CN109136169A - A kind of skin fibroblasts are changed into the system and its application method of artificial intervertebral disk - Google Patents
A kind of skin fibroblasts are changed into the system and its application method of artificial intervertebral disk Download PDFInfo
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- CN109136169A CN109136169A CN201810902378.9A CN201810902378A CN109136169A CN 109136169 A CN109136169 A CN 109136169A CN 201810902378 A CN201810902378 A CN 201810902378A CN 109136169 A CN109136169 A CN 109136169A
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- 238000004113 cell culture Methods 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 229940116351 sebacate Drugs 0.000 claims 1
- 206010061246 Intervertebral disc degeneration Diseases 0.000 abstract description 5
- 208000018180 degenerative disc disease Diseases 0.000 abstract description 5
- 208000021600 intervertebral disc degenerative disease Diseases 0.000 abstract description 5
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
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- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
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- Microbiology (AREA)
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Abstract
The present invention relates to systems and its application method that a kind of skin fibroblasts are changed into artificial intervertebral disk, including growth factor and artificial intervertebral disk cell culture fluid.System through the invention is injected into interverbebral disc after skin fibroblasts are changed into artificial intervertebral disk cell, promote the progress of interverbebral disc fibrosis, to maintain disc height, improve interverbebral disc signal, achieve the purpose that regulate and control intervertebral disc degeneration, while interverbebral disc being made to obtain new mechanical strength.
Description
Technical field
The invention belongs to intervertebral disc degeneration field, in particular to a kind of skin fibroblasts are changed into artificial intervertebral disk
System and its application method.
Background technique
With the extension of mankind's life expectancy, the accelerating rhythm of life, the increasing of working method of bending over of bowing causes lumbar vertebrae
The disease incidence of degenerative disease is increasing.And the regression of intervertebral disc of lumbar vertebra is to cause back pain, the main original of lower limb rediating pain
Cause.And interverbebral disc is maximum main no blood vessel, impassivity, the structure without lymph in body.And interverbebral disc is for backbone
Normal function it is most important because it is being compressed axially, is providing toughness and mechanical stability in bending and stretching.Interverbebral disc by
Several specific connective tissues compositions: the hyaline cartilage of 1. cartilage endplates, the cartilage endplate covering be located above interverbebral disc and
The surface of the vertebrae of lower section encapsulates nucleus pulposus and fibrous ring；2. the gelatinous nucleus pulposus in center, although it contains osteoblast-like cells,
It is not hyaline cartilage.Transitional areas is identified, just as the term implies, between fibrous ring and nucleus pulposus.Fibrous ring
It is made of concentric collagen layer, the bone edge connection of the concentric collagen layer and centrum.In aging and degenerative processes, interverbebral disc hair
Raw significant matrix changes.
At present usually using Autologous Chondrocyte, potential cell therapy is detected to realize the dog of regression and repairing for human disc
Multiple research has been reported.Cell used is the cartilage cell harvested from the healthy nucleus pulposus of same species and is then implanted into again
Into the interverbebral disc of defect.The shortcomings that the method, is: the cell for the purposes need from neighbouring healthy disc or
From other donor harvestings of same species.Nucleus pulposus cell is taken to destroy the fibrous ring and marrow of normal disc from healthy disc
The overall structure of core, in some instances it may even be possible to accelerate the regression of normal disc.Secondly, primary cartilage or nucleus pulposus cell in vitro culture number generation
Aging is obvious afterwards, it is difficult to guarantee to re-inject the quantity and quality of cartilage or nucleus pulposus cell in intervertenral space.This method application prospect
Obviously there is limitation.
Summary of the invention
Technical problem to be solved by the invention is to provide what a kind of skin fibroblasts were changed into artificial intervertebral disk to be
System and its application method, system through the invention are injected into vertebra after skin fibroblasts are changed into artificial intervertebral disk cell
Disk, promotes the progress of interverbebral disc fibrosis, to maintain disc height, improve interverbebral disc signal, reaches regulation interverbebral disc
The purpose of regression, while interverbebral disc being made to obtain new mechanical strength.
The present invention provides the systems that a kind of skin fibroblasts are changed into artificial intervertebral disk, including growth factor and people
Work intervertebral disc cells culture solution.
The growth factor is transforming growth factor β.
The artificial intervertebral disk cell culture fluid is cultivated under high pressure and anoxia condition.
The system also includes cellular matrix constructions.
The cellular matrix construction is polymer.
The polymer is polyglycolic acid, polylactic acid, polylactic acid-glycollic acid, poly- 6-caprolactone or polyglycereol-decanedioic acid
Ester.
The present invention also provides the application method that a kind of skin fibroblasts are changed into the system of artificial intervertebral disk, packets
It includes:
(1) skin histology is cut into fragment, is placed in alcohol and impregnates, be then placed in dispase solution and stay overnight for 4 DEG C；?
Skin histology was taken out in two days, tears epidermal tissue off, then tissue is shredded, is subsequently placed into clostridiopetidase A and digests；Digestion is completed
After filter, be centrifuged, washing then is centrifuged to get skin fibroblasts；
(2) skin fibroblasts that step (1) obtains are inoculated in sterile petri dish, artificial intervertebral disk cell is added
Culture solution is placed in 37 DEG C of incubators (containing 5%CO2) in culture, the culture solution of replacement in every 3 days, when growth and proliferation of cell is to connecing
When nearly converging state, had digestive transfer culture is carried out using trypsase, skin fibroblasts inoculation is then contained into transforming growth factor β
Culture solution in, cultivated.
The concentration of dispase solution in the step (1) is 0.25%；The concentration of clostridiopetidase A is 0.25%.
Digestion time in the step (1) is 37 DEG C；Digestion time is 3~5 hours.
The concentration of trypsase in the step (2) is 0.05%.
Beneficial effect
Skin fibroblasts are changed into after artificial intervertebral disk cell by system through the invention is injected into interverbebral disc, promotees
Into the progress of interverbebral disc fibrosis, to maintain disc height, improve interverbebral disc signal, reach the mesh of regulation intervertebral disc degeneration
, while interverbebral disc being made to obtain new mechanical strength.
Detailed description of the invention
Fig. 1 is preoperative to rat tails X-ray, MRI radiation picture；
Fig. 2 is to radiate picture to rat tails X-ray, MRI in postoperative 2 months；
Fig. 3 a-g tests to be injected into the biomechanical property after interverbebral disc after artificial intervertebral disc cells.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
Skin histology is cut into fragment (0.5cm*0.5cm), is placed in 75% alcohol and impregnates 30min, be then placed in
It is stayed overnight for 4 DEG C in 0.25% dispase solution；Skin histology was taken out in second day, epidermal tissue is torn off, then by tissue shear
It is broken, it is subsequently placed into 0.25% clostridiopetidase A and digests 4 hours for 37 DEG C；After 4 hours, whether observation tissue digests completely, does not digest
Digestion time can completely be appropriately extended；(unicellular strainer) is filtered after the completion of digestion, is centrifuged (5min, 1500rpm, 37 DEG C), training
Nutrient solution is washed one time, then is centrifuged and (is washed away clostridiopetidase A) to get skin fibroblasts.
The skin fibroblasts of the present embodiment be changed into the system of artificial intervertebral disk include transforming growth factor β, it is artificial
Intervertebral disc cells culture solution and cellular matrix construction polylactic acid.
Wherein, the composition of artificial intervertebral disk cell culture fluid are as follows: 15% fetal calf serum Hyclone, 110U/mL penicillin
Sigma, 110 μ g/mL streptomysin Sigma, high sugar DMDM culture medium Gibco, 0.2mM ascorbate-2-phosphate.
Obtained skin fibroblasts are inoculated in the sterile petri dish of diameter 10cm, artificial intervertebral disk cell is added
Culture solution is placed in 37 DEG C of 5%CO2It is cultivated in incubator, the culture solution of replacement in every 3 days, when growth and proliferation of cell converges to close
When conjunction state, using 0.05% trypsase carry out had digestive transfer culture, then by skin fibroblasts inoculation containing conversion growth because
In the culture solution of sub- β, cultivated.
Needle thorn is carried out to rat tail bone and makes intervertebral disc degeneration model, by 6 weeks SD rat anesthesias, successively cuts rat tails skin
Skin selects 4 section interverbebral discs, makes intervertebral disc degeneration model using the syringe needle puncture of 20G thickness to 3 sections thereafter.After modeling, according to original
The channel of formation is punctured, using two segments of micro syringe is injected separately into 10,000 and 100,000 thirds through the present embodiment culture
For rat dermal fibroblasts, then layer-by-layer suture tail skin notch, shown in result figure 1 and Fig. 2.
Fig. 3 a is 4 groups of change in displacement under stress, puncture modeling group under same pressure obvious mobility greater than other three
Group illustrates to relax after interverbebral disc is punctured modeling, once bearing pressure, height is lost at once.Fig. 3 b is the tool of pressure testing.
Fig. 3 c is 4 groups to be changed in bending force bottom offset, is punctured modeling group obvious flexion degree under same pressure and is less than
Other three groups, illustrate that spur is formed after obvious regression around after interverbebral disc is punctured, flexion degree is impacted.Fig. 3 d figure is curved
The tool of song test.
Fig. 3 e is 4 groups of Atomic Mechanics microscope figure, from it is microcosmic it is upper from four groups of interverbebral discs, modeling group is obviously in identical pressure
Whole Young's modulus is bigger under power.Fig. 3 f is all the points average of Fig. 3 e, and interverbebral disc is softer after illustrating modeling, structure disturbance.
Fig. 3 g is the Young's modulus for taking each point of Fig. 3 e diagonal line, and Puncture is relatively higher.
Claims (10)
1. the system that a kind of skin fibroblasts are changed into artificial intervertebral disk, it is characterised in that: including growth factor and manually
Intervertebral disc cells culture solution.
2. the system that a kind of skin fibroblasts according to claim 1 are changed into artificial intervertebral disk, it is characterised in that:
The growth factor is transforming growth factor β.
3. the system that a kind of skin fibroblasts according to claim 1 are changed into artificial intervertebral disk, it is characterised in that:
The artificial intervertebral disk cell culture fluid is cultivated under high pressure and anoxia condition.
4. the system that a kind of skin fibroblasts according to claim 1 are changed into artificial intervertebral disk, it is characterised in that:
The system also includes cellular matrix constructions.
5. the system that a kind of skin fibroblasts according to claim 4 are changed into artificial intervertebral disk, it is characterised in that:
The cellular matrix construction is polymer.
6. the system that a kind of skin fibroblasts according to claim 5 are changed into artificial intervertebral disk, it is characterised in that:
The polymer is polyglycolic acid, polylactic acid, polylactic acid-glycollic acid, poly- 6-caprolactone or polyglycereol-sebacate.
7. the application method that a kind of skin fibroblasts are changed into the system of artificial intervertebral disk, comprising:
(1) skin histology is cut into fragment, is placed in alcohol and impregnates, be then placed in dispase solution and stay overnight for 4 DEG C；Second day
Skin histology is taken out, epidermal tissue is torn off, then tissue is shredded, is subsequently placed into clostridiopetidase A and digests；Mistake after the completion of digestion
Filter is centrifuged, washing, then is centrifuged to get skin fibroblasts；
(2) skin fibroblasts that step (1) obtains are inoculated in sterile petri dish, artificial intervertebral disk cell culture is added
Liquid is placed in 37 DEG C of incubators and cultivates, the culture solution of replacement in every 3 days, when growth and proliferation of cell is to close to converging state, makes
Had digestive transfer culture is carried out with trypsase, then skin fibroblasts are inoculated in the culture solution containing transforming growth factor β, is carried out
Culture.
8. a kind of skin fibroblasts according to claim 7 are changed into the application method of the system of artificial intervertebral disk,
It is characterized by: the concentration of the dispase solution in the step (1) is 0.25%；The concentration of clostridiopetidase A is 0.25%.
9. a kind of skin fibroblasts according to claim 7 are changed into the application method of the system of artificial intervertebral disk,
It is characterized by: the digestion time in the step (1) is 37 DEG C；Digestion time is 3~5 hours.
10. a kind of skin fibroblasts according to claim 7 are changed into the application method of the system of artificial intervertebral disk,
It is characterized by: the concentration of the trypsase in the step (2) is 0.05%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810902378.9A CN109136169A (en) | 2018-08-09 | 2018-08-09 | A kind of skin fibroblasts are changed into the system and its application method of artificial intervertebral disk |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810902378.9A CN109136169A (en) | 2018-08-09 | 2018-08-09 | A kind of skin fibroblasts are changed into the system and its application method of artificial intervertebral disk |
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| CN109136169A true CN109136169A (en) | 2019-01-04 |
Family
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| Application Number | Title | Priority Date | Filing Date |
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| CN201810902378.9A Pending CN109136169A (en) | 2018-08-09 | 2018-08-09 | A kind of skin fibroblasts are changed into the system and its application method of artificial intervertebral disk |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104684591A (en) * | 2012-08-10 | 2015-06-03 | 先进医疗技术有限责任公司 | Generation of cartilage ex vivo from fibroblasts |
| CN105640992A (en) * | 2008-03-21 | 2016-06-08 | 组织基因公司 | Treatment of intervertebral disc degeneration |
| WO2017123951A1 (en) * | 2016-01-14 | 2017-07-20 | Spinacyte, Llc | Cellular blend for the regeneration of chondrocytes or cartilage type cells |
| US20180195044A1 (en) * | 2017-01-11 | 2018-07-12 | Spinalcyte, Llc | Methods of Enhancing Fibroblast Therapeutic Activity |
-
2018
- 2018-08-09 CN CN201810902378.9A patent/CN109136169A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105640992A (en) * | 2008-03-21 | 2016-06-08 | 组织基因公司 | Treatment of intervertebral disc degeneration |
| CN104684591A (en) * | 2012-08-10 | 2015-06-03 | 先进医疗技术有限责任公司 | Generation of cartilage ex vivo from fibroblasts |
| WO2017123951A1 (en) * | 2016-01-14 | 2017-07-20 | Spinacyte, Llc | Cellular blend for the regeneration of chondrocytes or cartilage type cells |
| US20180195044A1 (en) * | 2017-01-11 | 2018-07-12 | Spinalcyte, Llc | Methods of Enhancing Fibroblast Therapeutic Activity |
Non-Patent Citations (1)
| Title |
|---|
| CHEN CHEN，ET AL.: ""Autologous fibroblasts induce fibrosis of the nucleus pulposus to maintain the stability of degenerative intervertebral discs"", 《BONE RESEARCH》 * |
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