CN113995888B - Tissue engineering tendon and preparation method thereof - Google Patents

Tissue engineering tendon and preparation method thereof Download PDF

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Publication number
CN113995888B
CN113995888B CN202111117608.9A CN202111117608A CN113995888B CN 113995888 B CN113995888 B CN 113995888B CN 202111117608 A CN202111117608 A CN 202111117608A CN 113995888 B CN113995888 B CN 113995888B
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tendon
collagen
rich plasma
fibrin
platelet
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CN113995888A (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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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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Dermatology (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Materials For Medical Uses (AREA)
  • Prostheses (AREA)

Abstract

The invention relates to a preparation method of tissue engineering tendons, which comprises 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 nanofiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare tendon fibers; wrapping a plurality of tendon fibers by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare tendon fiber bundles; and wrapping a plurality of tendon fiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare the tissue engineering tendon. The tendon organoid is constructed, can be used for exploring molecular mechanisms of tendon injury in vitro, analyzing signal regulation and control, and further preparing tissue engineering tendons by using the tendon organoid to repair and regenerate tissue 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., muscle, tendon, ligament, etc.) are very common in all levels of exercise. In teenager sport 50% of injuries are ligament sprains; in college sports, ankle sprains occur quite commonly, at least 1 occurrence per 100 athletes. Meanwhile, the incidence of Anterior Cruciate Ligament (ACL) injury has increased by 1.3% over the last decade. Except for the sports population, the probability of tendon injury for diabetics is ten times that for non-diabetics. With age, 80% of the elderly experience tendon rupture and tendon injury. Although musculoskeletal injuries are very common and can have a significant impact on individuals, families and economies, there has been limited progress in preventing and repairing after injury.
At present, the prevention and treatment of tendon injury mainly comprises brake fixation, drug treatment and operation repair. The brake fixation is to fix the affected limb through gypsum, so as to avoid the tendon from being damaged again by the activity; the drug treatment mainly promotes the repair of tendon tissues or relieves pain through pharmaceutical actions; surgical repair is the promotion of healing by surgically restoring the physical structure of the damaged tendon. However, current treatments often lead to scar healing, even disunion, of tendon tissue, mainly because the current mechanisms for healing and regeneration of tendon injuries are not well understood, and the current repair of tendon injuries lacks efficient and quality tendon grafts or tendon prostheses.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides a tissue engineering tendon and a preparation method thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the present invention provides a method for preparing a tissue engineering tendon, comprising the steps of:
s1, coating platelet-rich plasma-fibrin gel on one surface of an oriented collagen nanofiber membrane to prepare a collagen-platelet-rich plasma-fibrin composite membrane;
s2, wrapping 1-100 oriented collagen nanofiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare tendon fibers;
s3, wrapping 1-100 tendon fibers by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare tendon fiber bundles;
s4, wrapping 1-100 tendon fiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare the tissue engineering tendon.
Preferably, the oriented collagen nanofiber membrane is prepared from collagen by an electrostatic spinning technology; the oriented collagen nanofiber bundle is prepared by compounding collagen through electrostatic spinning and twisting. Preferably, the thickness of the oriented collagen nanofiber membrane is 0.01mm-1mm; the diameter of the oriented collagen nanofiber bundles is 50nm-50 mu m.
Preferably, the thickness of the oriented collagen nanofiber membrane is 0.02mm-0.2mm; the diameter of the oriented collagen nanofiber bundles is 500nm-5 mu m. Preferably, the platelet rich plasma-fibrin gel is prepared by mixing platelet rich plasma with fibrin.
Preferably, the platelet rich plasma is prepared from peripheral blood by in vitro centrifugation after anticoagulation.
Preferably, the collagen-platelet rich plasma-fibrin composite film is coated with the platelet rich plasma-fibrin gel on one side thereof when curled.
Preferably, the platelet rich plasma-fibrin gel of tendon fibers is seeded with tendon fibroblasts.
Preferably, the diameter of the tendon fiber is 0.05mm-0.2mm; the diameter of the tendon fiber bundles is 0.5mm-1mm; the diameter of the tissue engineering tendon is 2mm-10mm. In a second aspect, the present invention provides a tissue engineering tendon prepared by the preparation method as described above.
Compared with the prior art, the invention has the following technical effects:
the tendon organoid is constructed, can be used for exploring molecular mechanisms of tendon injury in vitro, analyzing signal regulation and control, and further preparing tissue engineering tendons by using the tendon organoid to repair and regenerate tissue of tendon injury in animal experiments.
Drawings
FIG. 1 is a cell morphology diagram of application example 1 of the present invention;
FIG. 2 is a graph showing the results of gene expression in practical example 2 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.
Examples
The embodiment provides a tissue engineering tendon, which comprises the following preparation steps:
s1, taking 50mL of peripheral blood, and performing in-vitro centrifugation for 15-20min (thrombin is not added) after anticoagulation to prepare platelet-rich plasma; mixing the platelet-rich plasma with 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-0.2mm; 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 10-25 oriented collagen nanofiber bundles by adopting the collagen-platelet-rich plasma-fibrin composite membrane, and curling (one surface coated with the platelet-rich plasma-fibrin gel is included, and tendon fibroblasts are inoculated in the platelet-rich plasma-fibrin gel) to prepare tendon fibers, wherein the diameter of the tendon fibers is 0.05-0.2 mm;
s3, wrapping 10-25 tendon fibers by adopting the collagen-platelet-rich plasma-fibrin composite membrane, and then curling to prepare tendon fiber bundles, wherein the diameter of the tendon fiber bundles is 0.5-1 mm;
s4, wrapping 10-25 tendon fiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to obtain the tissue engineering tendon, wherein the diameter of the tissue engineering tendon is 2-10 mm.
Application example 1
Tendon stem cells were seeded onto the collagen-platelet rich plasma-fibrin composite membrane as described in the examples, and after 24 hours, the stem cells adhered to the material in the form shown in fig. 1A; after 72 hours the stem cell morphology changed to a long spindle morphology, as shown in figure 1B, similar to that of typical fibroblasts/tenocytes. The collagen-platelet rich plasma-fibrin composite membrane has the function of promoting the differentiation of adhered stem cells to fibroblasts/tenocytes.
Application example 2
Tendon stem cells were inoculated on blank plates (i.e., control group) and Tissue Engineering Tendons (TETs) as described in example 1, respectively, and cultured in normal culture solution for 7 days, and expression of collagen I (COL I) and transforming growth factor beta (TGF-beta) was detected in the two groups of cells, respectively, as shown in FIG. 2, and expression of two genes in the TET group of cells was significantly increased compared to the Control group, indicating that TET has a better function of promoting differentiation of stem cells into tendon cells/fibroblasts.
In summary, the present invention constructs an organoid structure simulating tendon in vitro, which can induce the differentiation of mesenchymal stem cells into tenocytes and promote the secretion of type I collagen tissue (the main component of tendon tissue) by tenocytes; by virtue of this tendon organoid structure, specific molecular mechanisms of tendon tissue injury can be studied and effective drugs or methods (including but not limited to material structure) to promote tenocyte collagen differentiation can be explored;
the invention relies on the tendon organ membrane to prepare bionic tendon fiber, so as to synthesize tendon bundles, further prepare tissue engineering tendon and realize repair and regeneration of tendon injury.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the present invention, and are intended to be included within the scope of the present invention.

Claims (9)

1. The preparation method of the tissue engineering tendon is characterized by comprising the following steps:
s1, coating platelet-rich plasma-fibrin gel on one surface of an oriented collagen nanofiber membrane to prepare a collagen-platelet-rich plasma-fibrin composite membrane;
s2, wrapping a plurality of oriented collagen nanofiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare tendon fibers;
s3, wrapping a plurality of tendon fibers by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare tendon fiber bundles;
s4, wrapping a plurality of tendon fiber bundles by adopting the collagen-platelet rich plasma-fibrin composite membrane, and then curling to prepare the tissue engineering tendon;
when the collagen-platelet rich plasma-fibrin composite membrane is curled, one surface coated with the platelet rich plasma-fibrin gel is included.
2. The method of claim 1, wherein the oriented collagen nanofiber membrane is prepared from collagen by electrospinning; the oriented collagen nanofiber bundle is prepared by compounding collagen through electrostatic spinning and twisting.
3. The method of claim 1, wherein the oriented collagen nanofiber membrane has a thickness of 0.01mm to 1mm; the diameter of the oriented collagen nanofiber bundles is 50nm-50 mu m.
4. A method of preparation according to claim 3, wherein the thickness of the oriented collagen nanofiber membrane is 0.02mm-0.2mm; the diameter of the oriented collagen nanofiber bundles is 500nm-5 mu m.
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 according to claim 5, wherein the platelet-rich plasma is obtained by in vitro centrifugation of peripheral blood after anticoagulation.
7. The method of claim 1, wherein tendon fiber platelet rich plasma-fibrin gel is seeded with tendon fibroblasts.
8. The method of claim 1, wherein the tendon fiber has a diameter of 0.05mm to 0.2mm; the diameter of the tendon fiber bundles is 0.5mm-1mm; the diameter of the tissue engineering tendon is 2mm-10mm.
9. A tissue engineered tendon produced by the production method of any one of claims 1 to 8.
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