CN115721781B - 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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- CN115721781B CN115721781B CN202211114799.8A CN202211114799A CN115721781B CN 115721781 B CN115721781 B CN 115721781B CN 202211114799 A CN202211114799 A CN 202211114799A CN 115721781 B CN115721781 B CN 115721781B
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- 210000002435 tendon Anatomy 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000000661 sodium alginate Substances 0.000 claims abstract description 22
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 22
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 22
- 239000002775 capsule Substances 0.000 claims abstract description 20
- 239000001509 sodium citrate Substances 0.000 claims abstract description 12
- 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
- 238000000034 method Methods 0.000 claims abstract description 10
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- 230000008569 process Effects 0.000 claims abstract description 6
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 5
- 125000004122 cyclic group Chemical group 0.000 claims abstract description 4
- 238000005096 rolling process Methods 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 41
- 239000007864 aqueous solution Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- 239000008188 pellet Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 230000002792 vascular Effects 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 239000003102 growth factor Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
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- 229960005322 streptomycin Drugs 0.000 claims description 3
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 2
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- 238000005119 centrifugation Methods 0.000 claims description 2
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- 230000017423 tissue regeneration Effects 0.000 abstract description 3
- 239000010408 film Substances 0.000 description 11
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- 108090000623 proteins and genes Proteins 0.000 description 6
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- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000010963 304 stainless steel Substances 0.000 description 1
- 101150066718 FMOD gene Proteins 0.000 description 1
- 229910000589 SAE 304 stainless steel Inorganic materials 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 101150092686 TNMD gene Proteins 0.000 description 1
- 102100033740 Tenomodulin Human genes 0.000 description 1
- 101710114852 Tenomodulin Proteins 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
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- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
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: (1) preparing PCL grid by using electrofluid jet printing technology; (2) rolling the PCL into a tube; (3) Forming a film on the periphery of the tubular PCL bracket by utilizing the characteristic of crosslinking 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 stent capsule by utilizing the decrosslinking characteristic of sodium alginate and sodium citrate; (6) After the culture was performed for a predetermined period of time, the cyclic stretching was started for a predetermined period of time. The cell concentration of the artificial tendon prepared by the technology can reach 3 multiplied by 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 tissue repair treatment by using biological materials for artificial tendons, and various artificial tendons have been prepared. The prior art mainly has the following problems:
1. biological materials are needed for preparing the tendon tissue engineering fiber scaffold, and currently, the popular materials comprise synthetic high polymer materials such as PLA, PCL, PLGA and natural materials such as silk and sodium alginate, and the biological materials have biocompatibility, but the microstructure and activity of the biological materials are greatly different from those of the natural tendon matrix.
2. The scaffold is prepared by taking up most of the space of the scaffold by biological material, so that the density of cells on the scaffold is low (e.g. 1×10 6 Individual cells/mL) cell-cell interactions are weak, which is detrimental to cell expansion, proliferation, 3D culture, and ECM formation.
3. The structure body manufactured based on the cell cluster technology only comprises cells, and has high cell density, but the structure body can not be applied to repair of tendon tissues because of the lack of support of biological materials and the need of long-time culture to have high strength (compression modulus is approximately equal to 1.1 MPa), but the structure body still has difficulty in meeting the requirements of the strength of the tendon tissues (different tendon positions and different strength, and the whole range is approximately 65-820MPa of Young modulus and 11-80MPa of ultimate strength).
The prior art CN201610534817.6 discloses a method for manufacturing artificial tendons, wherein the cell concentration of the artificial tendon inoculation is 1×10 6 cells/mL, but the number of inoculated cells remains to be increased.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a preparation process of an artificial tendon with cell density and mechanical strength, comprising the following steps:
(1) PCL grids are prepared by using electrofluidic 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, preparing PCL grid with the size of 30 x 20 mm;
(2) Rolling PCL into a tube shape;
(3) The film is formed on the periphery of the tubular PCL bracket by utilizing the characteristic of crosslinking sodium alginate and calcium ions, and the preferable technical scheme is as follows: soaking in 0.8% sodium alginate aqueous solution to ensure that sodium alginate solution is quickly placed in 4% calcium chloride aqueous solution after uniform adhesion, and forming a film on the periphery of the PCL bracket;
(4) The cells are injected into the stent capsule, and the preferable technical scheme is as follows: tendon differentiation medium prepared from 2.5mg ascorbic acid +5 mug TGF-beta 3 growth factor +75ml bovine serum albumin (Corning) +5ml penicillin-streptomycin +500ml DMEM medium (FBS) is used for differentiating human mesenchymal stem cell tendons for 7 days, then cell mass is obtained through trypsin digestion and centrifugation, the cell mass is sucked by a microinjector and injected into a bracket capsule, and two ends are clamped by a medium vascular clamp, and the bracket capsule is placed into a culture dish (Corning) with 60mm for starting culture, wherein liquid is changed every two days, and 6-8ml of liquid can be used each time;
(6) After the culture is carried out for a certain time, the film outside the stent capsule is dissolved by utilizing the decrosslinking characteristic of sodium alginate and sodium citrate, and the preferable technical scheme is as follows: placing into water solution of sodium citrate with mass concentration of 4%, shaking for 5min at speed of 100rpm by using a shaking table, and dissolving out film outside the bracket capsule by using the decrosslinking property of sodium alginate and sodium citrate;
(6) After culturing for a certain time, starting to circularly stretch for a certain time, and adopting the preferable technical scheme as follows: the stretching frequency was 0.1Hz and the radial stretching amplitude was 3%.
Through a great deal of research, it was found that: the sodium alginate aqueous solution with the mass concentration of 0.8% is selected, so that the sodium alginate aqueous solution on the tubular PCL bracket can be uniformly distributed at all positions of the bracket, and the thickness is proper. If the concentration of the solution is too high, the viscosity of the solution will be too high to form large droplets on the tubular PCL stent and the thickness of the fabricated film will be too thick, and if the concentration of the solution is too low, the fabricated film will not be easily defective because of the locations on the completely segmented tubular PCL stent.
And, through extensive research, it was found that: the calcium chloride aqueous solution with the mass concentration of 4% is selected to enable the film to be crosslinked more quickly, 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 quick crosslinking removal, the film can be dissolved out quickly and better, the time of artificial tendons in the sodium citrate aqueous solution is shortened, the damage to cells is avoided, and in addition, the damage to the cells due to the concentration is small.
As a preferable embodiment of the invention, the culturing time in the step (5) is from the 7 th day.
As a preferable embodiment of the invention, the culturing time in the step (6) is from the 14 th day of culturing, and the cyclic stretching is carried out for seven days.
The beneficial effects of the invention compared with the prior art include:
1. compared with the traditional tendon scaffold preparation technology, the process has higher cell concentration and is close to natural human tissues. For example, CN201610534817.6 in the patent, the cell concentration of artificial tendon inoculation made of the same is 1X 10 6 cell/mL, 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 the use of cyclic stretching culture can bring new stimulus to cells, and can further promote the expression of tendon-related genes (Tnmd, scx) and tendon-related proteins Col I and tenomodulin and tendon-related matrix genes (Col I, fmod, fn, tnC, and Thbs 4) of the prepared artificial intratendon cells.
Drawings
FIG. 1 is a flow chart of a preparation process of an artificial tendon;
FIG. 2, a flowchart for preparing a PCL grid using an electrofluid jet printing technique;
FIG. 3, PCL grid schematic;
FIG. 4, rolling PCL into a tubular schematic;
FIG. 5, a thin film schematic diagram is formed on the periphery of a tubular PCL bracket by utilizing the characteristic of crosslinking of sodium alginate and calcium ions;
fig. 6 shows a schematic diagram of the structure of the stent capsule.
FIG. 7, schematic representation of injection of cells into a stent capsule;
FIG. 8, injection completed, two ends clamped with vascular clamps;
FIG. 9 is a schematic of cell aggregation for a period of time in culture;
FIG. 10, schematic view of dissolution of the membrane outside the stent capsule by the decrosslinking property of sodium alginate and sodium citrate after culturing to seventh day;
FIG. 11, schematic drawing of the cycle stretching culture for seven days, starting from day 14 of culture.
Detailed Description
The present invention will be further described with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 referring to fig. 1 to 11, a process for preparing an artificial tendon having both cell density and mechanical strength comprises the steps of:
1) Using the electrofluidic jet printing technique (print parameter settings: printing needle 21G, printing voltage 3kv, injection speed: 1 microliter/min), PCL grids with a size of 30 x 20mm (transverse grid gaps 0.5mm, longitudinal grid gaps 1mm, which are formed by stacking 1 layer of coarse fiber grids with a diameter of about 100 microns and 3 layers of fine fiber grids with a diameter of about 20 microns) are prepared.
2) It was rolled into a tube using a 0.8mm diameter 304 stainless steel rod.
3) The steel bar and the tubular PCL bracket on the bar are put into an aqueous solution of sodium alginate with the mass concentration of 0.8% together for soaking, so that 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 a bracket capsule) is formed on the periphery of the tubular PCL bracket by utilizing the characteristic of crosslinking of sodium alginate and calcium ions.
4) Tendon differentiation medium prepared from 2.5mg ascorbic acid +5 μg TGF-beta 3 growth factor +75ml bovine serum albumin (Corning) +5ml penicillin-streptomycin +500ml DMEM medium (FBS) was used to differentiate human mesenchymal stem cell tendon for 7 days, then trypsinized, centrifuged (2000 rpm,5 min) to obtain cell pellet, the cell pellet was aspirated by a microinjector and injected into a stent capsule, and both ends were clamped by vascular clamps, and cell concentration was 3X 10 8 The cells/ml are placed in a culture dish (Corning) with the thickness of 60mm to start culture, wherein the liquid is changed every two days, and the volume is 6-8ml each time.
5) After seven days of culture, the whole structure is put into an aqueous solution of sodium citrate with the mass concentration of 4%, the shaking table is used for shaking for 5 minutes at the speed of 100rpm, and the film outside the bracket capsule is dissolved by utilizing the decrosslinking characteristic of sodium alginate and sodium citrate, and then the culture is continued.
6) After 7 days of culture, the artificial tendon is subjected to stretching culture for 7 days by using a stretching culture bioreactor, expression of tendon-related genes and proteins of cells in the artificial tendon and arrangement of proteins are further promoted, and finally the preparation of the artificial tendon is completed, wherein the stretching frequency is 0.1Hz, and the radial stretching amplitude is 3%.
The detailed diagram of the scaffold capsule structure obtained by the previous steps is shown in FIG. 6, and the cell concentration can reach 3×10 8 Individual cells/ml.
Note that: sterility needs to be ensured in the whole process of the bracket preparation technology.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Claims (7)
1. A preparation process of an artificial tendon with cell density and mechanical strength is characterized by comprising the following steps: (1) preparing PCL grid by using electrofluid jet printing technology; (2) rolling the PCL into a tube; (3) Forming a film on the periphery of the tubular PCL bracket by utilizing the characteristic of crosslinking of sodium alginate and calcium ions; (4) Injecting cells into the stent capsule, wherein the method for obtaining the cells comprises the following steps: tendon differentiation medium prepared from 2.5mg ascorbic acid +5 μg TGF-beta 3 growth factor +75ml bovine serum albumin +5ml penicillin-streptomycin +500ml DMEM medium is used to differentiate human mesenchymal stem cell tendon for 7 days, and then cell mass is obtained by trypsin digestion and centrifugation; (5) After culturing for a certain time, dissolving the film outside the stent capsule by utilizing the decrosslinking 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, the stretching frequency is 0.1Hz, and the radial stretching amplitude is 3%.
2. The manufacturing process according to claim 1, characterized in that the printing parameters are set: printing needle type is 21G, printing voltage 3kv, injection speed: 1 microliter/min, PCL grids with a size of 30 x 20mm were prepared.
3. The preparation process according to claim 1, wherein the sodium alginate solution is soaked in an aqueous solution of sodium alginate with the mass concentration of 0.8%, so that the sodium alginate solution is ensured to be quickly placed in an aqueous solution of calcium chloride with the mass concentration of 4% after being uniformly adhered, and a film is formed on the periphery of the PCL bracket.
4. The preparation process according to claim 1, wherein the cell pellet is sucked by a microinjector and injected into a stent capsule, and both ends are clamped by a medium-sized vascular clamp, and the culture is started by placing the cell pellet in a culture dish of 60mm, wherein the cell pellet is changed every two days, and 6-8ml of cell pellet is used each time.
5. The preparation process according to claim 1, wherein the film outside the stent capsule is dissolved out by the decrosslinking property of sodium alginate and sodium citrate by placing the stent capsule in an aqueous solution of sodium citrate with a mass concentration of 4% and shaking the stent capsule for 5 minutes at a speed of 100rpm using a shaking table.
6. The preparation process according to claim 1, wherein the cultivation time in the step (5) is from the 7 th day.
7. The process according to claim 1, wherein the culturing in the step (6) is carried out for a period of time from day 14 to seven days.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Application Number | Priority Date | Filing Date | Title |
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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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| CN115721781A CN115721781A (en) | 2023-03-03 |
| CN115721781B true CN115721781B (en) | 2023-11-07 |
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| CN1454995A (en) * | 2002-04-29 | 2003-11-12 | 天津市肝胆疾病研究所 | Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule |
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