CN113995888A - Tissue engineering tendon and preparation method thereof - Google Patents

Tissue engineering tendon and preparation method thereof Download PDF

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Publication number
CN113995888A
CN113995888A CN202111117608.9A CN202111117608A CN113995888A CN 113995888 A CN113995888 A CN 113995888A CN 202111117608 A CN202111117608 A CN 202111117608A CN 113995888 A CN113995888 A CN 113995888A
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tendon
collagen
rich plasma
platelet
fibrin
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CN113995888B (en
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赵金忠
李亚民
崔文国
蒋佳
赵松
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Shanghai Sixth Peoples Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/225—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3616—Blood, e.g. platelet-rich plasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Transplantation (AREA)
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  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Dermatology (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
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  • Urology & Nephrology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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  • Materials For Medical Uses (AREA)
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Abstract

The invention relates to a preparation method of tissue engineering tendon, comprising the following steps: coating platelet-rich plasma-fibrin gel on one surface of the oriented collagen nanofiber membrane to prepare a collagen-platelet-rich plasma-fibrin composite membrane; wrapping a plurality of oriented collagen nano-fibers with the collagen-platelet rich plasma-fibrin composite membrane, and then curling to obtain tendon fibers; wrapping a plurality of tendon fibers by using the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare a tendon fiber bundle; and wrapping a plurality of tendon fiber bundles by the collagen-platelet rich plasma-fibrin composite membrane, and then curling to obtain the tissue engineering tendon. The invention constructs a tendon organoid which can be used for in vitro exploration of a molecular mechanism of tendon injury, analysis of signal regulation and control, and further preparation of a tissue engineering tendon by using the tendon organoid for tissue repair and regeneration of tendon injury in animal experiments.

Description

Tissue engineering tendon and preparation method thereof
Technical Field
The invention relates to the field of medical treatment, in particular to a tissue engineering tendon and a preparation method thereof.
Background
Soft tissue injuries (e.g., muscles, tendons, ligaments, etc.) are very common in all levels of exercise. Ligament sprains are 50% of injuries in adolescent sports; in college sports, the incidence of ankle sprain is quite common, with at least 1 occurrence per 100 athletes. At the same time, the incidence of Anterior Cruciate Ligament (ACL) injury increased by 1.3% over the past decade. Except for the sports population, the probability of tendon injury in diabetic patients is ten times that in non-diabetic patients. With age, 80% of the elderly experience tendon rupture and tendon injury. Although musculoskeletal injuries are very common and have a tremendous impact on individuals, families, and economics, there have been limited advances in prevention and post-injury repair.
Currently, the prevention and treatment of tendon injury mainly includes brake fixation, drug treatment and surgical repair. The brake fixation is to fix the affected limb by plaster to avoid the tendon from being injured again by the movement; the drug therapy is mainly to promote the repair of tendon tissues or relieve pain through the pharmaceutical action; surgical repair is the surgical restoration of the physical structure of the injured tendon to promote its healing. However, the current treatment means often causes scar healing of tendon tissue, even does not heal, mainly because the current mechanism for tendon injury healing and regeneration is not deeply understood, and the high-efficiency and high-quality tendon graft or tendon prosthesis for repairing tendon injury is lacking at present.
Disclosure of Invention
The invention aims to provide a tissue engineering tendon and a preparation method thereof aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the first aspect of the invention provides a method for preparing tissue engineered tendon, comprising the following steps:
s1, coating platelet-rich plasma-fibrin gel on one surface of the oriented collagen nanofiber membrane to prepare a collagen-platelet-rich plasma-fibrin composite membrane;
s2, wrapping 1-100 oriented collagen nanofibers by the collagen-platelet rich plasma-fibrin composite membrane, and then crimping to obtain tendon fibers;
s3, wrapping 1-100 tendon fibers with the collagen-platelet rich plasma-fibrin composite membrane, and curling to obtain a tendon fiber bundle;
s4, wrapping 1-100 tendon fiber bundles with the collagen-platelet rich plasma-fibrin composite membrane, and then crimping to obtain the tissue engineering tendon.
Preferably, the oriented collagen nanofiber membrane is prepared from collagen by an electrospinning technique; the oriented collagen nanofiber bundle is prepared by compounding collagen through electrostatic spinning and twisting. Preferably, the oriented collagen nanofiber membrane has a thickness of 0.01mm to 1 mm; the diameter of the oriented collagen nanofiber bundle is 50nm-50 μm.
Preferably, the oriented collagen nanofiber membrane has a thickness of 0.02mm to 0.2 mm; the diameter of the oriented collagen nanofiber bundle is 500nm-5 μm. Preferably, the platelet rich plasma-fibrin gel is prepared by mixing platelet rich plasma with fibrin.
Preferably, the platelet rich plasma is prepared by in vitro centrifugation of peripheral blood after anticoagulation.
Preferably, the collagen-platelet rich plasma-fibrin composite membrane is curled with the side coated with the platelet rich plasma-fibrin gel inside.
Preferably, the platelet rich plasma-fibrin gel of tendon fibers is seeded with tendon fiber cells.
Preferably, the tendon fibers have a diameter of 0.05mm to 0.2 mm; the diameter of the tendon fiber bundle is 0.5mm-1 mm; the diameter of the tissue engineering tendon is 2mm-10 mm. In a second aspect, the present invention provides a tissue engineered tendon prepared by the above-mentioned preparation method.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention constructs a tendon organoid which can be used for in vitro exploration of a molecular mechanism of tendon injury, analysis of signal regulation and control, and further preparation of a tissue engineering tendon by using the tendon organoid for tissue repair and regeneration of tendon injury in animal experiments.
Drawings
FIG. 1 is a cell morphology map of application example 1 of the present invention;
FIG. 2 is a graph showing the results of gene expression in application example 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Examples
This embodiment provides a tissue engineered tendon, which is prepared by the following steps:
s1, taking 50mL of peripheral blood, performing anticoagulation and then performing in-vitro centrifugation for 15-20min (without adding thrombin), and preparing platelet-rich plasma; mixing the platelet-rich plasma and fibrin according to the proportion of 1:1 to prepare platelet-rich plasma-fibrin gel; respectively preparing an oriented collagen nanofiber bundle and an oriented collagen nanofiber membrane from collagen by adopting an electrostatic spinning and twisting composite technology, wherein the diameter of the oriented collagen nanofiber bundle is 50nm-50 mu m, and the thickness of the oriented collagen nanofiber membrane is 0.02mm-0.2 mm; coating the platelet-rich plasma-fibrin gel on one surface of the oriented collagen nanofiber membrane to prepare a collagen-platelet-rich plasma-fibrin composite membrane;
s2, wrapping 10 to 25 aligned collagen nanofibers with the collagen-platelet rich plasma-fibrin composite membrane, and then crimping the collagen nanofibers (including the surface coated with the platelet rich plasma-fibrin gel, wherein tendon fibroblasts are seeded in the platelet rich plasma-fibrin gel) to obtain tendon fibers, wherein the diameter of the tendon fibers is 0.05mm to 0.2 mm;
s3, wrapping 10-25 tendon fibers with the collagen-platelet rich plasma-fibrin composite membrane, and crimping to obtain a tendon fiber bundle, wherein the diameter of the tendon fiber bundle is 0.5-1 mm;
s4, wrapping 10-25 tendon fiber bundles with the collagen-platelet rich plasma-fibrin composite membrane, and then crimping to obtain the tissue engineering tendon, wherein the diameter of the tissue engineering tendon is 2-10 mm.
Application example 1
The tendon stem cells are inoculated on the collagen-platelet rich plasma-fibrin composite membrane as described in the example, and after 24 hours, the stem cells are adhered on the material, and the shape is shown in figure 1A; the stem cell morphology changed to a long spindle morphology after 72 hours, as shown in fig. 1B, similar to that of a typical fibroblast/tenocyte cell. The collagen-platelet rich plasma-fibrin composite membrane is shown to have the function of promoting the differentiation of the adhered stem cells to fibroblasts/tenocytes.
Application example 2
The tendon stem cells were inoculated on a blank culture plate (i.e., Control group Control) and the Tissue Engineering Tendon (TET) as described in example 1, cultured in a normal culture solution for 7 days, and the expression of collagen (COL I) and transforming growth factor beta (TGF-beta) in the two groups of cells was detected, respectively, as shown in fig. 2, the expression of the two genes in the TET group cells was significantly higher than that in the Control group, indicating that TET has a better function of promoting the differentiation of stem cells into tendon cells/fibroblasts.
In conclusion, the invention constructs an organoid structure simulating tendon in vitro, which can induce mesenchymal stem cells to differentiate into tendon cells and promote the tendon cells to secrete type I collagen tissues (main components of tendon tissues); by virtue of this tendon organoid structure, it is possible to study the specific molecular mechanisms of tendon tissue injury and to explore effective drugs or methods (including but not limited to material structures) that promote tenocyte collagen differentiation;
the invention can prepare bionic tendon fiber by relying on the tendon organ membrane, further synthesize the tendon, further prepare the tissue engineering tendon and realize the repair and regeneration of tendon injury.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (10)

1. A method for preparing tissue engineering tendon is characterized by comprising the following steps:
s1, coating platelet-rich plasma-fibrin gel on one surface of the oriented collagen nanofiber membrane to prepare a collagen-platelet-rich plasma-fibrin composite membrane;
s2, wrapping a plurality of oriented collagen nanofibers by the collagen-platelet rich plasma-fibrin composite membrane, and then curling to obtain tendon fibers;
s3, wrapping a plurality of tendon fibers with the collagen-platelet rich plasma-fibrin composite membrane, and curling to obtain a tendon fiber bundle;
s4, wrapping a plurality of tendon fiber bundles with the collagen-platelet rich plasma-fibrin composite membrane, and then curling to obtain the tissue engineering tendon.
2. The method for preparing the collagen nanofiber membrane according to claim 1, wherein the oriented collagen nanofiber membrane is prepared from collagen by an electrospinning technique; the oriented collagen nanofiber bundle is prepared by compounding collagen through electrostatic spinning and twisting.
3. The method for preparing the collagen nanofiber membrane according to claim 1, wherein the oriented collagen nanofiber membrane has a thickness of 0.01mm to 1 mm; the diameter of the oriented collagen nanofiber bundle is 50nm-50 μm.
4. The method for preparing according to claim 3, wherein the oriented collagen nanofiber membrane has a thickness of 0.02mm to 0.2 mm; the diameter of the oriented collagen nanofiber bundle is 500nm-5 μm.
5. The method of claim 1, wherein the platelet rich plasma-fibrin gel is prepared by mixing platelet rich plasma with fibrin.
6. The method for producing according to claim 5, wherein the platelet-rich plasma is produced by in vitro centrifugation of peripheral blood after anticoagulation.
7. The method according to claim 1, wherein the collagen-platelet-rich plasma-fibrin composite membrane is rolled on one side thereof with the platelet-rich plasma-fibrin gel coated thereon.
8. The method according to claim 1, wherein the platelet rich plasma-fibrin gel of tendon fiber is seeded with tendon fiber cells.
9. The method for preparing the tendon fiber according to claim 1, wherein the tendon fiber has a diameter of 0.05mm to 0.2 mm; the diameter of the tendon fiber bundle is 0.5mm-1 mm; the diameter of the tissue engineering tendon is 2mm-10 mm.
10. A tissue engineered tendon obtainable by the method according to any one of claims 1 to 9.
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