for seven days, putting the whole structure into an aqueous solution of sodium citrate with the mass concentration of 4%, shaking for 5 minutes by using a shaking table at the speed of 100rpm, dissolving the film outside the bracket capsule by utilizing the de-crosslinking characteristic of sodium alginate and sodium citrate, and then continuing culturing.
6) After 7 days of culture, the artificial tendon is subjected to stretching culture for 7 days by using a stretching culture bioreactor, so as to further promote expression of tendon-related genes and proteins of cells in the artificial tendon and arrangement of the proteins, and finally complete preparation of the artificial tendon, wherein the stretching frequency is 0.1Hz, and the radial stretching amplitude is 3%.
The detailed diagram of the structure of the bracket capsule obtained by the above steps is shown in FIG. 6, and the cell concentration can reach 3X 10 8 Individual cells/ml.
Note: sterility is required to be ensured in the whole process of the stent preparation process.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (8)
1. A preparation process of an artificial tendon with cell density and mechanical strength is characterized by comprising the following steps:
(1) Preparing a PCL grid by using an electrofluid jet printing technology;
(2) Rolling PCL into a tube shape;
(3) Forming a film on the periphery of the tubular PCL support by utilizing the cross-linking characteristic of sodium alginate and calcium ions;
(4) Injecting cells into the scaffold capsule;
(5) After culturing for a certain time, dissolving the film outside the bracket capsule by utilizing the de-crosslinking characteristic of sodium alginate and sodium citrate;
(6) After the culture is carried out for a certain period of time, cyclic stretching is started for a certain period of time.
2. The manufacturing process according to claim 1, characterized in that the print parameter setting: printing needle model number is 21G, printing voltage is 3kv, injection speed: 1 microliter/min, PCL mesh of 30 × 20mm in size was prepared.
3. The preparation process according to claim 1, wherein the PCL scaffold is immersed in an aqueous solution of sodium alginate having a mass concentration of 0.8%, ensuring that the sodium alginate solution is rapidly immersed in an aqueous solution of calcium chloride having a mass concentration of 4% after being uniformly attached, and forming a thin film on the periphery of the PCL scaffold.
4. The process according to claim 1, wherein the tendon differentiation medium is prepared by using 2.5mg ascorbic acid +5 μ g tgf- β 3 growth factor +75ml bovine serum albumin (corning) +5ml penicillin-streptomycin (corning) +500ml DMEM medium (corning), differentiating the human mesenchymal stem cell tendon for 7 days, then digesting with trypsin, centrifuging to obtain cell mass, sucking the cell mass with a micro-syringe, injecting into the scaffold capsule, clamping both ends with a medium-sized vascular clamp, placing into a culture dish (corning) of 60mm to start culturing, wherein 6-8ml of the solution is changed every two days.
5. The preparation process according to claim 1, wherein the membrane is placed in an aqueous solution of sodium citrate with a mass concentration of 4%, and shaken for 5 minutes by using a shaker at a speed of 100rpm to dissolve the membrane outside the stent capsule by utilizing the de-crosslinking property of sodium alginate and sodium citrate.
6. The process according to claim 1, wherein the stretching frequency is 0.1Hz and the radial stretching amplitude is 3%.
7. The production process according to claim 1, wherein the culturing time in the step (5) is from the 7 th day of culturing.
8. The process according to claim 1, wherein the cultivation time in the step (6) is from the cultivation to the 14 th day, and the cyclic stretching is performed for seven days.
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| CN202211114799.8A CN115721781B (en) | 2022-09-09 | 2022-09-09 | Preparation process of artificial tendon with cell density and mechanical strength |
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| CN202211114799.8A CN115721781B (en) | 2022-09-09 | 2022-09-09 | Preparation process of artificial tendon with cell density and mechanical strength |
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| CN115721781B CN115721781B (en) | 2023-11-07 |
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