CN104399130B - A kind of porous cell free tissue engineering bone/cartilage support and preparation method - Google Patents
A kind of porous cell free tissue engineering bone/cartilage support and preparation method Download PDFInfo
- Publication number
- CN104399130B CN104399130B CN201410669747.6A CN201410669747A CN104399130B CN 104399130 B CN104399130 B CN 104399130B CN 201410669747 A CN201410669747 A CN 201410669747A CN 104399130 B CN104399130 B CN 104399130B
- Authority
- CN
- China
- Prior art keywords
- cartilage
- tissue engineering
- engineering bone
- tissue
- sheet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a kind of porous cell free tissue engineering bone/cartilage support, i.e. by by tissue engineering bone/cartilage material through punching process after, utilize tissue engineering bone/cartilage support prepared by method for removing cells so that it is not only have three-dimensional porous property, and there is stronger biomechanical property and biocompatibility.This tissue engineering bone/cartilage support is applicable to cartilages in loaded parts defect repair, will become the important breakthrough of organizational project reparation cartilage defect.
Description
Technical field
The present invention relates to medical material tech field, be specifically related to novel tissue engineered cartilage support and preparation method.
Background technology
The articular cartilage damage caused because of reasons such as wound, inflammation, regression, tumor resections is the most common disease
Sick.Regeneration of Articular Cartilage and repair ability are extremely limited, once after damage, it is difficult to self repairs, and the state of an illness continues development,
Necessarily cause osteoarthritis.Current Therapeutic Method, such as microfrature, autologous or allosome periosteum, perichondrium, bone
Cartilage block transplanting etc., all also exist many disadvantages, thus limit clinical practice.Along with 19th-century organizational project skill
The appearance of art, application organizational project technique construction tissue engineering bone/cartilage or osteochondral composites are considered to solve this and ask
Inscribe best technological means.
Tissue engineering technique comes across 19th-century, refers to utilize bioactive substance, by In vitro culture or structure
Method, reproduce or repair the technology of organ and tissue.Tissue engineering technique three elements: " seed cell, support
And signal factor ".By seed cell is cultivated on biomaterial scaffolds, then it is transplanted to defect and is formed newly
Tissue, through reinventing shape and combine with body tissue, be the most square of organizational project repair tissue damage
Formula.Timbering material, except providing the 3 dimensional coil geometry of tissue, also provides the microenvironment of existence for cell, supports thin
The adhesion of born of the same parents, breed, break up and ultimately form cambium.So how obtain three-dimensional, porous, biological
The support of activity is the key link of organizational project.
It is currently used for the tissue engineering bracket that cartilage injury repairs and mainly has two classes, be i.e. synthetic material and natural
Extracellular matrix.
The polymer of synthetic has the following characteristics that good mechanical strength, satisfied biocompatibility, controlled
The degradation rate of system.But, the surface of synthetic polymer is hydrophobic, lacks inherent biological activity.And, PLGA
Inflammatory reaction may be caused Deng the acid degradation products of some polymer.
Relative to artificial-synthetic copolymer, tradition natural material cartilage frame is dry by freezing after being mostly to pulverize cartilage
The preparations such as dry and cross-linking method become the support having three-dimensional porous structure, although remain or bionical natural tissues
Main biochemical composition, there is more preferable biological activity and functional, can be beneficial to the identification of seed cell, adhesion,
Propagation and differentiation.But its initial mechanical intensity is relatively low, compound cells incubation occurs " hollow " phenomenon,
The properties of natural cartilage can not be simulated completely.
Summary of the invention
For the problems referred to above, it is an object of the invention to provide a kind of porous cell free tissue engineering bone/cartilage support,
I.e. prepared the de-cellular system engineering cartilage frame of porous by cheesing techniques so that it is not only have three-dimensional porous property, and
And there is stronger biomechanical property and biocompatibility, this tissue engineering bone/cartilage support is applicable to cartilages in loaded parts and lacks
Damage and repair, the important breakthrough of organizational project reparation cartilage defect will be become.
A further object of the present invention is to provide the preparation method of above-mentioned tissue engineering bone/cartilage support.
For achieving the above object, the present invention is by the following technical solutions:
A kind of porous cell free tissue engineering bone/cartilage support, be by by tissue engineering bone/cartilage material at punching
After reason, utilize tissue engineering bone/cartilage support prepared by method for removing cells.
Further, its porosity is made to reach 50-98%, preferably through punching process tissue engineering bone/cartilage material
85%.For reaching above-mentioned requirements, the degree of depth of punching, aperture and interval, need to according to required difformity,
The tissue engineering bone/cartilage of thin and thick is adjusted, and makes hole can be uniformly distributed on tissue engineering bone/cartilage.
Higher porosity is conducive to propagation and the differentiation of cell, and the porosity of support is the highest, the increment of internal cell
Number is the most, and the surface area of timbering material is big, material and cell can contact area bigger, can carry out more
Become the absorption of cartilage-inducing factor and ion exchange, promote chondrocyte sticking and breeding and cartilage in the material
The generation of tissue.But porosity crosses conference affects the mechanical property of material, also can affect its degradation rate.
Further, above-mentioned tissue engineering bone/cartilage material can be allogeneic or heterozoic shoulder joint, knee joint
The intraarticular cartilage such as joint, hip joint.
Further, described intraarticular cartilage carries out separating, clean and being prepared as cartilage sheet, institute before punching processes
Cartilage sheet can be thick with subchondral bone (i.e. osteochondral composites) or without, a diameter of 1-40mm of cartilage sheet
Degree is 0.1-20mm.
Further, the pore size that punching processes is 10-1000 μm, and pitch of holes is 10-1000 μm.
Further, above-mentioned method for removing cells can be chemical-agent technique (acid-base method, osmosis, Detergents method,
Polyethylene glycol method, alcohols method etc.), Physical (freeze-thaw method, pressure application, electrical breakdown method etc.), biological reagent method (enzyme
Facture, non-enzymatic reagent method).
The preparation method of a kind of porous cell free tissue engineering bone/cartilage material, comprises the following steps:
1) cartilaginous tissue thin slice is prepared in in-vitro separation, cleaning；
2) the tissue engineering bone/cartilage thin slice of porous it is prepared as after the cartilaginous tissue thin slice punching of preparation being processed；
3) the tissue engineering bone/cartilage thin slice of porous carries out de-cell to process, eliminate its immunogenicity.
Above-mentioned steps 1) in the cartilaginous tissue thin slice of indication, may be from the joint of allogeneic, heterogenous animal or people
Cartilage, cartilaginous tissue wafer diameters 1-40mm, thickness is 0.1-20mm, is used sterile purified water spray gun high pressure
Rinse, remove blood constituent and fatty tissue as far as possible.
Above-mentioned steps 2) described in punching process refer to, application card punch to cartilaginous tissue thin slice punch, Ke Yiying
Directly punchinging with sharp-pointed mechanical force card punch, such as borer, fine needle etc., or application laser instrument carries out laser and beats
Hole, can be gas laser, liquid laser, semiconductor laser etc..Punching shape can be taper,
The shape such as ellipse, cylinder, square, rectangle, pore size is 10-1000 μm, and pitch of holes is
10-1000μm。
Above-mentioned steps 3) in de-cell process refer to by various method for removing cells (chemical method, Physical, life
Thing reagent etc.) the tissue engineering bone/cartilage sheet after punching is carried out de-cell process, prepare the tissue work of non-immunogenicity
Journey cartilage frame.
Above-mentioned preparation method, also includes: gone out by the tissue engineering bone/cartilage support Gamma x ray irradiation x prepared
Bacterium, irradiation dose is 25kGy, exposure time 5-10 hour.
The porous cell free tissue engineering bone/cartilage support of the present invention have compared with normal tissue engineered cartilage support with
Lower advantage:
1) porous cell free tissue engineering bone/cartilage support, the most close with normal cartilage, has preferable biomechanics
Performance, it is adaptable to the cartilage defect repair in heavy burden district, it is possible to have the most raw with the cartilaginous tissue around host
The thing compatibility gives mechanical support.
2) porous cell free tissue engineering bone/cartilage support with compared with other tissue engineering brackets can more retain
Endochondral matrix components, closer to normal cartilage, can be more beneficial for seed cell and grow in support, point
Change and keep the phenotype of chondrocyte.
3) the de-cellular system engineering cartilage of porous, material obtains easily, and technical need is simple to operation, to cartilage
Reparation has big advantage.
Accompanying drawing explanation
Fig. 1. the substantially photo of the tissue engineering bone/cartilage sheet of laser boring of the present invention, Fig. 1 (a) shows diameter, figure
1 (b) display thickness (being 1cm between numeral 1 and 2, each little lattice scale is 1mm).
Fig. 2. porous of the present invention takes off the Hoechst 33258 of cellular system engineering cartilage and dyes.
Fig. 3. porous of the present invention takes off the HE dyeing of cellular cartilage sheet.
Fig. 4. porous of the present invention takes off the Toluidine blue staining of cellular system engineering cartilage sheet.
Fig. 5. porous of the present invention takes off the electron microscope scanning photo of cellular system engineering cartilage, and Fig. 5 (a) shows
For punching cartilage sheet cross section electromicroscopic photograph, Fig. 5 (b) is punching cartilage sheet longitudinal section electromicroscopic photograph.
Fig. 6. porous of the present invention takes off cellular system engineering cartilage and compares figure with normal articular cartilage Biomechanics test.
Fig. 7. porous of the present invention takes off cellular system engineering chondrocyte toxicity inspection growth curve, * P > 0.05.
Fig. 8. primary rabbit cartilage cell is inoculated in the live/dead colored graph after support of the present invention upper 7 day.
Detailed description of the invention
Embodiment 1
(1) take the cartilage of fresh adult pig condyle of femur, remove surrounding tissue, sterile purified water spray gun high pressure rinse,
Remove blood constituent and fatty tissue as far as possible.
(2) acquirement cartilage trepan is made diameter 4mm, thickness 2mm cylindrical cartilage sheet 5 (see
Fig. 1).
(3) it is placed in-24 DEG C after 24h, takes out after-80 DEG C of frozen 72h of refrigerator.
(4) the tissue engineering bone/cartilage sheet prepared is soaked in the middle of 0.01MPBS buffer, through carbon dioxide
Laser boring punches on the cartilage sheet of above-mentioned immersion, aperture 300 μm, pitch of holes 300 μm, porosity
It is 85%.
(5) by the multi-porous tissue engineering cartilage sheet of preparation in step (4) after punching, aseptic tri-distilled water is used
Rinse after soaking, proceed to after-20 ° of freezing 24h-80 DEG C are taken out after freezing 72h.
(6) after cleaning, standby tissue engineering bone/cartilage sheet is placed in the Tris-HCl of 10mM (containing protease suppression
Agent) in liquid, 7500rpm is centrifuged 20min, cleans 3 times；Tissue engineering bone/cartilage sheet after being centrifuged adds
In 3%SDS cleanout fluid (inner protein enzyme inhibitor), on 37 DEG C of isothermal vibration machines, shake 48h, remove thin
Born of the same parents' composition, takes out at latter 4 DEG C with aseptic PBS liquid, 8000rmp, 20min, centrifugal elutriation 3 times；After flushing
Tissue engineering bone/cartilage is in the digestive enzyme liquid containing 1U/ml RNase A and 50U/ml DNase I, and 37 DEG C digest 24h
After, 7500rpm, 7min, take out after centrifugal 3 times.
(7) after the tissue engineering bone/cartilage sheet of the laser boring of taking-up being soaked in distilled water continuous flushing 72h, will
The tissue engineering bone/cartilage support Gamma x ray irradiation x sterilizing prepared, irradiation dose is 25kGy, during irradiation
Between 5-10 hour, 4 DEG C of refrigerators are standby.
Embodiment 2
By the support frozen section of preparation, row Hoechst 33258 dyes (see Fig. 2), and acellular core colours, and says
Bright cartilage sheet takes off cell success；HE dyes (see Fig. 3), Toluidine blue staining (see Fig. 4), it is seen that tissue work
The microstructure of journey cartilage frame；Scanning electron microscope shows the internal structure (see Fig. 5) of tissue engineering bone/cartilage support.
The cartilaginous tissue of the tissue engineering bone/cartilage support of preparation Yu formed objects is carried out Biomechanics test, is made
The modulus of elasticity in comperssion of standby cartilage frame is 0.5734 ± 0.021Mpa, with normal articular cartilage (0.6875 ±
Though 0.013Mpa) there being significant difference (* P < 0.05), but it is closer to, it was demonstrated that the cartilage frame of preparation has preferably
Biomechanical strength, can be used for repair heavy burden district cartilage defect (see Fig. 6).
L cell L929 is seeded in 96 orifice plates, sets up three groups, i.e. negative control group, test group,
Positive controls, adds the lixiviating solution of tissue engineering bone/cartilage in negative control group in adding normal culture fluid, experimental group,
Positive controls add dimethyl sulfoxide, draw CCK-8 cell growth curve (see Fig. 7), draw test group with
Negative control group no difference of science of statistics (* P>0.05), has significant difference (* P<0.05) with positive controls,
The support no cytotoxicity of preparation is described.
After primary rabbit cartilage cell is inoculated in support upper 7 day, can be found by live/dead (see Fig. 8) dyeing
Cell can be good at growing in cartilage hole and breeding, and has preferable biopotency.
Above three experiment specific experiment method can be found in thesis for the doctorate:
Yang Qiang. the experiment building tissue engineering bone/cartilage and osteochondral composites based on cartilage cell epimatrix is ground
Study carefully: [D]. Beijing: Military Surgeon Further Education College, PLA .2008
Prove that the tissue engineering bone/cartilage support of porous that we prepare takes off cell by above-mentioned experiment relatively successful, without immunity
Originality and cytotoxicity, have higher biopotency and good histocompatibility, can be described as tissue engineering bone/cartilage and repair
Compound joint cartilage injury provides new means.
Claims (2)
1. a preparation method for porous cell free tissue engineering bone/cartilage material, comprises the following steps:
1) take the cartilage of fresh adult pig condyle of femur, remove surrounding tissue, sterile purified water spray gun high pressure rinse,
Remove blood constituent and fatty tissue as far as possible；
2) acquirement cartilage trepan is made the cylindrical cartilage sheet of diameter 4mm, thickness 2mm；
3) it is placed in-24 DEG C after 24h, takes out after-80 DEG C of frozen 72h of refrigerator；
4) the cartilaginous tissue sheet prepared is soaked in the middle of 0.01MPBS buffer, through carbon dioxide laser
Card punch punches on the cartilage sheet of above-mentioned immersion, aperture 300 μm, and pitch of holes is 300 μm, hole
Rate is 85%；
5) by step 4 after punching) the aseptic tri-distilled water of multi-porous tissue engineering cartilage sheet prepared rinses and soaks
After bubble, proceed to after-20 ° of freezing 24h-80 DEG C are taken out after freezing 72h；
6) after cleaning, standby multi-porous tissue engineering cartilage sheet is placed in the 10mM containing protease inhibitor
In Tris-HCl liquid, 7500rpm is centrifuged 20min, cleans 3 times；In tissue engineering bone/cartilage sheet after being centrifuged
Add in the 3%SDS cleanout fluid of inner protein enzyme inhibitor, on 37 DEG C of isothermal vibration machines, shake 48h, go
Except cell component, take out at latter 4 DEG C with aseptic PBS liquid, 8000rpm, 20min, centrifugal elutriation 3 times；
After flushing, tissue engineering bone/cartilage is in the digestive enzyme liquid containing 1U/ml RNase A and 50U/ml DNase I, 37 DEG C
After digestion 24h, 7500rpm, 7min, take out after centrifugal 3 times；
7) after the tissue engineering bone/cartilage sheet of the laser boring of taking-up being soaked in distilled water continuous flushing 72h, will
The tissue engineering bone/cartilage support Gamma x ray irradiation x sterilizing prepared, irradiation dose is 25kGy, spoke
According to 5-10 hour time, 4 DEG C of refrigerators were standby.
2. use porous cell free tissue engineering bone/cartilage support prepared by the preparation method described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410669747.6A CN104399130B (en) | 2014-11-20 | 2014-11-20 | A kind of porous cell free tissue engineering bone/cartilage support and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410669747.6A CN104399130B (en) | 2014-11-20 | 2014-11-20 | A kind of porous cell free tissue engineering bone/cartilage support and preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104399130A CN104399130A (en) | 2015-03-11 |
| CN104399130B true CN104399130B (en) | 2016-10-12 |
Family
ID=52636835
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410669747.6A Active CN104399130B (en) | 2014-11-20 | 2014-11-20 | A kind of porous cell free tissue engineering bone/cartilage support and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104399130B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108721698A (en) * | 2017-04-24 | 2018-11-02 | 上海市肺科医院 | A kind of tissue engineering bone/cartilage holder and its preparation method and application |
| EP3630213B1 (en) * | 2017-05-30 | 2023-07-05 | Trauma Care Consult Traumatologische Forschung Gemeinnützige Gesellschaft mbH | Cartilage graft scaffolds |
| CN108310466B (en) * | 2018-04-04 | 2021-04-06 | 浙江大学 | Preparation method of nucleus pulposus acellular material from natural tissue |
| CN112274698B (en) * | 2020-11-05 | 2021-07-06 | 四川大学 | Preparation method of bionic artificial temporomandibular joint disc |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9623051B2 (en) * | 2011-04-15 | 2017-04-18 | The Regents Of The University Of California | Decellularized extracellular matrix |
| CN102293690B (en) * | 2011-06-07 | 2014-07-09 | 天津市托福医用原子能科技有限公司 | Preparation method of freeze-thawing xenogenic laser microporous irradiated acellular dermal matrix and product thereof |
| CN102580141B (en) * | 2012-03-09 | 2013-10-09 | 潘银根 | Preparation method of acellular dermal matrix dressing |
-
2014
- 2014-11-20 CN CN201410669747.6A patent/CN104399130B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104399130A (en) | 2015-03-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2662331T3 (en) | Regenerative tissue matrices | |
| ES2886127T3 (en) | Cellulose nanofibrillar biotin for 3D bioprinting for cell culture, tissue engineering and regenerative medicine applications | |
| Halberstadt et al. | A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep | |
| Esposito et al. | PLDLA/PCL-T scaffold for meniscus tissue engineering | |
| Mellor et al. | Fabrication and evaluation of electrospun, 3D-bioplotted, and combination of electrospun/3D-bioplotted scaffolds for tissue engineering applications | |
| ES2672622T3 (en) | Preparation of regenerative tissue frames | |
| CN104399130B (en) | A kind of porous cell free tissue engineering bone/cartilage support and preparation method | |
| Lin et al. | In vitro and in vivo evaluation of the developed PLGA/HAp/Zein scaffolds for bone-cartilage interface regeneration | |
| JP2018527047A (en) | Preparation of cell-free cartilage graft and use thereof | |
| KR20180049712A (en) | Wet 3D cell printing using decellularized extracellular matrix | |
| CN105288737A (en) | Tissue engineering cartilage composite scaffold and preparation method thereof | |
| Zheng et al. | A rabbit model of osteochondral regeneration using three-dimensional printed polycaprolactone-hydroxyapatite scaffolds coated with umbilical cord blood mesenchymal stem cells and chondrocytes | |
| CN106492281B (en) | Biocompatible bone graft and preparation method thereof | |
| AU2017201708B2 (en) | Generation of cartilage ex vivo from fibroblasts | |
| JP2019518078A (en) | 3D printable biogel and method of use | |
| CN108888804B (en) | Soft tissue repair material and preparation method thereof | |
| CN111228574A (en) | Tissue engineering meniscus scaffold and preparation method thereof | |
| US20120022233A1 (en) | Collagen implant | |
| CN102178981A (en) | Method for preparing cartilage repairing scaffold material | |
| Gabler et al. | Biomechanical, biochemical, and cell biological evaluation of different collagen scaffolds for tendon augmentation | |
| Wasyłeczko et al. | Three-dimensional scaffolds for bioengineering of cartilage tissue | |
| Tran et al. | Decellularization of bone tissue | |
| CN104548203A (en) | Collagen-rich artificial nerve scaffold and preparation method thereof | |
| CN204601181U (en) | A kind of Meniscus scaffold | |
| Monzavi et al. | Extracellular matrix scaffold using decellularized cartilage for hyaline cartilage regeneration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |