CN110180025A - Cartilage-bone integrated porous bionic scaffold and preparation method thereof - Google Patents
Cartilage-bone integrated porous bionic scaffold and preparation method thereof Download PDFInfo
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- CN110180025A CN110180025A CN201910589504.4A CN201910589504A CN110180025A CN 110180025 A CN110180025 A CN 110180025A CN 201910589504 A CN201910589504 A CN 201910589504A CN 110180025 A CN110180025 A CN 110180025A
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- 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
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Abstract
The invention relates to a cartilage-bone integrated porous bionic scaffold and a preparation method thereof. The preparation method comprises the following steps: preparing cartilage acellular matrix powder, and preparing bone tissue decalcified bone matrix powder; preparing a gelatin or collagen solution; preparing a gelatin or collagen suspension containing a cartilage acellular matrix, and preparing a gelatin or collagen suspension containing a decalcified bone matrix; preparing a gelatin or collagen suspension containing a cartilage acellular matrix and a decalcified bone matrix; constructing a cartilage-bone integrated three-dimensional digital model through computer aided design, and performing 3D printing to form a cartilage-bone integrated core grid frame; fixing the cartilage-bone integrated core grid frame in a cylindrical mold, pouring the gelatin or collagen suspension containing the decalcified bone matrix to prepare a bone phase scaffold, pouring the gelatin or collagen suspension containing the cartilage acellular matrix and the decalcified bone matrix to prepare a boundary phase scaffold, and then pouring the gelatin or collagen suspension containing the cartilage acellular matrix to prepare a cartilage phase scaffold, thereby preparing the cartilage-bone integrated three-dimensional frozen scaffold.
Description
Technical field
The present invention relates to a kind of biomedical tissue engineering more particularly to a kind of soft bone-to-bone one with three-dimensional structure
Change porous biomimetic scaffolds and preparation method thereof.
Background technique
The joint part bone cartilage complex injury as caused by a variety of causes such as wound, disease, regression is that Orthopedic Clinical is common
Disease, and in China in the trend that rises year by year, the reparation of bone cartilage composite defects has important clinical demand.At present for this
The complex injury that articular cartilage merges subchondral bone treats difficulty, and effect is unsatisfactory, and it is ideal permanent to need research
Property reconstruction measure.Current clinically used treatment method includes bone and cartilage autotransplantation, allograph bone cartilage transplantation and people
Work joint replacement etc..Bone and cartilage autotransplantation art limited source, it is difficult to repair the defect of larger area；Allograph bone cartilage transplantation
There are immunological rejections and pathophorous possibility for art；And serious defect can only be treated using prosthetic replacement, but
There are the drawbacks such as somewhat expensive, abiology function, foreign material repulsion, abrasion loosening at a specified future date for currently used joint prosthesis.How
Realize that osteochondro tissue regeneration and the reconstruction of permanent joint physiological function are still the international headache for being difficult to capture.
The rapid development of tissue engineering technique, the Regeneration and Repair for joint bone cartilage complex injury provide new solution party
Case.Organizational engineering is to be related to a cross discipline of cell biology, material science, engineering science and bioreactor, benefit
With the basic principle and method of life science and engineering science, constructing needed by human body will be organized, for repairing, substituting because of wound, disease
Sick and non-functional tissue or organ.Once had and construct tissue engineered bone, cartilaginous tissue respectively using two kinds of different stent materials, so
Be assemblied together afterwards method building tissue engineered bone-cartilage complex, but the bone-cartilage complex constructed there are bone,
Cartilage portion combines not good enough problem.
Soft bone-to-bone complex tissue regeneration in situ is induced to be expected to become joint function restoration important means using biomimetic scaffolds,
But the current field there is no important breakthrough.Its key difficulties is how to complete soft bone-to-bone integrated bracket preparation and microenvironment
Human simulation realizes the building in biological joint.
There are many kinds of cartilages and bone holder material, preparation method, method also there are many kinds of.Wherein, natural material is excellent
Gesture is that good biocompatibility, degradable, catabolite easily absorb and toxic side effect is small, the disadvantage is that structure and performance are in the presence of poor
It is different.Artificial synthesized timbering material type is more, because having many advantages, such as that physical mechanical property is good, degradation is controllable, source is unrestricted
And by extensive concern, but polymeric biomaterial is easily inflamed, rejection there are certain immunogenicity, and hydrophily is poor,
Biocompatibility is not good enough；Metabolite after degradation has certain corrosivity etc..It is best to obtain according to bionics principle
Cartilage is repaired as a result, cartilage-bone tissue engineering scaffold similar as far as possible with cartilage and bone matrix composition will be selected.For this purpose, I
The compound transformation of ingredient is being carried out to cartilaginous tissue and bone tissue, preparation meets cartilage and the integrated bionic of bone matrix feature is more
Hole tissue engineering bracket material makes bracket adapt to cartilage and osteocyte biological requirement, to improve building organizational project integration
The quality of soft bone-to-bone.
Summary of the invention:
The purpose of the present invention is to provide a kind of to have three-dimensional structure based on cartilage acellular matrix and decalcified bone matrix
Porous biomimetic scaffolds of soft bone-to-bone integration and preparation method thereof.
Technical scheme is as follows:
A kind of preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration, includes the following steps:
(1) cartilaginous tissue is cut into small pieces, is pre-chilled in liquid nitrogen, is ground into carefully with cryogenic freezing beveller and historrhexis's machine
Graininess is prepared into cartilaginous tissue powder, and it is de- carefully that cartilage is made in the de- cell processing of cartilaginous tissue powder warp and vacuum freeze drying
Cytoplasmic matrix powder；
(2) bone tissue is cut into fritter, is pre-chilled in liquid nitrogen, is ground into carefully with cryogenic freezing beveller and historrhexis's machine
Graininess prepares osteogenic tissue's powder, then carries out decalcification, ungrease treatment, bone tissue decalcification bone is made through vacuum freeze drying
Matrix powder；
(3) gelatin or collagen for taking certain mass are dissolved in the gelatin or collagen that 1-5% (w/v) concentration is made into deionized water
Solution,
(4) the cartilage acellular matrix powder of certain mass is taken to be mixed into the gelatin or collagen solution prepared, 4 degree of low temperature
Pre-cooling stirs and evenly mixs, and is made into gelatin or collagen suspension that cartilage acellular matrix content accounts for 1-10% (w/v), is used to prepare soft
Bone photo bracket；
(5) the decalcified bone matrix powder of certain mass is taken to be mixed into the gelatin or collagen solution prepared, 4 degree of low temperature pre-coolings
It stirs and evenly mixs, is made into gelatin or collagen suspension that decalcified bone matrix content accounts for 1-10% (w/v), is used to prepare bone photo bracket；
(6) the cartilage acellular matrix powder of certain mass and decalcified bone matrix powder are mixed into the gelatin prepared simultaneously
Or in collagen solution, 4 degree of low temperature pre-coolings are stirred and evenly mixed, and are made into cartilage acellular matrix and decalcified bone matrix content respectively accounts for 1-10%
(w/v) gelatin of concentration or collagen suspension are used to prepare soft bone-to-bone boundary phase bracket；
(7) construct the soft integrated three-dimensional digital model of bone-to-bone through computer Computer Aided Design, divide cartilage phase, have a common boundary mutually and
Three layers of bone photo, cartilage phase is adjusted according to cartilage-bone structure and mechanical characteristics, have a common boundary phase and bone photo slice width, angle of deposit and aperture
Size；
(8) the soft bone-to-bone Monolithic Kernel grid frame of grade is prepared through 3D printing PCL substrate based on three-dimensional digital model
Frame；
(9) soft bone-to-bone integration grid framework is fixed in cylindrical mold, the base containing decalcification bone of certain volume is perfused
The gelatin or collagen suspension of matter prepare bone photo bracket, and freezing processing 6-12 hours, temperature was -20-0 degree, the certain body of Reperfu- sion
The gelatin or collagen suspension of long-pending acellular matrix containing cartilage and decalcified bone matrix prepare boundary phase bracket, freezing processing 6-
12 hours, temperature was -20-0 degree；Then, the gelatin or collagen suspension of the acellular matrix containing cartilage of certain volume is perfused, makes
Standby cartilage phase bracket, freezing processing 6-12 hours, temperature was -20-0 degree, and soft bone-to-bone integrated three-dimensional freezing bracket is made；
(10) soft bone-to-bone integrated three-dimensional freezing bracket is immersed in chemical cross-linking agent and is crosslinked after vacuum freeze drying,
Crosslinking completion deionized water repeated flushing is impregnated the remaining chemical cross-linking agent of removal, then is obtained finally through vacuum freeze drying
The porous biomimetic scaffolds of soft bone-to-bone integration.
Further, the cartilaginous tissue includes that the elasticity such as Ear cartilage, articular cartilage, costal cartilage, cartilago scapulae, meniscus is soft
Bone, hyaline cartilage and fibrocartilage tissue.
Further, the de- cell of the cartilaginous tissue powder is handled including at Hypotonic treatment, trypsin treatment, detergent
Reason and nucleic acid enzymatic treatment.
Further, the bone tissue includes cancellous bone and cortex bone.
Further, the bone tissue ungrease treatment includes alcohol, chloroform, methyl alcohol process.
Further, the decalcification processing of the bone tissue powder includes hydrochloric acid, EDTA processing.
Further, cartilage-inducing factor, blood vessel suppression are added in the gelatin or collagen suspension of the cartilage acellular matrix
The factor processed or drug.
Further, be added in the gelatin of the decalcified bone matrix or collagen suspension bone-inducing factor, promote vascularization because
Son or drug.
Further, the cross-linking reagent are as follows: 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, Huo Zhewu
Dialdehyde or Geniposide.
The porous biomimetic scaffolds of a kind of soft bone-to-bone integration, including the preparation side by the porous biomimetic scaffolds of soft bone-to-bone integration
The porous biomimetic scaffolds of soft bone-to-bone integration that method is prepared into.
Beneficial effects of the present invention:
The invention proposes the preparations of a kind of preparation method of cartilage acellular matrix powder and decalcified bone matrix powder
Method, and disclose a kind of soft bone-to-bone integration with three-dimensional structure based on cartilage acellular matrix and decalcified bone matrix
Porous biomimetic scaffolds and preparation method thereof, its advantages include:
(1) cartilaginous tissue and bone tissue are fine and close, and the de- cell of row handles, is de- again after cartilaginous tissue and bone tissue are thoroughly crushed
Rouge and decalcification processing, can be detached from completely, and then thoroughly eliminate immunogenicity.
(2) cartilage acellular matrix and decalcified bone matrix are as natural degradable material, good biocompatibility, immunogenicity
It is low, it is often more important that, the cartilage matrix components and bone matrix composition contained can provide regenerating bone or cartilage and osteanagenesis respectively
Microenvironment promotes Subchondral drilling and bon e formation, mitigates internal inflammatory reaction.
(3) simple cartilage acellular matrix and decalcified bone matrix difficult forming, compound a certain proportion of gelatin or collagen are made
For adjuvant, cross-linking properties is effectively improved, can be made into the three-dimensional rack for having certain shapes.
(4) aperture of bracket and porosity and Material cladding ratio and freeze-drying parameter it is closely related, scaffold degradation rate with
It is crosslinked relating to parameters, systematically controlled material compositely proportional, concentration, freeze-drying and crosslinking parameter accurately can control aperture hole
Rate and degradation rate.
(5) series techniques such as 3D printing integration grid framework, moulding by casting, freeze-drying are integrated and are prepared into accurate one
Change porous biomimetic scaffolds, has not only solved the problems, such as that natural material is unable to 3D printing and accurately controls form, but also keep to a greater extent
Its biological activity and cartilage and bone matrix microenvironment, while being more suitable for adding in moulding by casting and freezing dry process each
Class bioactie agent provides possibility further to promote the adjusting function of compound rest.
(6) high-intensitive, the slow degradation kernel theory applied, by the integrated polycaprolactone of 3D printing
(polycaprolactone, PCL) net grid support is fixed in casting mold as kernel, has been obviously improved integrated three-dimensional
The mechanical property of bracket.
(7) it is based on joint anatomy and microenvironment feature, passes through the accurate control biomimetics scaffold three-dimensional shape of 3D printing technique
State and lamination gradient porous structure are soft in biomimetic scaffolds respectively in conjunction with growth factor slow-releases technologies such as freeze-drying, microballoon packages
Bone area, soft bone-to-bone junctional area, zonc of ossification locally integrate cartilage/bone specific matrix ingredient or add simultaneously activity specific because
Son, preparation have gradient aperture, region particular substrate ingredient or the soft bone-to-bone one simultaneously with specific activity factor slow release function
Body biomimetic scaffolds and biological joint tissue regeneration support.
Detailed description of the invention
Fig. 1 is the SEM picture of cartilage phase bracket；
Fig. 2 is the SEM picture of boundary phase bracket；
Fig. 3 is the SEM picture of bone photo bracket.
Specific embodiment
The invention will be further described with embodiment with reference to the accompanying drawing.
Embodiment 1:
A kind of preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration, includes the following steps:
(1) fresh ox articular cartilage tissue is taken, excess surface tissue and fascia are rejected, obtains hyaline cartilage piece.By sample
Immerse 5min in liquid nitrogen, cartilage powder is made in chilled beveller, then carry out de- cell processing: 1. 0.5% trypsase/
37 DEG C of isothermal vibrations of PBS solution (w/v) for 24 hours, the trypsin solution that every 4h more renews；2. (the 50U/ml deoxidation of nucleic acid enzyme solutions
Ribalgilase, 1U/ml ribonuclease A, are dissolved in 10mM Tris-HCL, PH=7.5) 37 DEG C of stirring 4h；③10mM
37 DEG C of concussion 20h of Tris-HCL (Aprotinin containing 10U/ml)；4. 37 DEG C of 1%Triton X-100/PBS solution (v/v) concussions
24h；5. PBS solution washs 6 circulations, each 2h.It is finally freeze-dried that acellular matrix powder is made.
(2) fresh bulls bone tissue is taken, excess surface tissue, fascia, cortex bone and inherent marrow is rejected, obtains spongiosa
Bone tissue block.Sample is immersed into 5min in liquid nitrogen, spongiosa bone meal is made in chilled beveller, then carries out decalcification processing, degreasing
Processing and the processing of de- cell: 1. 70% alcohol is repeatedly impregnated, is rinsed；2. 0.5N HCL desalination, the every 2h replacement of HCL, totally 3 times；③
After deionized water is rinsed 3 times, it is placed in the solution containing 0.05% pancreatin and 0.02%EDTA, 37 degree of isothermal vibration 2h；4. go from
Sub- water rinses 3 times, -85 degree freezing 2h, and vacuum freeze drying is for 24 hours, final to obtain decalcified bone matrix powder.
(3) gelatin particle of certain mass is taken, isothermal vibration 1h dissolves in 37 DEG C of deionized waters, and being configured to concentration is 1%
(w/v) gelatin solution.The cartilage acellular matrix powder of certain mass is taken, it is 1% (w/v's) that deionized water, which is configured to concentration,
Suspension, taking gelatin solution and cartilage acellular matrix suspension, (1:1) concussion mixes by volume, 4 degree of low temperature pre-cooling stirrings 6
Hour mixes well, and certain density cartilage acellular matrix/gelatin suspension is made into, for constructing cartilage phase.
(4) the decalcified bone matrix powder of certain mass is taken, deionized water is configured to the suspension that concentration is 1% (w/v), takes
(1:1) concussion mixes by volume for gelatin solution and decalcified bone matrix suspension, and 4 degree of low temperature pre-cooling stirrings mix well for 6 hours,
It is made into certain density decalcified bone matrix/gelatin suspension, for constructing bone photo.
(5) the cartilage acellular matrix powder and decalcified bone matrix powder of certain mass are taken, by volume 1:1 deionized water
Be configured to the suspension of final concentration of 1% (w/v), take gelatin solution and cartilage acellular matrix and decalcified bone matrix suspension by
Volume ratio (1:1) concussion mixes, and 4 degree of low temperature pre-cooling stirrings mix well for 6 hours, is made into certain density cartilage acellular matrix
And decalcified bone matrix/gelatin suspension, for constructing boundary phase.
(6) construct the soft integrated three-dimensional digital model of bone-to-bone through computer Computer Aided Design, divide cartilage phase, have a common boundary mutually and
Three layers of bone photo, cartilage phase is adjusted according to cartilage-bone structure and mechanical characteristics, have a common boundary phase and bone photo slice width, angle of deposit and aperture
The parameters such as size.It is printed based on three-dimensional digital model through 3D printer (II FreeForm Fabrication System of MAM-)
PCL substrate prepares the soft bone-to-bone Monolithic Kernel grid framework of grade.By adjusting different slice widths, different angle of deposit (-
45 °/45 °, -60 °/60 ° or 0 °/60 °/120 °) and different-diameter stainless steel syringe needle (19G, 20G or 21G), setting is suitable
Material extrusion speed, print speed and the high print parameters of layer prepare the soft bone-to-bone integration net of different pore size size and internal structure
Case frame.
(7) soft bone-to-bone integration grid framework is fixed in special cylindrical mold, perfusion certain volume (1ml)
Gelatin or collagen suspension containing decalcified bone matrix, prepare bone photo bracket (see Fig. 3).- 10 degree are after freezing processing 12 hours, perfusion
The acellular matrix containing cartilage of certain volume (0.5ml) and the gelatin of decalcified bone matrix or collagen suspension prepare boundary phase branch
Frame (see Fig. 2).The gelatin or glue of the acellular matrix containing cartilage of certain volume (1ml) is perfused after freezing processing 12 hours in -10 degree
Former suspension prepares cartilage phase bracket (see Fig. 1).- 10 degree freezing processing 12 hours obtain soft bone-to-bone integrated three-dimensional freezing
Bracket.
(8) bracket is put into 95% wine containing 0.5%EDC after vacuum freeze drying 48 hours by three-dimensional freezing bracket
Full cross-linked 12 hours in smart solution.Deionized water soaking flushing three-dimensional rack 6 hours repeatedly remove the chemistry in three-dimensional rack
Crosslinking agent, then through vacuum freeze drying 24 hours, obtain the porous biomimetic scaffolds of soft bone-to-bone integration.
The foregoing is merely preferred embodiments of the invention, but scope of protection of the present invention is not limited thereto, for
This field is familiar with for technical staff, and the present invention can have various change and variation, should all cover in guarantor of the invention
Within the scope of shield.Therefore, protection scope of the present invention should be subject to the protection scope in claims.
Claims (10)
1. a kind of preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration, which comprises the steps of:
(1) cartilaginous tissue is cut into small pieces, is pre-chilled in liquid nitrogen, be ground into fine grained with cryogenic freezing beveller and historrhexis's machine
Shape, is prepared into cartilaginous tissue powder, and cartilaginous tissue powder is made cartilage through de- cell processing and vacuum freeze drying and takes off cell base
Matter powder；
(2) bone tissue is cut into fritter, is pre-chilled in liquid nitrogen, be ground into fine grained with cryogenic freezing beveller and historrhexis's machine
Shape prepares osteogenic tissue's powder, then carries out decalcification, ungrease treatment, bone tissue decalcified bone matrix is made through vacuum freeze drying
Powder；
(3) take that the gelatin of certain mass or collagen are dissolved in the gelatin that 1-5% (w/v) concentration is made into deionized water or collagen is molten
Liquid；
(4) the cartilage acellular matrix powder of certain mass is taken to be mixed into the gelatin or collagen solution prepared, 4 degree of low temperature pre-coolings
It stirs and evenly mixs, is made into gelatin or collagen suspension that cartilage acellular matrix content accounts for 1-10% (w/v), is used to prepare cartilage phase
Bracket；
(5) the decalcified bone matrix powder of certain mass is taken to be mixed into the gelatin or collagen solution prepared, 4 degree of low temperature pre-cooling stirrings
It mixes, is made into gelatin or collagen suspension that decalcified bone matrix content accounts for 1-10% (w/v), is used to prepare bone photo bracket；
(6) the cartilage acellular matrix powder of certain mass and decalcified bone matrix powder are mixed into the gelatin or glue prepared simultaneously
In original solution, 4 degree of low temperature pre-coolings are stirred and evenly mixed, and are made into cartilage acellular matrix and decalcified bone matrix content respectively accounts for 1-10% (w/
V) gelatin of concentration or collagen suspension are used to prepare soft bone-to-bone boundary phase bracket；
(7) the soft integrated three-dimensional digital model of bone-to-bone is constructed through computer Computer Aided Design, divides cartilage phase, have a common boundary phase and bone photo
Three layers, cartilage phase is adjusted according to cartilage-bone structure and mechanical characteristics, have a common boundary phase and bone photo slice width, angle of deposit and pore size；
(8) the soft bone-to-bone Monolithic Kernel grid framework of grade is prepared through 3D printing PCL substrate based on three-dimensional digital model；
(9) soft bone-to-bone integration grid framework is fixed in cylindrical mold, be perfused certain volume containing decalcified bone matrix
Gelatin or collagen suspension prepare bone photo bracket, and freezing processing 6-12 hours, temperature was -20-0 degree, Reperfu- sion certain volume
The gelatin or collagen suspension of acellular matrix containing cartilage and decalcified bone matrix, prepare boundary phase bracket, and freezing processing 6-12 is small
When, temperature is -20-0 degree；Finally, the gelatin or collagen suspension of the acellular matrix containing cartilage of perfusion certain volume, prepare soft
Bone photo bracket, freezing processing 6-12 hours, temperature was -20-0 degree, and soft bone-to-bone integrated three-dimensional freezing bracket is made；
(10) soft bone-to-bone integrated three-dimensional freezing bracket is immersed in chemical cross-linking agent and is crosslinked, is crosslinked after vacuum freeze drying
It completes deionized water repeated flushing, impregnate the remaining chemical cross-linking agent of removal, then obtain final cartilage-through vacuum freeze drying
The porous biomimetic scaffolds of bone integration.
2. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
Cartilaginous tissue includes that the elastic cartilages such as Ear cartilage, articular cartilage, costal cartilage, cartilago scapulae, meniscus, hyaline cartilage and fiber are soft
Bone tissue.
3. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
The de- cell processing of cartilaginous tissue powder includes Hypotonic treatment, trypsin treatment, detergent-treatment and nucleic acid enzymatic treatment.
4. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
Bone tissue includes cancellous bone and cortex bone.
5. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
The ungrease treatment of bone tissue powder includes through alcohol, chloroform, methyl alcohol process.
6. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
The decalcification processing of bone tissue powder includes through hydrochloric acid, EDTA processing.
7. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
Cartilage-inducing factor, Anti-angiogenesis factors or drug are added in the gelatin or collagen suspension of cartilage acellular matrix.
8. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
Bone-inducing factor is added in the gelatin or collagen suspension of decalcified bone matrix, promotees angiogenesis factor or drug.
9. the preparation method of the porous biomimetic scaffolds of soft bone-to-bone integration according to claim 1, it is characterised in that: described
Cross-linking reagent are as follows: 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride perhaps glutaraldehyde or Geniposide.
10. the porous biomimetic scaffolds of a kind of soft bone-to-bone integration, including by any soft bone-to-bone integration of claim 1-9
The porous biomimetic scaffolds of soft bone-to-bone integration that the preparation method of porous biomimetic scaffolds is prepared into.
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