CN106215238A - A kind of three-dimensional bone tissue engineering scaffold based on decalcification process and preparation method thereof - Google Patents
A kind of three-dimensional bone tissue engineering scaffold based on decalcification process and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2430/00—Materials or treatment for tissue regeneration
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention discloses a kind of three-dimensional bone tissue engineering scaffold based on decalcification process and preparation method thereof.First make Acellular bone, then use osseous tissue decalcifying solution that Acellular bone support carries out decalcification in various degree and process, obtain the three-dimensional bone tissue engineering stent material with different-stiffness.This invention can prepare the three-dimensional stent material with different-stiffness in the case of not changing three-dimensional rack micro structure, and this support has good biocompatibility, and the most different types of stem cell adheres to, breeds and differentiation, and cost of material is relatively low, it is easy to make.
Description
Technical field
The present invention relates to a kind of bone tissue engineering scaffold and preparation method thereof, a kind of have the de-thin of different-stiffness
Born of the same parents' decalcification three-dimensional bone support and preparation method thereof.
Background technology
The many factors such as the constituent of extracellular matrix, mechanical characteristics, porosity, aperture value are under the conditions of physiological and pathological
All it is proved the destiny of scalable cell.Wherein, simple substrate rigidity has been proved to induce people under two-dimensional condition
Mescenchymal stem cell be divided into neurocyte (0.1-1kPa), sarcoplast (8-17kPa) and osteoblast (25-respectively
40kPa)(Engler AJ,Sen S,Sweeney HL,Discher DE.Matrix elasticity directs stem
cell lineage specification.Cell.2006；126:677-689.).But, under three-dimensional condition, preparation has not
Research with the timbering material of substrate rigidity is the most fewer, is concentrated mainly under quasi-three-dimensional condition, and very difficult analog cell is in vivo
Residing true mechanics microenvironment.Such as, by changing the density of matrix of diverse location, the difference of regulation polymer cross-links close
Degree, change the various ways such as ratio of two or more mixed-matrix and change the mechanical characteristics of three-dimensional stent material.But, these
Method inevitably changes basic structure (the such as support shape of three-dimensional rack while changing support mechanical characteristics
State, internal connection, porosity and pore size etc.).Such as, utilize flat board vertical compression wedge shape collagen to be formed and there is rigidity gradient
Biomaterial (soft: 1057 ± 487kPa；In: 1835 ± 31kPa；Hard: 2305 ± 693kPa) (Hadjipanayi E,
Mudera V,Brown RA.Brown.Guiding cell migration in 3D:a collagen matrix with
graded directional stiffness.Cell Motil Cytoskeleton.2009；66:121-128.) it is a kind of
The effective ways of preparation substrate rigidity gradient material, but same along with substrate stiffness variation of the interior three-dimensional structure of collagen scaffold
Time also there occurs significant change.Recently, the computer MSR Information system of Chen leader utilizes differing heights (0.97 μm, 6.10 μm and 12.9 μ
The hardness (coefficient of elasticity: 1556nN/ μm, 18.16nN/ μm and 1.90nN/ μm) of elastic microtrabeculae regulation substrate m), finds this
The substrate of different hardness can affect the form of cell, adhesion and cytoskeleton and shrink, the mescenchymal stem cell of controllable people
Differentiation direction, high rigidity substrate be more conducive to promote mescenchymal stem cell Osteoblast Differentiation, soft substrate then promotes mesenchyme
One-tenth Adipose Differentiation (Fu J, Wang YK, Yang MT, Desai RA, Yu X, the Liu Z, Chen of stem cell
CS.Mechanical regulation of cell function with geometrically modulated
elastomeric substrates.Nat Methods.2010；7:733-736.).(Huebsch N, the Arany such as Huebsch
PR,Mao AS,Shvartsman D,Ali OA,Bencherif SA,Rivera-Feliciano J,Mooney
DJ.Harnessing traction-mediated manipulation of the cell/matrix interface to
control stem-cell fate.Nat Mater.2010；9:518-526.) be prepared for one there is different substrates rigidity
The synthetic water gel extracellular matrix of (2.5～110kPa), and detect its impact on Derived from Mesenchymal Stem Cells pedigree.They
Find that mescenchymal stem cell primary differentiation on the support of 11～30kPa is osteoblast.Early stage, we utilize different ratio
Collagen/hydroxyapatite is coated process acellular bone, defines a kind of three-dimensional bone tissue engineer with different substrates rigidity
Timbering material (Lv Yonggang, Chen Guobao, Zou Yang. one has different-stiffness three-dimensional bone tissue engineering stent material and preparation side thereof
Method. Chinese invention patent: ZL 201410153286.7.).But, prepare that micro structure is constant and rigidity is different at bone tissue engineer
Three-dimensional bone tissue engineering scaffold the most extremely difficult.
Summary of the invention
The technical problem to be solved in the present invention is the bone tissue engineering scaffold building under three-dimensional condition and having different-stiffness
Material, it is therefore an objective to make support have different-stiffness in the case of not changing three-dimensional rack micro structure.
For solving above-mentioned technical problem, the three-dimensional bone tissue engineering scaffold based on decalcification process of the present invention is by de-cell
Bone processes through decalcification in various degree and obtains；It is characterized in that, utilize Acellular bone to the micro-knot of three-dimensional keeping timbering material
Structure is consistent, is processed by decalcification in various degree and makes Acellular bone support have different rigidity, is beneficial to induce the one-tenth of stem cell
Bone breaks up.
In the present invention, Acellular bone can use the method for routine to prepare, and is to take multiple fresh pig capital in brief
Spongy bone, delivers under clean conditions with after 0.9% saline soak of 4 DEG C, removes the remaining muscle on sample and tissue, according to facing
Bed Cranial defect situation make a diameter of 2～20mm, a height of 2～the cylinder of 20mm or length × width × height be 2～20mm × 2～
The shaped samples such as the cube of 20mm × 2～20mm, rinse, are then immersed in 4 DEG C of distilled water overnight.With the Triton of 1%
X-100 processes sample 48h.With methanol, sample is carried out defat 24h.After 37 DEG C of incubated samples 2h of DNA enzymatic/RNase, use PBS
Clean and persistently rock.Support is immersed 4h in dehydrated alcohol, to remove the cell rests thing on support.Use substantial amounts of deionization
Water cleans support 2h, drying bracket, it is thus achieved that Acellular bone, is saved under 4 DEG C of environment.
Acellular bone has three-dimensional micropore structure, and bore dia can be controlled in 300～800 μ m, and porosity can reach
To 70%～95%, and having good connectivity, range in stiffness is generally 1-10Gpa.Acellular bone is carried out in various degree
Decalcification process after can obtain the three-dimensional bone tissue engineering stent material with different-stiffness.
Present invention also offers and a kind of prepare the described method with different-stiffness three-dimensional bone tissue engineering stent material,
Specifically comprise the following steps that
1) multiple Acellular bone three-dimensional rack is obtained；
2) by step 1) multiple timbering materials of obtaining immerse and process different time in osseous tissue decalcifying solution；
3) by step 2) the Acellular bone support of different calcium depletion that obtains is through deionized water cleaning treatment；37 DEG C of bakings
Dry；
4) by step 3) three-dimensional stent material with different-stiffness that obtains passes through60After Co (25k Gay) radiosterilization,
Kept dry after sterile sealing.
Preferably, step 1) described in the preparation method of Acellular bone three-dimensional rack be: take the capital spongiosa of fresh pig
Bone, delivers under clean conditions with after 0.9% saline soak of 4 DEG C, removes the remaining muscle on sample and tissue, according to clinical bone
Defect situation makes required form sample, rinses, is then immersed in 4 DEG C of distilled water overnight.At the Triton X-100 of 1%
Reason sample 48h.With methanol, sample is carried out defat 24h.After 37 DEG C of incubated samples 2h of DNA enzymatic/RNase, PBS also continues
Rock.Support is immersed 4h in dehydrated alcohol, to remove the cell rests thing on support.Support is cleaned with substantial amounts of deionized water
2h, drying bracket, it is thus achieved that Acellular bone, it is saved under 4 DEG C of environment.
Preferably, step 1) described in Acellular bone three-dimensional rack be prepared as a diameter of 2～20mm, a height of 2～20mm
Cylinder or length × width × height are 2～the shaped samples such as the cube of 20mm × 2～20mm × 2～20mm.
Preferably, step 2) described in decalcification process the time can be chosen as 1 day～14 days according to the learned rigidity value of support.
Preferably, step 2) described in decalcification process optional constant-temperature table and process or microwave treatment mode.
In sum, the three-dimensional bone tissue engineering stent material with different-stiffness of the present invention passes through Acellular bone support
Ensure that the three-dimensional microstructures of material is consistent, utilize the different calcium depletion of Acellular bone to make Acellular bone support have difference
Rigidity, formed and there is the three-dimensional stent material of different-stiffness.Compared with existing three-dimensional bone tissue engineering stent material, overcome
Most of bone tissue engineering stent materials have also been changed the defect of micro structure while changing substrate rigidity, and this is also this
The key of bright proposition.The three-dimensional bone tissue engineering stent material that the present invention provides has good biocompatibility, it is ensured that bone
The native micro-structures of support and composition, and there is the adhesion of good connectivity, beneficially cell, grow and breed, it is beneficial to battalion
Support the entrance of material and the timely discharge of cellular metabolism refuse.Utilize the different stiffness of support that different calcium depletion is formed,
It is beneficial to the adhesion of different dry cell, breeds and differentiation, the generation of new bone can be promoted.For preparing three-dimensional bone tissue engineering scaffold
The Acellular bone of material may select the bone of the different piece of different animals as material, as long as meeting the requirement of bone to be repaired
?.For brevity, the present invention is mainly illustrated by as a example by the capital spongy bone of pig, and the bone of other animals and part is also
Identical principle can be used.
Accompanying drawing explanation
Fig. 1 is the compression of the three-dimensional bone tissue engineering scaffold obtained after Acellular bone carries out the process of decalcification in various degree
Young's modulus；
Fig. 2 is Acellular bone aperture value before and after complete decalcification and porosity.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
Take the capital spongy bone of multiple fresh pig, deliver under clean conditions with after 0.9% saline soak of 4 DEG C, remove
Remaining muscle on sample and tissue, make a diameter of 2～20mm, a height of 2～the cylinder of 20mm according to clinical Cranial defect situation
Body or length × width × height are 2～the shaped samples such as the cube of 20mm × 2～20mm × 2～20mm, rinse, are then immersed in
In 4 DEG C of distilled water, 4 DEG C overnight.Triton X-100 with 1% processes sample 48h.With methanol, sample is carried out defat 24h.With
After 37 DEG C of incubated samples 2h of DNA enzymatic/RNase, PBS also persistently rocks.Support is immersed 4h in dehydrated alcohol, props up to remove
Cell rests thing on frame.Clean support 2h, drying bracket, it is thus achieved that Acellular bone with substantial amounts of deionized water, be saved in 4 DEG C of rings
Under border.Acellular bone has three-dimensional micropore structure, and bore dia can be controlled in 300～800 μ m, and porosity can reach
70%～95%, and there is good connectivity, range in stiffness is generally 1-10Gpa.
The multiple Acellular bone timbering materials prepared are immersed the disodiumedetate decalcifying solution of 12%,
Jiggle different duration with constant-temperature table at 37 DEG C, during processing, change a decalcifying Fluid every 12h.The process time can root
Adjusting according to the final rigidity value of the support needing preparation, scope is typically between 1 day～14 days.Such as need to prepare rigidity and be
The three-dimensional rack of about 10Mpa, optional decalcification processes 14 days to decalcification terminal.The de-cell of different calcium depletion that will obtain
Bone support is after deionized water cleaning treatment, and 37 DEG C of drying, the three-dimensional stent material with different-stiffness obtained passes through60Co
After (25k Gay) radiosterilization, kept dry after sterile sealing.
The decalcification of Acellular bone processes and also may select microwave treatment mode.Acellular bone support is immersed the second equipped with 12%
In the small beaker of edetate disodium decalcifying solution, insert in the beaker added with 200mL frozen water, be then placed in microwave oven,
Making decalcifying Fluid temperature control within 37 DEG C, change the frozen water in a large beaker at interval of 2min, every 1h changes a decalcification
Liquid.
By the feasibility of the method that checking is used, it is prepared for the Acellular bone support of different calcium depletion, then utilizes
Electronic universal material sound state experimental system (Electropuls E1000) test instrunment of American I nstron company is measured and is propped up
Frame young's modulus in compression.As it is shown in figure 1, along with the increase of decalcification rate, the elastic modelling quantity of support is gradually lowered.As in figure 2 it is shown, with
Completely as a example by the Acellular bone support of decalcification, micro-CT experiment confirms the aperture value before and after Acellular bone support decalcification and hole
Rate does not the most occur significantly to change.
Processed, in the situation not changing support micro structure by the decalcification multiple Acellular bone supports carried out in various degree
The three-dimensional porous rack material of different-stiffness can be formed down.Just because of these composite factors so that the three-dimensional that the present invention provides
Bone tissue engineering stent material has the adhesion of good biocompatibility, beneficially different dry cell, breeds and differentiation, can promote
Enter the generation of new bone.Preparation method is simple and low cost, has significantly than conventional three-dimensional bone tissue engineering stent material
Advantage.
Claims (5)
1. the three-dimensional bone tissue engineering scaffold processed based on decalcification, it is processed through decalcification in various degree by Acellular bone
Obtain, it is characterised in that: described Acellular bone can be made a diameter of 2 according to clinical Cranial defect situation by the capital spongy bone of pig
～20mm, a height of 2～the cylinder of 20mm or length × width × height are 2～20mm × 2～20mm × 2～the cube of 20mm,
Bore dia is 300～800 μm, and porosity is 70%～95%, utilizes osseous tissue decalcifying solution to carry out described Acellular bone not
Process with the decalcification of degree.
2. prepare the method for three-dimensional bone tissue engineering scaffold processed based on decalcification described in claim 1, including following
Step:
1) multiple Acellular bone three-dimensional rack is prepared；
2) by step 1) multiple timbering materials of obtaining immerse and process different time in osseous tissue decalcifying solution；
3) by step 2) the Acellular bone support of different calcium depletion that obtains is through deionized water cleaning treatment, 37 DEG C of drying；
4) by step 3) three-dimensional stent material with different-stiffness that obtains passes through60Co radiosterilization, after its sterile sealing
Kept dry.
Preparation method the most according to claim 2, it is characterised in that: step 2) described in decalcification in various degree process side
Method is, is immersed by Acellular bone support in the disodiumedetate decalcifying solution of 12%, shakes gently at 37 DEG C of constant-temperature tables
The different duration of rolling, changes a decalcifying Fluid every 12h during process.
Preparation method the most according to claim 2, it is characterised in that: step 2) described in decalcification in various degree process side
Method is, immerses Acellular bone support equipped with in the small beaker of the disodiumedetate decalcifying solution of 12%, insert added with
In the beaker of 200mL frozen water, it is then placed in microwave oven, makes decalcifying Fluid temperature control within 37 DEG C, change at interval of 2min
Frozen water in large beaker, every 1h changes a decalcifying Fluid.
Preparation method the most according to claim 2, it is characterised in that: step 2) described in decalcification process the time can according to
Needed for frame, rigidity value is chosen as 1 day～14 days.
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| JP2021523749A (en) * | 2019-01-17 | 2021-09-09 | 浙江大学医学院附属邵逸夫医院Ｓｉｒ Ｒｕｎ Ｒｕｎ Ｓｈａｗ Ｈｏｓｏｉｔａｌ Ｚｈｅｊｉａｎｇ Ｕｎｉｖｅｒｓｉｔｙ Ｓｃｈｏｏｌ ｏｆ Ｍｅｄｉｃｉｎｅ | Method for preparing extracellular matrix material for gradient mineralized bone |
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| US11684696B2 (en) * | 2019-01-17 | 2023-06-27 | Sir Run Run Shaw Hospital Zhejiang University School Of Medicine | Preparation method of gradient mineralized cancellous bone matrix material |
| CN110639061A (en) * | 2019-08-27 | 2020-01-03 | 中南大学湘雅医院 | Double bionic bone tendon interface page support |
| CN111494719A (en) * | 2019-12-31 | 2020-08-07 | 中南大学湘雅医院 | Novel bone tissue engineering scaffold and preparation method thereof |
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| CN116328039A (en) * | 2023-02-24 | 2023-06-27 | 浙江狄赛生物科技有限公司 | Natural bone repair material with specific mineralization degree and capable of regulating inflammatory metabolism, and preparation method and application thereof |
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