CN106421915A - Method of repairing human articular cartilage based on 3D bioprinting - Google Patents
Method of repairing human articular cartilage based on 3D bioprinting Download PDFInfo
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- A61L27/38—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 containing added animal cells
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- 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
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
The invention relates to a method for repairing human articular cartilage based on 3D bioprinting. The method comprises the following steps: a) PEGDMA is prepared from PEG (polyethylene glycol)and methacryloyl chloride and purified; b) human mesenchymal stem cells are separated; c) the human mesenchymal stem cells are subjected to passage and adjusted in density, an induction medium is replaced for culture after cell attachment; d) a bovine femur is washed, a full-thickness cartilage defect is formed at the center of an osteochondral embolism by a sterile living body perforator, and the osteochondral embolism is placed in a DMEM (dulbecco's modified eagle medium) and cultivated into 3D biopaper; e) the purified PEGDMA is dissolved, a photoinitiator 2959 is added, and chondrocytes obtained through induction in the step c) are re-suspended in a filtered and sterilized PEGDMA solution for preparation of bio-ink; f) the bio-ink is added to a disinfected and sterilized bioprinter, and the 3D cartilage is printed on the 3D biopaper according to the designed shape and size and is cultured in the induction medium. The method has the advantages of being simple and quick to operate, accurate, low in cytotoxicity and is expected to become a clinical cartilage repair method.
Description
Technical field
The present invention relates to tissue engineering technique field, particularly to a kind of, human articular cartilage is repaired based on 3D biometric print
Method.
Background technology
The cartilage defect that the reasons such as osteoarthritis, aging and joint injury lead to is the main of arthralgia and chronic disability
One of reason.Due to lacking blood vessel, nerve and lymph, ripe cartilage is unable to spontaneous recovery.It is most commonly used to late period cartilage degradation at present
Therapeutic Method is joint replacement, but this operation complex operation itself, often cause surrounding tissue to infect, and surgery cost
Very expensive.Although being introduced into before more than 20 years based on the organizational project therapy of cell transplantation, cartilaginous tissue work at present
Journey strategy can't produce the tissue the same with natural cartilage, including hierarchical structure, extracellular matrix and mechanical performance.Separately
Outward, almost all of knee cartilage restorative procedure has a step：Healthy cartilaginous tissue around damage location is removed, with
Produce the artificial defect of definite shape, be easy to treatment and the transplanting in later stage.This step actually causes volume to existing cartilage
Outer necrosis has simultaneously ultimately resulted in cartilage degradation and tissue transplantation's failure.
Direct repair of cartilage method based on organizational project is very attractive, because the method is directly to defect portion
Position is repaired, without leading to the extra damage of surrounding health tissue.Preferably transplanting organization need and natural cartilage are fine
Integration and the damage location of different size and thickness is repaired.Accordingly, it would be desirable to the new technology of exploitation, make engineering tissue
Shape that can be different from damage location and property match.
Inkjet printing is contactless printing technique, replicates digital-to-analogue information by the form of micro- ink droplet in substrate.Print
Front heating produces heat, and then the ink that gasifies produces bubble, and consequent pressure makes ink droplet with different volumes（10-150 pL）
Injection.In print procedure, although the heating element heater at each nozzle makes local temperature rise to 300 DEG C and continue several microseconds,
But mammalian cell is only heated 2 microseconds, temperature exceedes external environment 4-10 DEG C, and 90% cell is appointed and can be maintained vigour.With
Development and the hi-Fix to cell, somatomedin and biomaterial scaffolds of high flux Numeric Control Technology, had at present
The method of seminar's biometric print is successfully manufactured that the human microvascular with biological function.
Producing of biomaterial scaffolds typically less for viscosity bio-ink is ejected on the larger biochargeable paper of viscosity.
Research shows, by Polyethylene Glycol（PEG）The hydrogel of macromonomer synthesis can keep the vigor of chondrocyte and promote thin
The deposition of extracellular matrix, such as Dan Baiduotang proteoglycan PG and II Collagen Type VI.The modulus of compressibility phase of the modulus of compressibility of PEG-hydrogel and human cartilage
Imitate, and natural tissues have biocompatibility, will not cause strong immunologic rejection.In addition, PEG is water miscible, viscosity is low, permissible
Modified by photo-crosslinking method.Therefore, in biometric print, PEG can be used for spontaneous polymerization as biologic bracket material.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of method repairing human articular cartilage based on 3D biometric print, should
Method can quickly and accurately manufacture corresponding tissue according to cartilage position defect shape, size and property, eventually for
Repair human cartilage defect.
For solving above-mentioned technical problem, the technical scheme is that：A kind of soft based on 3D biometric print reparation person joint
The method of bone, its innovative point is：Comprise the following steps：
a）PEG prepares PEGDMA purification with methacrylic chloride reaction overnight；
b）Using full bone marrow adherent method separation human mesenchymal stem cell；
c）Human mesenchymal stem cell good for growth conditions is passed on and adjusts density, change inducing culture after cell attachment
Culture；
d）Clean fresh bulls bone, make full-thickness cartilage defects in the center of bone cartilage thromboembolism with aseptic live body perforator,
Bone cartilage thromboembolism is placed in DMEM and is trained 3D biochargeable paper；
e）By the PEGDMA dissolving of purification, add light trigger 2959, by step c）Induction gained chondrocyte is resuspended in filtration
Bio-ink is made in degerming PEGDMA solution；
f）Will after biometric print machine sterilization add bio-ink, according to design shapes and sizes on 3D biochargeable paper layer by layer
Print 3D cartilage, 3D cartilage is placed in culture in inducing culture.
Further, step a）The PEG of middle employing 3kDa and methacrylic chloride reaction overnight under a nitrogen atmosphere, then lead to
Cross ether precipitation and the method for overnight lyophilizing carries out purification, make the PEGDMA macromer purity of synthesis be more than 95%.
Further, step b）The middle human mesenchymal stem cell that separates includes aseptically taking health adult's bone marrow fluid,
Rinsed with PBS and count, by the cell culture after processing containing 20% (v/v) hyclone and 1% (v/v) Pen .- Strep-
In the α-MEM basal medium of gentamycin, it is placed in 37 DEG C, 5% (v/v) CO2Cell culture incubator culture, change liquid after 48 hours,
Change liquid every 3 days afterwards.
Further, step c）Middle human mesenchymal stem cell density adjusts to 5 × 103Individual/cm2, described inducing culture contains
1 × Insulin-Transferrin-selenium-Sodium Pyruvate, 0.1mmol/L ascorbic acid phosphate, 1.25mg/mL human seralbumin egg
In vain, 10-7mol/L dexamethasone, 1% (v/v) Pen .- Strep-gentamycin, 10 ng/mL TGF-b1, will be described
Inducing culture be placed in 37 DEG C, 5%（v/v）CO2Cell culture incubator, every 3 days change an inducing culture, culture 2-6 week.
Further, step d）In a diameter of 4mm of aseptic live body perforator, the depth of defect is 2-5mm, and described DMEM contains
There are 10% (v/v) calf serum and 1% (v/v) Pen .- Strep-gentamycin, 37 DEG C of cultivation temperature, DMEM is placed in 5%
(v/v)CO2Cell culture incubator.
Further, step e）Middle PEGDMA is dissolved in PBS or deionized water, and concentration is 10 % (w/v) and 20 % (w/
V), the addition of light trigger 2959 is 0.05 % (w/v), and in described bio-ink, every milliliter of solution contains 5 × 106Individual cell.
Further, step f）Middle biometric print machine is sterilized with ultra-vioket radiation, prints pen 70% (v/v) ethanol disinfection, beats
During print by UV intensity be 4.5 mW/cm2Long-wave ultra violet lamp be placed at 25cm above print platform, print pen in fill it up with step e）
Prepared bio-ink is simultaneously covered with aluminium foil to prevent ultra-vioket radiation, and the 3D cartilage that goes out printing is trained in inducing culture respectively
Support 2,4,6 weeks, change a subculture within every 3 days.
It is an advantage of the current invention that：1）Suitable cartilage can be produced according to the size and shape at tissue damaged position to move
Plant；2）The position of individual cells and biomaterial scaffolds can accurately be controlled；3）Little to cell injury be conducive to remaining soft
Bone phenotype；4）Using thermal ink jet printers and spontaneous photopolymerization reaction, the time that organizational project manufactures is greatly shortened.The present invention
Simple to operate fast and accurately cytotoxicity is little, the method promising to be very much clinical repair of cartilage.
Brief description
Fig. 1 is a kind of process schematic of the method repairing human articular cartilage based on 3D biometric print of the present invention.
Fig. 2 is the COL2A1 gene expression dose block diagram of biometric print articular cartilage of the present invention.
Fig. 3 is the ACAN gene expression dose block diagram of biometric print articular cartilage of the present invention.
Fig. 4 is the COL1A1 gene expression dose block diagram of biometric print articular cartilage of the present invention.
Specific embodiment
As shown in figure 1, the invention discloses a kind of method that human articular cartilage is repaired based on 3D biometric print, including below
Step：
The preparation of PEGDMA
PEG by 3kDa（Polyethylene Glycol）It is dissolved in oxolane, make PEG under a nitrogen atmosphere with methacrylic chloride overnight
Reaction.PEGDMA with the method purification synthesis of overnight lyophilizing is separated out by ether（Polyethylene glycol dimethacrylate）Big point
Sub- monomer, using proton nmr detection it is ensured that the PEGDMA macromer purity of synthesis is more than 95%.
Human mesenchymal stem cell separates
Aseptic condition Xia Qu health adult bone marrow fluid, adds equal-volume PBS, blows and beats cell dispersion with suction pipe.300g is taken under room temperature
Bone marrow PBS mixed liquor is centrifuged 5min, collects cell.Use PBS re-suspended cell, counted.300g bone marrow PBS is taken to mix under room temperature
Liquid is centrifuged 5min, collects cell, with containing 20% (v/v) hyclone and 1% (v/v) PSG（Pen .- Strep-gentamycin）
α-MEM basal medium re-suspended cell, by cell with 1.6 × 105Individual/cm2Density be inoculated in culture bottle, be placed in 37 DEG C,
5%(v/v)CO2Cell culture incubator culture, change liquid after 48 hours, changed liquid every 3 days afterwards.Amplification in vitro, will to after the second filial generation
Stem cell is passed on, adjustment density to 5 × 103Individual/cm2, cultivating makes cell attachment in 24 hours.Basal medium is replaced by induction
Culture medium DMEM, is placed in 37 DEG C, 5% (v/v) CO2Cell culture incubator culture, every 3 days change an inducing culture, cultivate 2-
6 weeks.Described inducing culture DMEM contains 1 × Insulin-Transferrin-selenium-Sodium Pyruvate (ITS-A), and 0.1mmol/L is anti-bad
Hematic acid phosphate, 1.25mg/mL human serum albumin, 10-7mol/L dexamethasone, 1% (v/v) Pen .- Strep-
Gentamycin, 10 ng/mL TGF-b1.
3D biochargeable paper and the preparation of bio-ink
Take fresh bulls bone, got into the cave with the rustless steel of diameter 8mm under aseptic condition, bone cartilage thromboembolism is clear with the DMEM containing antibiotic
Wash 3 times.Make full-thickness cartilage defects in the aseptic live body perforator of the center diameter 4mm of bone cartilage thromboembolism, defect
Depth about 2-5mm, is specifically dependent upon the thickness of cartilage.The culture of the bone prepared by said method cartilage thromboembolism is being contained 10%
（v/v）Calf serum and 1% (v/v) PSG（Pen .- Strep-gentamycin）DMEM in, in 37 DEG C, 5% (v/v) CO2's
It is trained 3D biochargeable paper in cell culture incubator.Aforementioned prepared purification PEGDMA is dissolved in PBS or deionized water, makes solution
Final concentration of 10% (w/v) and 20 % (w/v).0.05% (w/v) light trigger 2959 is added in solution.Gained cartilage will be induced
Cell is resuspended in the PEGDMA solution of filtration sterilization, and every milliliter of solution contains 5 × 106Individual cell.
3D biometric print
Biometric print machine is sterilized with ultra-vioket radiation, by 70% (v/v) ethanol disinfection of the printing pen containing 50 ink-jet storehouses.Beat
During print, long-wave ultra violet lamp is placed at 25cm above print platform, UV intensity is 4.5 mW/cm2.Print in pen and fill it up with aforementioned system
The bio-ink obtaining, is covered with aluminium foil and prints pen to prevent ultra-vioket radiation.Design the big of cartilage defect with Adobe Photoshop
Little and shape, is printed layer by layer with printer, produces 3D cartilage, and it is cultivated in inducing culture respectively 2,4,6 weeks, training
Foster base is changed once for every 3 days.
For being better understood upon and characterizing PEGDMA and 3D cartilage performance, we have carried out corresponding performance test work(to it
Can identification.
（1）PEGDMA measuring mechanical property
Before test, the acellular hydrogel of PEGDMA of preparation is placed 48 hours in 37 DEG C of DMEM, surveyed with twin shaft controller
The thickness of amount hydrogel.By the way of step-by-step movement compression, with the time dependent loading of answering that test speed is 0.1mm/s, hydrogel is compressed
To maximum compression tension force 20%.After the circulation of each load, all DMEM solution equilibria is carried out to cartilage.Modulus of compressibility according to pressure-
The slope of tension curve calculates.Result shows that printed cartilaginous tissue mechanical strength reaches or the Biological Strength better than natural cartilage
Learn characteristic.
（2）PEGDMA swelling coefficient measures
Take a certain amount of PEGDMA hydrogel to weigh after placing 48 hours in 37 DEG C of DMEM, obtain swelling rear quality Ws；Will
PEGDMA hydrogel lyophilizing, weighs and obtains dry weight Wd.Equilibrium swelling is than Q=Ws/Wd, water content M=（Ws -Wd）/ Ws.Result
Show that printed articular cartilage tissue and natural tissues are closely similar.
（3）3D biometric print cartilage related gene expression level
With the 3D cartilage thing of liquid nitrogen flash freezer preparation and by its powdered, the RNA reverse transcription extracting above-mentioned sample obtains cDNA, with real-time
The method detection cartilage related gene of quantitative PCR（COL1A1, COL2A1, ACAN, GAPDH are as internal reference）Expression, such as schemes
2nd, shown in Fig. 3, Fig. 4, respectively COL2A1, the expression of ACAN, COL1A1.Result shows to print the gene expression water of cartilage
The flat requirement reaching natural cartilage.
（4）3D biometric print cartilage biochemical analysises
Cultivated 2 weeks, 4 weeks, the 3D cartilage lyophilizing of 6 weeks 48 hours respectively, added 1ml papain respectively in cartilage samples
Solution（Containing 125 mg/mLIII type papains, 10mM L-Cysteine, 100mM phosphate buffer and 10mM EDTA,
pH 6.4）, process 16 hours at 60 DEG C.Backward solution in add 1ml pepsin solution（Concentration 100 mg/mL, stomach egg
White enzyme is dissolved in 0.05M acetic acid）, solution is placed in 4 DEG C and processes 6 days.With CyQUANT Cell Proliferation Assay
Kit measurement DNA content, measures I type and II Collagen Type VI content with ELISA kit.Result shows to print the albumen life of cartilage
Product level is excellent.
（5）3D biometric print cartilaginous tissue credit is analysed
The 3D cartilage of preparation is fixed overnight with 10% formalin solution, transfers it to 70%（v/v）Ethanol solution
In, the 3D cartilage that then processed through above-mentioned steps with paraffin embedding.It is fabricated to the paraffin section of 6 μ m-thick after embedding and use Huang red
The fast green dyeing of O/, observes the Dan Baiduotang proteoglycan PG of hydrogel secretion.Result shows that the Dan Baiduotang proteoglycan PG generation level printing cartilage reaches sky
So cartilage levels.
（6）The interfacial failure stress test of natural cartilage
PEG-hydrogel is printed on 3D biometric print paper, when culture 2,4,6 weeks in inducing culture respectively, collects sample
Product.Careful is separated 3D biometric print paper with subchondral bone with the cartilage portion of graft, and gained cartilage sample is placed in specially
The cell that can support outer ring portion in.Using the piston being driven by SMAC voice coil motor（Diameter is slightly smaller than 3.5mm）Push away soft
Bone center of a sample, speed is 0.1mm/s, measures axial force and record when 1kHz.The thickness vernier calliper of core
Chi measures, and interfacial failure stress is the ratio recording maximum, force and center side area in experiment.Result shows natural cartilage and beats
The power that be combined with each other of print cartilage strengthens with the growth of incubation time.
Ultimate principle and the principal character of the present invention have been shown and described above.It should be understood by those skilled in the art that,
The present invention is not restricted to the described embodiments, merely illustrating the principles of the invention described in above-described embodiment and description,
Without departing from the spirit and scope, the present invention also has various changes and modifications, and these changes and improvements all fall
Enter in scope of the claimed invention.Claimed scope is by appending claims and its equivalent thereof.
Claims (7)
1. a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Comprise the following steps：
PEG prepares PEGDMA purification with methacrylic chloride reaction overnight；
Using full bone marrow adherent method separation human mesenchymal stem cell；
Human mesenchymal stem cell good for growth conditions is passed on and adjusts density, change inducing culture training after cell attachment
Support；
Clean fresh bulls bone, make full-thickness cartilage defects in the center of bone cartilage thromboembolism with aseptic live body perforator, will
Bone cartilage thromboembolism is placed in DMEM and is trained 3D biochargeable paper；
By the PEGDMA dissolving of purification, add light trigger 2959, by step c）Induction gained chondrocyte was resuspended in and filtered
Bio-ink is made in the PEGDMA solution of bacterium；
Bio-ink will be added, the shapes and sizes according to design are beaten on 3D biochargeable paper layer by layer after biometric print machine sterilization
Print off 3D cartilage, 3D cartilage is placed in culture in inducing culture.
2. as claimed in claim 1 a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Step
A）The PEG of middle employing 3kDa and methacrylic chloride reaction overnight under a nitrogen atmosphere, then separated out and overnight lyophilizing by ether
Method carry out purification, make synthesis PEGDMA macromer purity be more than 95%.
3. as claimed in claim 1 a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Step
B）The middle human mesenchymal stem cell that separates includes aseptically taking health adult's bone marrow fluid, is rinsed with PBS and counts, will process
Cell culture afterwards is in the α-MEM basis training containing 20% (v/v) hyclone and 1% (v/v) Pen .- Strep-gentamycin
In foster base, it is placed in 37 DEG C, 5% (v/v) CO2Cell culture incubator culture, change liquid after 48 hours, changed liquid every 3 days afterwards.
4. as claimed in claim 1 a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Step
C）Middle human mesenchymal stem cell density adjusts to 5 × 103Individual/cm2, described inducing culture contain 1 × Insulin-Transferrin-
Selenium-Sodium Pyruvate, 0.1mmol/L ascorbic acid phosphate, 1.25mg/mL human serum albumin, 10-7mol/L ground plug rice
Pine, 1% (v/v) Pen .- Strep-gentamycin, 10 ng/mL TGF-b1, by described inducing culture be placed in 37 DEG C,
5%（v/v）CO2Cell culture incubator, every 3 days change an inducing culture, culture 2-6 week.
5. as claimed in claim 1 a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Step
D）In a diameter of 4mm of aseptic live body perforator, the depth of defect is 2-5mm, described DMEM contain 10% (v/v) calf serum and
1% (v/v) Pen .- Strep-gentamycin, 37 DEG C of cultivation temperature, DMEM is placed in 5% (v/v) CO2Cell culture incubator.
6. as claimed in claim 1 a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Step
E）Middle PEGDMA is dissolved in PBS or deionized water, concentration be 10 % (w/v) and 20 % (w/v), light trigger 2959 add
Entering amount is 0.05 % (w/v), and in described bio-ink, every milliliter of solution contains 5 × 106Individual cell.
7. as claimed in claim 1 a kind of based on 3D biometric print repair human articular cartilage method it is characterised in that：Step
F）Middle biometric print machine with ultra-vioket radiation sterilize, print pen 70% (v/v) ethanol disinfection, during printing by UV intensity be 4.5 mW/
cm2Long-wave ultra violet lamp be placed at 25cm above print platform, print pen in fill it up with step e）Prepared bio-ink simultaneously uses aluminum
Paper tinsel covers to prevent ultra-vioket radiation, and the 3D cartilage that goes out printing is cultivated 2,4,6 weeks in inducing culture respectively, changes within every 3 days
One subculture.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108130313A (en) * | 2017-12-28 | 2018-06-08 | 杭州枫霖科技有限公司 | A kind of method that three-dimensional samples of human glioma is built based on biological 3D printing |
| CN110450401A (en) * | 2019-09-05 | 2019-11-15 | 上海交通大学医学院附属第九人民医院 | A kind of method of biological 3 D-printing joint prosthesis in support liquid medium |
| CN115212002A (en) * | 2022-06-15 | 2022-10-21 | 苏州大学 | 3D biological printing support for repairing cartilage defect and preparation thereof |
| CN115944781A (en) * | 2021-10-09 | 2023-04-11 | 上海软馨生物科技有限公司 | Cartilage tissue engineering compound based on 3D printing and application thereof |
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2016
- 2016-10-12 CN CN201610890438.0A patent/CN106421915A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108130313A (en) * | 2017-12-28 | 2018-06-08 | 杭州枫霖科技有限公司 | A kind of method that three-dimensional samples of human glioma is built based on biological 3D printing |
| CN108130313B (en) * | 2017-12-28 | 2021-04-30 | 杭州枫霖科技有限公司 | Method for constructing three-dimensional glioma tissue based on biological 3D printing |
| CN110450401A (en) * | 2019-09-05 | 2019-11-15 | 上海交通大学医学院附属第九人民医院 | A kind of method of biological 3 D-printing joint prosthesis in support liquid medium |
| CN115944781A (en) * | 2021-10-09 | 2023-04-11 | 上海软馨生物科技有限公司 | Cartilage tissue engineering compound based on 3D printing and application thereof |
| CN115212002A (en) * | 2022-06-15 | 2022-10-21 | 苏州大学 | 3D biological printing support for repairing cartilage defect and preparation thereof |
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