CN110384820A - It is a kind of that de- cell tendon line and its construction method is lyophilized - Google Patents
It is a kind of that de- cell tendon line and its construction method is lyophilized Download PDFInfo
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- CN110384820A CN110384820A CN201910608048.3A CN201910608048A CN110384820A CN 110384820 A CN110384820 A CN 110384820A CN 201910608048 A CN201910608048 A CN 201910608048A CN 110384820 A CN110384820 A CN 110384820A
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- tendon
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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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
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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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
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- Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Materials For Medical Uses (AREA)
Abstract
It is specifically a kind of that de- cell tendon line and its construction method is lyophilized the present invention relates to biological fibrous material field.Tissue freeze-drying tendon fiber bunch prepared by the present invention is a kind of novel natural biologic material, main component is Type I collagen albumen, the intrinsic mechanical structure unit of tendon collagenous fibres and cell attaching structure are remained with, there is good mechanical property and biocompatibility.Tendon collagenous fibres or tow material are from a wealth of sources, and amount is big and cheap, and the tendon of the mammals such as pig, ox can be used as material source.De- cell tendon fiber line is lyophilized because of its characteristic with fibrous material, can be widely applied to other biological medical domain, social required quantity is big.
Description
Technical field
The present invention relates to biological fibrous material fields, are a kind of new natural biologic materials, specifically, being a kind of freeze-drying
De- cell tendon line (collagenous fiber material) and its construction method.
Background technique
Medical bio polymer fiber material is the biomaterial for medical purpose being most widely used at present, it can derive from day
Right substance, can also be artificial synthesized.This material is in addition to it should meet general physics, chemical property requires, it is necessary to have enough
Good biocompatibility.It has degradable and non-degradable type two types.Non-degradable type has to long in biotic environment
Phase keeps stablizing, non-degradable, is not crosslinked, the significant changes of physical abrasion and physical property, available high molecular material do not occur
There are polyethylene, polypropylene, polyacrylate, aromatic polyester etc.；Degradable then has collagen, linear aliphatic adoption ester, first
Shell element, cellulose, amino acid, polylactic acid etc..Medical bio polymer fiber material be mainly used as cardiovascular system, soft tissue and
Win the repair materials of tissue.
Medical regeneration biological fibrous material refers to the medical bio material for taking natural biological tissue form by specially treated
Material, therefore also referred to as regeneration biological material, it is mainly used for heart valve prosthesis, vascular prostheses, skin applying film, fibrin system
Product, bone repair, mantle dummy, nasal cartilages planting body, hemodialysis membrane etc..Biocompatibility, it is to influence biomaterial
An important factor for success or failure.Biocompatibility refers to that material is received and accommodated by other surrounding tissues and entire body system, at least
It cannot cause the excitement of surrounding tissue, evoke infection and allergy, not cause cell tissue that cancer occurs.
Summary of the invention
It is specifically a kind of that de- cell tendon line is lyophilized the purpose of the present invention is to provide a kind of new natural biologic material
(collagenous fiber material) and its construction method.
To achieve the goals above, the present invention uses following methods:
The de- cell tendon line of freeze-drying of the invention is a kind of novel biomaterial, and it is intrinsic to remain with Tenocyte cell epimatrix
Mechanical structure unit and cell attaching structure, eliminate cell component, have good mechanical property and biocompatibility.Freeze
Dry de- cell tendon line is different from conventional collagen fiber suture such as catgut.Although the animal that all has drawn from is (as small in derived from sheep
Intestinal submucosa, ox are with tendinous tissue), but conventional collagen fiber suture is by the tropocollagen molecule of animal collagen dissolution purification, In
For polymerization in vitro at fibrinogen, the process for purifying tropocollagen molecule destroys the space structure and ingredient of its extracellular matrix completely,
It is destroyed in terms of biomethanics.The de- cell tendon line of freeze-drying is separated into after tendon fibre bundle by animal tendon and is twisted into line,
It is obtained after carrying out de- cell again, remains with the intrinsic mechanical structure unit of Tenocyte cell epimatrix and cell attaching structure.Cause
Its characteristic with fibrous material, can be widely applied to field of biomedicine, and social required quantity is big.
The braiding of freeze-drying tendon line is substantially to become freeze-drying tendon fiber by oriented and ordered from rambling state
The fiber of the process of arrangement, separation early period is dispersion, and every fiber does not have certain orientation, and the cohesive force of fiber interfascicular
Difference, common resultant yarn textile technology are difficult to make fibre bundle at line.But after the tendon fiber humidifying of freeze-drying, interfibrous cohesive force
It dramatically increases, twisting, de- cell etc. are easier to be in long linear fiber material under the conditions of humidifying, and linear tendon fiber is without complete
The wrapping of fibre bundle film, internal cell component can be removed by the methods of trypsase, ultrasonic vibration, and retain tendon fiber
The collagen fiber structure of beam.It is convenient for tendon, ligament, fascia and other Various Tissues engineering branch at the de- cell tendon fibre bundle of line
The braiding of frame, it is widely used.
The first aspect of the present invention provides a kind of construction method that de- cell tendon line is lyophilized, comprising the following steps:
A) outer membrane and the synovial tissue on fresh animal tendon surface are removed；
B) tendon is placed in -56 DEG C of vacuum freeze-dryings for 24 hours；
C) freeze-drying tendon fibre bundle is obtained by the methods of physics shredding, combing；
D) freeze-drying tendon fibre bundle is by being combed into item；
E) tendon fibre bundle distilled water humidifying in stripes, the cohesive force of reinforcing fiber interfascicular；
F) by twisting, the tendon fibre bundle in stripes of humidifying is prepared into growth line, is stored in distilled water；
G) the tendon fiber line of humidifying carries out de- cell processing: removing tendon by the methods of trypsase, ultrasonic vibration
Fiber line surface and internal cell component；
H) cell free tendon fiber line is placed in -56 DEG C of vacuum freeze-drying 3h, the braiding of standby biomaterial is used.
Further, animal described in step a is the mammals such as pig, ox.
Further, physics opening process described in step c refers to the methods of anti-quick-fried gas shock of extruding, hammering, vacuum.
Further, the step d be by 1000mg/ meters, 500mg/ meters and 250mg/ meters freeze-drying tendon fibre bundles at
Item.
Further, de- cellular processes described in step g are as follows: the tendon fiber line that step f is obtained is put into Tris
0.05% serpin of mass concentration is added in solution, and shaking table shakes 12h under room temperature；Outwell solution, PBS solution
It rinses 3 times, 0.05% trypsase of mass concentration is added, 37 DEG C of shaking tables shake 2h；Solution is outwelled, after PBS is rinsed 3 times, is added
90U/mL nuclease, 37 DEG C of shaking tables shake 2h；Solution is outwelled, after PBS is rinsed 3 times, using sonicator, ultrasonic probe is outstanding
It floats on above sample at 0.5cm, 240 μm of ultrasonic amplitude, 20 hertz of frequency, receives the 5 minutes ultrasonication (U.S.
Qsonica Q700 sonicator).
Further, all steps of step g are carried out at shaking table (150RPM), plus 5ml/L penicillin/streptomycin solution
(10000U/ml/10000mg/ml) Pollution protection completes each step and PBS is used to rinse 3 times.
The second aspect of the present invention provides a kind of be lyophilized and takes off cell tendon line, uses construction method system as described above
It is standby to obtain.
The third aspect of the present invention provides the de- cell tendon line of one kind freeze-drying as described above and is preparing medical regeneration biological
Application in fibrous material.
The invention has the advantages that:
1, freeze-drying of the invention takes off cell tendon fiber line, and from the collagenous fibres of the tendon of animal, materials are abundant, honest and clean
Valence is free of cell component, has good biomechanics characteristic, biocompatibility and practicability.
2, low temperature drying influences Tenocyte cell external structure small, can reduce its antigenicity, and convenient for physics shredding, comb
The method of reason separates collagenous fibres.It is experimentally confirmed that collagenous fibres or fibre bundle without aponeurosis (aponeuroses) wrapping, pass through low concentration trypsase
And ultrasonic vibration can remove the cell component for being adhered to collagenous fibres surface, and to its biomethanics and histocompatbility without bright
Development is rung, be lyophilized made of braiding the collagens lines such as de- more traditional catgut of cell tendon line have better histocompatbility and
Biomimetic features.
3, ideal biological braided material should have good biomechanics characteristic, biocompatibility.Group of the invention
Knitting freeze-drying tendon fiber bunch is a kind of novel natural biologic material, and main component is Type I collagen albumen, remains with tendon glue
The intrinsic mechanical structure unit of fibrinogen and cell attaching structure have good mechanical property and biocompatibility.Tendon glue
Fibrinogen or tow material are from a wealth of sources, and amount is big and cheap, and the tendon of the mammals such as pig, ox can be used as material source.
De- cell tendon fiber line is lyophilized because of its characteristic with fibrous material, can be widely applied to other biological medical domain, society
Meeting demand is big.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1: the preparation of de- cell tendon line is lyophilized
1) fresh ox tendon is taken, outer membrane and the synovial tissue on tendon surface are removed.
2) tendon is placed in -56 DEG C of vacuum freeze-drying instrument, for 24 hours.
3) freeze-drying tendon collagenous fiber bundle is obtained by the methods of physics shredding, combing separation.
4) by 1000mg/ meters, 500mg/ meters and 250mg/ meters freeze-drying tendon fibre bundles at item.
5) tendon fibre bundle distilled water humidifying in stripes, twisting remain in distilled water at line
6) the tendon fiber line of humidifying takes off cell: hypotonic tris buffer+serpin of 10mM
(0.05% phenylmethyl sulfonylfluoride alcohol solvent, 35ml/L 50ml ethyl alcohol+25mgPMSF), shaking table shakes 12h under room temperature.
Solution is outwelled, PBS solution is rinsed 3 times, and 0.05% trypsase is added, and 37 DEG C of shaking tables shake 2h.Outwell solution, PBS solution punching
It washes 3 times, is added 90U/mL nuclease (type II ox), 37 DEG C of shaking tables shake 2h.Solution is outwelled, after PBS is rinsed 3 times, using super
Sonication instrument, ultrasonic probe are suspended in above sample at 0.5cm, 240 μm of ultrasonic amplitude, 20 hertz of frequency, are received 5 minutes
Ultrasonication (U.S.'s Qsonica Q700 sonicator).All steps shaking table (150RPM) carry out, plus
5ml/L penicillin/streptomycin solution (10000U/ml 10000mg/ml) solution Pollution protection, completes each step and uses
PBS is rinsed 3 times.
7) de- cell tendon fiber line is placed in -56 DEG C of vacuum freeze-drying instrument, kept dry is stand-by after 3h.
Embodiment 2: de- cell tendon linear diameter measurement is lyophilized
The diameter of the de- cell tendon fiber line of freeze-drying of different mg/m freeze-drying tendon fibre bundle preparations in stripes.Such as the following table 1.
The diameter of the de- cell tendon fiber line of freeze-drying of 1 difference mg/m of table freeze-drying tendon fibre bundle preparation in stripes
Embodiment 3: the Biomechanics test of de- cell tendon line is lyophilized
The Biomechanics test of the de- cell tendon fiber line of freeze-drying of different mg/m freeze-drying tendon fibre bundle preparations in stripes.
Such as the following table 2:
The biomethanics of the de- cell tendon fiber line of freeze-drying of 2 difference mg/m of table freeze-drying tendon fibre bundle preparation in stripes is surveyed
Examination
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (7)
1. a kind of construction method that de- cell tendon line is lyophilized, which comprises the following steps:
A) outer membrane and the synovial tissue on fresh animal tendon surface are removed；
B) tendon is placed in -56 DEG C of vacuum freeze-dryings for 24 hours；
C) freeze-drying tendon fibre bundle is obtained by the method for physics shredding, combing；
D) freeze-drying tendon fibre bundle is by being combed into item；
E) tendon fibre bundle distilled water humidifying in stripes；
F) by twisting, the tendon fibre bundle in stripes of humidifying is prepared into growth line, is stored in distilled water；
G) the tendon fiber line of humidifying carries out de- cell processing: the tendon fiber line that step f is obtained being put into Tris solution, is added
0.05% serpin of mass concentration, shaking table shakes 12h under room temperature；Solution is outwelled, PBS solution is rinsed 3 times, added
Enter 0.05% trypsase of mass concentration, 37 DEG C of shaking tables shake 2h；Solution is outwelled, after PBS is rinsed 3 times, 90U/mL nucleic acid is added
Enzyme, 37 DEG C of shaking tables shake 2h；Solution is outwelled, after PBS is rinsed 3 times, using sonicator, ultrasonic probe is suspended on sample
At square 0.5cm, 240 μm of ultrasonic amplitude, 20 hertz of frequency, receive 5 minutes ultrasonications；
H) cell free tendon fiber line is placed in -56 DEG C of vacuum freeze-drying 3h.
2. the construction method according to claim 1 that de- cell tendon line is lyophilized, which is characterized in that moved described in step a
Object is pig, ox.
3. the construction method according to claim 1 that de- cell tendon line is lyophilized, which is characterized in that object described in step c
Reason opening process refers to the anti-quick-fried gas shock of extruding, hammering or vacuum.
4. the construction method according to claim 1 that de- cell tendon line is lyophilized, which is characterized in that the step d is
By 1000mg/ meters, 500mg/ meters and 250mg/ meters freeze-drying tendon fibre bundles at item.
5. the construction method according to claim 1 that de- cell tendon line is lyophilized, which is characterized in that all steps of step g
It is carried out in shaking table, plus 5ml/L penicillin/streptomycin solution Pollution protection, completes each step and PBS is used to rinse 3 times.
6. a kind of be lyophilized de- cell tendon line, construction method a method as claimed in any one of claims 1 to 5 is used to be prepared.
7. a kind of de- cell tendon line of freeze-drying as claimed in claim 6 is preparing answering in medical regeneration biological fibrous material
With.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910608048.3A CN110384820A (en) | 2019-07-08 | 2019-07-08 | It is a kind of that de- cell tendon line and its construction method is lyophilized |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910608048.3A CN110384820A (en) | 2019-07-08 | 2019-07-08 | It is a kind of that de- cell tendon line and its construction method is lyophilized |
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| CN110384820A true CN110384820A (en) | 2019-10-29 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111407926A (en) * | 2020-03-13 | 2020-07-14 | 深圳兰度生物材料有限公司 | Biological repairing mesh and preparation method and application thereof |
| CN115089757A (en) * | 2022-07-28 | 2022-09-23 | 单县华宇缝合制品有限公司 | Processing method of self-degradation type suture line |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831724A (en) * | 2010-04-16 | 2010-09-15 | 陈雄生 | Acellular tendon or ligament collagenous fiber material and preparation method thereof |
| US20140065238A1 (en) * | 1999-06-07 | 2014-03-06 | Lifenet Health | Process for Devitalizing Soft-Tissue Engineered Medical Implants and Devitalized Soft-Tissue Medical Implants Produced |
| CN104587522A (en) * | 2015-02-15 | 2015-05-06 | 黑龙江力海鑫生物科技股份有限公司 | Nutria tail collagen surgical suture and preparation method thereof |
| CN109331229A (en) * | 2018-11-28 | 2019-02-15 | 上海白衣缘生物工程有限公司 | A kind of medical bio line, medical bio repair mesh and preparation method thereof |
-
2019
- 2019-07-08 CN CN201910608048.3A patent/CN110384820A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140065238A1 (en) * | 1999-06-07 | 2014-03-06 | Lifenet Health | Process for Devitalizing Soft-Tissue Engineered Medical Implants and Devitalized Soft-Tissue Medical Implants Produced |
| CN101831724A (en) * | 2010-04-16 | 2010-09-15 | 陈雄生 | Acellular tendon or ligament collagenous fiber material and preparation method thereof |
| CN104587522A (en) * | 2015-02-15 | 2015-05-06 | 黑龙江力海鑫生物科技股份有限公司 | Nutria tail collagen surgical suture and preparation method thereof |
| CN109331229A (en) * | 2018-11-28 | 2019-02-15 | 上海白衣缘生物工程有限公司 | A kind of medical bio line, medical bio repair mesh and preparation method thereof |
| CN109621011A (en) * | 2018-11-28 | 2019-04-16 | 上海白衣缘生物工程有限公司 | A kind of tendon Biological Repair mesh sheet and application thereof and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 钱闯等: "牛肌腱冻干脱细胞支架的生物力学特性", 《中国组织工程研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111407926A (en) * | 2020-03-13 | 2020-07-14 | 深圳兰度生物材料有限公司 | Biological repairing mesh and preparation method and application thereof |
| CN111407926B (en) * | 2020-03-13 | 2022-04-08 | 深圳兰度生物材料有限公司 | Biological repairing mesh and preparation method and application thereof |
| CN115089757A (en) * | 2022-07-28 | 2022-09-23 | 单县华宇缝合制品有限公司 | Processing method of self-degradation type suture line |
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