CN105013016B - A kind of Cranial defect Regeneration and Repair tissue engineered bone and its construction method and application - Google Patents
A kind of Cranial defect Regeneration and Repair tissue engineered bone and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of Cranial defect Regeneration and Repair tissue engineered bone and its construction method and application, composite layered structure that tissue engineered bone is made up of three layers of mesenchymal stem cells MSCs condensate and two layers of bone matrix particle；The complex that composite layered structure is made up of mesenchymal stem cells MSCs condensate bone matrix particle mesenchymal stem cells MSCs condensate bone matrix particle mesenchymal stem cells MSCs condensate；Mesenchymal stem cells MSCs condensate include mesenchymal stem cells MSCs and by mesenchymal stem cells MSCs secretion and be distributed in the extracellular matrix around mesenchymal stem cells MSCs.The tissue engineered bone while compound living cells and active factors formed using construction method of the present invention, reconstruction process is quick, area of new bone is combined complete, consistency of thickness with periphery autologous bone, suitable for Cranial defect Regeneration and Repair, realize the physiological regeneration of bone and improve the repairing and treating effect of Cranial defect.
Description
Technical field
The invention belongs to organizational project and regenerative medicine medical biomaterial technical field, and in particular to one kind has physiology
Property regeneration biological active mass Engineering Bone and its construction method.The tissue engineered bone can be used for the Regeneration and Repair of Cranial defect.
Background technology
In current orthopaedics therapy field, to various wounds, infection, tumor resection and some congenital developmental deformities
The bone defects of limbs Deng caused by, braincap Cranial defect are still a great problem that Orthopedic Clinical is most common, most intractable.Cranial defect is controlled
Treatment method mainly includes bone collection, bone lengthening, tissue engineering technique etc..
The structure of tissue engineered bone is more and more developed, and its advantage also increasingly highlights, but different construction strategies
Effect exist very big difference.Wherein, the bone grafting material built by tissue engineering technique generally comprises one or more
Functional component, that is, include the host material with osteoacusis, and host material provides support for new bone growth；With osteoinductive
Biomolecule or chemicals form the microenvironment with regeneration potential, and seed cell is divided into induction structure and repair process
Gegenbaur's cell；Suitable seed cell is screened, quickly generates new bone tissue under appropriate circumstances.
（1）Bone grafting matrix material
The bone holder material of natural bone material working process mainly includes：1. autologous bone, its major advantage is to have bone concurrently
Conductibility, osteoinductive and osteogenic action；Shortcoming be mainly reflected in Qu Gu areas complication (Qu Gu areas pain and other potentially cut
Mouthfeel dye, hemotoncus, fracture etc.), take that bone amount is limited, amount of bleeding increase and operating time extend etc..2. allograph bone, allograph bone
Graft can provide osteoconductive scaffold, and retain most of osteoinductive protein composition, have osteoacusis and self-bone grafting ability concurrently, but exempt from
Epidemic disease rejection can make the cell of donor source in bone graft be killed, therefore lack osteogenic ability.In addition, allogenic bone transplantation thing should
Security receives much concern always, and transmission is still one of greateset risk of allogenic bone transplantation thing application.3. remove mineral matter
Bone matrix (demineralized bone matrix, DBM), also known as decalcified bone matrix, are by allosome or autologous bone tissue
The bone grafting material obtained after different demineralization thing process processing, can not only filling bone defects, also have potential osteoacusis
And bone inductive effect.DBM can promote revascularization, and induce host stem cells to be divided into Gegenbaur's cell.It is assumed that DBM life
Thing activity is relevant with stromatin remaining after the processing of demineralization thing and growth factor.
（2）Osteanagenesis microenvironment
Regeneration microenvironment is mainly what is formed by growth factor, is played a significant role during osteanagenesis.Growth factor
It is to be produced by specific cells, some polypeptides played effectiveness in different physiology courses, it is played during bone tissue reparation
Important regulating and controlling acts on.Mainly regulation cartilage matrix closes for IGF (IGF-1) and TGF (TGF- Β)
Into.Basic fibroblast growth factor (bFGF) is then strength mitogenic factor, can promote Chondrocyte Differentiation.Blood platelet
Derivative growth factor (PDGF) and VEGF（VEGF）Show the effect of promotion union.People's Bones morphology is sent out
Raw albumen (rhBMP-2) and tricalcium phosphate the compound more simple tricalcium phosphate in bone defect healing are preferred.
In order to obtain promising result, above-mentioned biomolecule needs carrier to deliver to required position and discharged at the appropriate time, also
Some confactors are needed farthest to play effectiveness.These requirements greatly limit the clinic of growth factor
Use, while also need more researchs to have shown that the clinical efficacy of growth factor, reliability and safety.
（3）Seed cell
Suitable seed cell is the important foundation of physiological osteanagenesis.Stem cell is careful as the kind that organizational project is built
Born of the same parents have multi-lineage potential, updating ability it is fast, almost non-immunogenicity many advantages, such as, played in bone tissue engineer important
Effect.For organizational project bone collection, best selection is to be divided into the mescenchymal stem cell of the tissues such as bone and cartilage.
Preferable Bone Defect Repari regeneration graft materials support should possess good biocompatibility, Integrated implant ability, can shape
Into osteanagenesis microenvironment to promote endogenous and external source seed cell osteanagenesis, can be perfectly combined after reparation with host bone.Further,
Preferable Bone Defect Repari regeneration graft materials support also needs to meet that physiologic function and mechanics function reach nature level, realizes
The requirement such as physiological regeneration, cheap, easy to manufacture.Research of the people to bone grafting material concentrates on the following aspects：①
Improve the biomechanical property and biocompatibility of host material, bone grafting material is possessed the drop to match with bon e formation speed
Solve speed；2. strengthening the affinity of host material and bioactive substance, and it is set to discharge process control；3. bioactive substance
Selection and acquisition, and optimal collocation between each material etc..In a word, bioactive substance is combined with preferable host material
Composite bone graft material is built, optimizing and degrade and regenerate synchronize etc. in bionic structure, Integrated implant ability obtains
More much progress, it is the developing direction of following bone grafting material.
Existing Bone Defect Repari graft materials and its application on bone defect healing, belong to non-physiologic regeneration.Its entirety
Show material can not or should not degrade upon, poor biocompatibility, it is almost poor without ductility, the capacity of heat transmission after repairing to cause, and repaiies
After multiple with original synosteosis is imperfect, shape, size, thickness with many defects such as autologous bone is variant.From osteanagenesis physiology
From the point of view of process, using simple bone material, material and the factor is compound, Material cladding cell technology forms the ability of bone and to bone
Defect repair effect is also undesirable.Therefore, the structure of tissue engineered bone stills need to make further lifting from the following aspect：Repair
Multiple process of reconstruction quickly with surrounding bone fusion；The factor of biotic factor and stem cell secretion promotes autogenous repairing to participate in cell simultaneously
Biological behaviour；Physiological osteanagenesis is realized in quick participation reparation；Bone after reparation has good mechanical property, biology
Performance, structural stability（It is consistent with nature bone, sutura can be formed）；It is consistent with periphery autologous bone combination full-thickness.
The content of the invention
It is an object of the invention to construct a kind of Cranial defect Regeneration and Repair tissue engineered bone, it can be used for the life of Cranial defect
Rationality Regeneration and Repair.The present invention can realize the physiological regeneration of bone and improve the repairing and treating effect of Cranial defect.
To reach above-mentioned purpose, the present invention takes following technical scheme：
The present invention constructs a kind of Cranial defect Regeneration and Repair tissue engineered bone, is polymerize by three layers of mesenchymal stem cells MSCs
The composite layered structure that body and two layers of bone matrix particle are formed, wherein, bone matrix particle is as regeneration support, medulla mesenchyma
Stem cell condensate sticks parcel regeneration support.It is emphasized that mesenchymal stem cells MSCs condensate is by being filled between marrow
Matter stem cell is quickly rised in value formation, and contains the substantial amounts of extracellular matrix secreted by mesenchymal stem cells MSCs, extracellular matrix
In containing promoting the active factors of New born formation.
The composite layered structure is to wrap up two layers of bone matrix granulated by three layers of mesenchymal stem cells MSCs condensate
Into Cranial defect Regeneration and Repair product, be to use " mesenchymal stem cells MSCs condensate -- bone matrix particle -- medulla mesenchyma
Reparation product prepared by the composite mode of stem cell condensate -- bone matrix particle -- mesenchymal stem cells MSCs condensate ".Tool
For body, the composite layered structure is followed successively by the mesenchymal stem cells MSCs condensate for being laid in bottom from the bottom to top,
The even bone matrix particle being laid on bottom mesenchymal stem cells MSCs condensate, tiled on bone matrix particle and be located at intermediate layer
Mesenchymal stem cells MSCs condensate, it is evenly laid out in the bone matrix on the mesenchymal stem cells MSCs condensate of intermediate layer
Grain, and the mesenchymal stem cells MSCs condensate positioned at upper strata being laid on bone matrix particle.
Further, the mesenchymal stem cells MSCs condensate includes mesenchymal stem cells MSCs and by being filled between marrow
Matter stem cell secretion and be distributed in the extracellular matrix around mesenchymal stem cells MSCs.Mesenchymal stem cells MSCs is secreted big
The extracellular matrix of amount forms osteanagenesis bone microenvironment, and endogenous and external source seed cell can be induced to participate in bone remoulding.Here kind
Daughter cell refers to mesenchymal stem cells MSCs of the present invention.
In order to which the thickness with Cranial defect matches, while create and be beneficial to the micro-loop that mesenchymal stem cells MSCs participates in bone remoulding
Border, every layer of mesenchymal stem cells MSCs condensate of the composite layered structure are both preferably two mesenchymal stem cells MSCs
Condensate, every mesenchymal stem cells MSCs condensate have 6~10 layers of mesenchymal stem cells MSCs layer.Filled between marrow is built
During matter stem cells are polymeric, extracellular matrix is uniformly distributed and arrangement form 6~10 with mesenchymal stem cells MSCs
Confluent monolayer cells diaphragm sample condensate, its thickness reach 75~150 μm.
Regeneration support of the bone matrix particle as tissue engineered bone as described above, it is preferable that the bone matrix particle
Be from autologous bone prepare bone matrix particle, nonantigenic rejection after transplanting.Further, autologous bone or decalcified bone matrix
The particle diameter of particle is 0.2~0.5cm.It is 0.2~0.5cm of diameter particulate by autologous bone or decalcified bone matrix processing, is advantageous to
Form the proportional space of osteanagenesis support.
On the other hand, present invention also offers the construction method of the Cranial defect Regeneration and Repair tissue engineered bone, including bone
Bone marrow-drived mesenchymal stem is separately cultured, mesenchymal stem cells MSCs polymeric structure, bone matrix particle preparation and organizational project
The steps such as the structure of bone.Specifically, comprise the following steps：
Step 1：Mesenchymal stem cells MSCs is separately cultured
Marrow blood sample is filtered, it is 5.0 × 10 to adjust mesenchymal stem cells MSCs concentration with nutrient solution I5~1.0 × 106
Individual/mL, and be inoculated in blake bottle, 12~18h is stood, culture medium I is replaced by training after mesenchymal stem cells MSCs is adherent
Nutrient solution II；
In 37 ± 1 DEG C, 4.5~5.5%CO2, 48~72h is cultivated under the conditions of moisture-saturated, abandon nutrient solution II, use 0.1mol/
The culture medium II that L PBS are washed twice and more renewed, later every 72~96h change nutrient solution II 1 times, treat that cell confluency reaches
When 70%~85%, carry out passage and expand culture to P3 for cell.
Step 2：The polymeric structure of mesenchymal stem cells MSCs
By P3 for mesenchymal stem cells MSCs, cell concentration is configured to as 5.0 × 10 with nutrient solution III5~1.0 × 107Individual/
ML mesenchymal stem cells MSCs suspension, 2mL~8mL mesenchymal stem cells MSCs suspensions are taken to be inoculated in a diameter of 3cm~10cm
In culture dish, 37 ± 1 DEG C, 4.5~5.5%CO are statically placed in2Cultivated in incubator；Nutrient solution III was changed every 22~26 hours once,
Culture is formed for 7~10 days to mesenchymal stem cells MSCs condensate, and nutrient solution III is replaced by into nutrient solution IV, continues culture 3 days；
Mesenchymal stem cells MSCs condensate is cleaned with physiological saline, mesenchymal stem cells MSCs condensate is taken out from culture dish and is used in combination
Physiological saline rinses 3 times, standby.
Step 3：Bone matrix particle preparation
Pretreated autologous bone or decalcified bone matrix are placed in -80 ± 5 DEG C of degree refrigerators and carry out stored frozen；37 DEG C of water
Bath is thawed, and aseptically crushes autologous bone or decalcified bone matrix, the bone matrix that screening grain diameter is 0.2~0.5cm
Sterile storage, 2~8 DEG C save backup.
Step 4：The structure of tissue engineered bone
2 bottom mesenchymal stem cells MSCs condensates are tiled, uniformly equalled autologous bone or decalcifed bone matrix
It is laid on bottom mesenchymal stem cells MSCs condensate；2 intermediate layer mesenchymal stem cells MSCs condensates are laid in certainly again
On body bone or decalcifed bone matrix, autologous bone or decalcifed bone matrix are uniformly laid between the marrow of intermediate layer by continuation fills
On matter stem cell condensate；2 bottom mesenchymal stem cells MSCs condensates are finally laid in autologous bone or decalcified bone matrix
On particle, required tissue engineered bone is formed.
In the construction method of Cranial defect Regeneration and Repair tissue engineered bone, used 4 kinds of nutrient solutions, respectively nutrient solution I,
Nutrient solution II, nutrient solution III and nutrient solution IV.
Wherein, nutrient solution I is DMEM nutrient solutions, can buy the product of sigma companies.
Nutrient solution II is the DMEM nutrient solutions containing FBS, and FBS volume fraction is 10% in DMEM nutrient solutions.
Nutrient solution III is by FBS, DMEM nutrient solution, vitamin C, 8- isopentene groups naringenin, licochalcone A and excessive sheep
Leaves of pulse plants glycosides forms；Wherein, FBS volume fraction is 10%, and ascorbic concentration is 50~100mg/L, 8- isopentene group naringenins
Concentration be 10-5~10-8Mol/L, the concentration of licochalcone A is 5 × 10-4~5 × 10-6Mol/L, the concentration of icariin
For 10-5~10-7mol/L。
Nutrient solution IV is by FBS, DMEM nutrient solution, β-phosphoglycerol sodium, dexamethasone, vitamin C, 8- isopentene group mandarin oranges
Tangerine element, licochalcone A and icariin composition；Wherein, FBS volume fraction is that the concentration of 10%, β-phosphoglycerol sodium is 10
~20mmol/L, the concentration of dexamethasone is 5~20nM, and ascorbic concentration is 50~100 μ g/ml, 8- isopentene group citruses
The concentration of element is 10-5~10-8Mol/L, the concentration of licochalcone A is 5 × 10-4~5 × 10-6Mol/L, icariin it is dense
Spend for 10-5~10-7mol/L。
For the construction method of above-mentioned Cranial defect Regeneration and Repair tissue engineered bone, it is also necessary to explanation, tissue engineered bone
Be made up of mesenchymal stem cells MSCs condensate and bone matrix particle, mesenchymal stem cells MSCs it is polymeric structure and
The sequencing of bone matrix particle preparation, it can adjust.The construction method that the present invention provides, simply a kind of preferable implementation
Mode, the preparation process of nutrient solution can also be included.
Further, the present invention gives application of the described tissue engineered bone in Cranial defect Regeneration and Repair.
The present invention has carried out the innovation of methodology to the structure of Cranial defect Regeneration and Repair tissue engineered bone, has prepared bone base
Matter particle is as regeneration support, while the tissue engineered bone of compound living cells and active factors.Containing big in the tissue engineered bone
The bioactie agents such as the abundant growth of amount, vascularization, self-bone grafting, more simple bone material, material and the factor is compound, material is multiple
Close cell, more bone formation ability.The preparation of bone matrix particle avoids the discarded of autologous bone at defect using autologous bone as raw material
With cause unnecessary rejection, there is provided be more beneficial to growth of mesenchymal stem cells, propagation, differentiation microenvironment.Move
Large area Cranial defect can be quickly repaired after planting tissue engineered bone of the present invention, reconstruction process is quickly melted with surrounding bone
Close, while the factor of biotic factor and stem cell secretion promotes autogenous repairing to participate in the biological behaviour of cell, quickly participates in repairing
Multiple, area of new bone has good mechanical property, biology performance, structural stability（It is consistent with nature bone, sutura can be formed）And
Periphery autologous bone combination full-thickness is consistent.
The a large amount of active factorses secreted by cell aggregation body technique mesenchymal stem cells MSCs are gathered in extracellular base
In matter, communication and interaction between cell and cell are remained, pancreatin digestion in existing cell transplantation procedure is avoided and leads
Cause extracellular matrix to scatter and disappear and the appearance of terminal situation is communicated between cell, farthest remain cell propagation, differentiation and hair
Wave the microenvironment of function.
Cord blood technology realizes bone or the long-time of Acellular bone organization bracket preserves, and reduces single in clinical treatment
The pure expense with bone material, and physiological osteanagenesis is realized, while carried by using the tissue engineered bone containing living cells
Height simultaneously ensure that the quality of bone defect healing.
It is carrier-free mesenchymal stem cells MSCs film disclosed in 201210090795.0 patent document with number of patent application
The preparation method of piece is compared, and Cell-aggregates technical operation is simpler, without using timbering material and temperature sensing material.In cell membrane
In the case of plate shape and area identical, using the polymeric thickness of mesenchymal stem cells MSCs of cell aggregation body technique preparation
It is 1.5~3 times of mesenchymal stem cells MSCs diaphragm prepared by patent document 201210090795.0, in addition, Cell-aggregates
Toughness and the accumulation of extracellular matrix significantly improve.Meanwhile the micromolecular compound in condensate induction system（Culture
Component in liquid）Promote Osteoblast Differentiation, and suppress mesenchymal stem cells differentiation into fat.With being lured using vitamin C merely
Lead mesenchymal stem cell membrane（Referring to the patent document that number of patent application is 201110210523.5）Compare, it is provided by the invention
The construction method of Cranial defect Regeneration and Repair tissue engineered bone is easier to participate in new bone formation and reconstruction.
The compound mesenchymal stem cells MSCs condensate of tissue engineered bone of the present invention and bone or decalcifed bone matrix, and
Good self-bone grafting microenvironment is created simultaneously, mesenchymal stem cells MSCs condensate is on the one hand come from and largely abundant work is provided
Sex factor, promote surviving and breaking up for mesenchymal stem cells MSCs, participate in bone remoulding；The advantage of another aspect come from osseous granules or
Bone matrix scaffold derives from autologous bone, and no rejection, security is improved, while ensure that its osteogenic ability and skeletonization
Environment.Therefore, the present invention adds postdigestive stem cell repairing bone defect more quickly and effectively compared with current material, more existing synthesis high score
Sub- material, biomaterial, nano material have more reparation advantage.
Compared with existing bone defect healing product, the Cranial defect Regeneration and Repair tissue engineered bone prepared by the present invention is easy to root
Formed according to defect and matched with defect shape, size, thickness.
The present invention is elaborated further below with reference to embodiment.
Brief description of the drawings
Fig. 1 is the Rat calvarial defect repair figure of embodiment 1.Wherein, Figure 1A is rat 2cm skull limit defect model figures；
Figure 1B is that mesenchymal stem cells MSCs condensate spreads in endocranium；Fig. 1 C are that bone matrix particle is uniformly distributed in defect；Figure
1D is that mesenchymal stem cells MSCs condensate spreads in coated stent material on osseous granules.
Fig. 2 is the repairing effect figure of Rat calvarial limit defect described in embodiment 1.
Fig. 3 A are mesenchymal stem cells MSCs cell aggregations of the rabbit P3 of embodiment 2 for Marrow Mesenchymal Stem Cells
Body.
Fig. 3 B are that the tissue engineered bone that embodiment 2 is built is used for bone defect healing figure.
Fig. 3 C are that the organizational project diostosis that embodiment 2 is built migrates to the subcutaneous ectopic bone formation design sketch of rabbit.
Fig. 3 D be 2 bone defect healing of embodiment after 12 weeks CT surface sweepings identify bone defect healing design sketch.
Fig. 4 a are 3 big animal models of embodiment with 1.5 years old long informal voucher pig.
Fig. 4 b are diameter 5cm holostromes skull defeci figures in embodiment 3.
Fig. 4 c are depth 3cm holostromes skull defeci figures in embodiment 3.
Fig. 4 d are structure product skin grafing and mending skull defeci figures in embodiment 3.
Fig. 4 e are CT analysis charts after repair of cranial defects in embodiment 3.
Fig. 4 f are that answer is taken pictures figure after repair of cranial defects in embodiment 3.
Embodiment
Embodiment 1：Rat calvarial limit defect repair
The present embodiment is repaired to its skull limit defect using rat as examination biology, specifically includes following steps.
Step 1: the preparation of nutrient solution
The present embodiment has used 4 kinds of nutrient solutions, respectively nutrient solution I, nutrient solution II, nutrient solution III and nutrient solution IV.Its
In, nutrient solution I is DMEM nutrient solutions, purchased from sigma companies.Nutrient solution II is the DMEM nutrient solutions containing FBS, and DMEM is cultivated
FBS volume fraction is 10% in liquid.
Nutrient solution III is by FBS, DMEM nutrient solution, vitamin C, 8- isopentene groups naringenin, licochalcone A and excessive sheep
Leaves of pulse plants glycosides forms.Wherein, FBS volume fraction is 10%, and ascorbic concentration is 100mg/L, 8- isopentene group naringenins it is dense
Spend for 10-6Mol/L, the concentration of licochalcone A is 5 × 10-5Mol/L, the concentration of icariin is 10-7mol/L。
Nutrient solution IV is by FBS, DMEM nutrient solution, β-phosphoglycerol sodium, dexamethasone, vitamin C, 8- isopentene group mandarin oranges
Tangerine element, licochalcone A and icariin composition；Wherein, FBS volume fraction is for the concentration of 10%, β-phosphoglycerol sodium
20mmol/L, the concentration of dexamethasone is 10nM, and ascorbic concentration is for the concentration of 50 μ g/ml, 8- isopentene group naringenins
10-8Mol/L, the concentration of licochalcone A is 5 × 10-5Mol/L, the concentration of icariin is 10-7mol/L。
Step 2: the separation and culture of mesenchymal stem cells MSCs
Dislocated and put to death from 2 week old rats, 75% alcohol-pickled sterilization, taken femur to obtain marrow and blown repeatedly with syringe
Break into monokaryon layer suspension；Strainer filtering and counting with 150 mesh, it is 5.0 × 10 to adjust cell concentration with nutrient solution I5Individual/
ML, is inoculated in 75mL blake bottles, and nutrient solution is nutrient solution II.In 37 DEG C, 5%CO2, cultivate 48 h under the conditions of moisture-saturated, abandon
Nutrient solution II, the phosphate buffer for being 0.1mol/L with concentration（PBS）Wash twice and more renew nutrient solution II.It is later every
72 h change nutrient solution II 1 times, when cell confluency reaches 80%, carry out passage and expand culture, take P3 to be used as structure for cell
Build the material of mesenchymal stem cells MSCs Cell-aggregates.
Step 3: the single treatment of bone matrix
3 months old rats are taken to carry out skull diameter 0.8cm limit defect modelings, at the osteocomma that will be taken out at skull modeling
Reason, with containing penicillin and streptomysin without Ca2+、Mg2+PBS skull osteocomma, discard skull osteocomma remaining blood, knot
Tissue, bone particle etc. are formed, by the skull osteocomma of collection（Bone tissue material）Carry out 3~5 times to rinse and screen repeatedly, by acquisition
Bone piece（Skull osteocomma）It is placed in sterile 5mL centrifuge tubes, sealed membrane sealing is placed in -80 DEG C of refrigerators and carries out stored frozen.
Step 4: mesenchymal stem cells MSCs Cell-aggregates are built
The mesenchymal stem cells MSCs in the P3 generations of step 2 culture is taken, contains 5.0 × 10 per 1mL nutrient solutions III6Individual cell,
It is prepared into mesenchymal stem cells MSCs suspension.By mesenchymal stem cells MSCs suspension using 2mL volume be inoculated in bore dia as
3.5cm six orifice plates（Culture dish）In, it is statically placed in 4.5-5.5% CO2, cultivate in 37 ± 1 DEG C of incubators.Training was changed every 24 hours
Nutrient solution III once, co-cultures 10 days, the visible milky film spline structure of naked eyes（Diaphragm or mesenchymal stem cells MSCs cell aggregation
Body）, when six orifice plate rims have slight tilting, now mesenchymal stem cells MSCs Cell-aggregates are formed diaphragm.Change culture
Liquid IV, continue culture 3 days.Mesenchymal stem cells MSCs Cell-aggregates are cleaned with physiological saline, are gently torn with tweezers from ware bottom
Under, physiological saline rinses 3 times（5min/ times）It is standby.
Step 5: the preparation of bone matrix particle
The bone tissue material or decalcified bone matrix for the single treatment that step 3 freezes are taken out, 37 DEG C of water-baths are thawed, sterile
Under the conditions of bone or decalcified bone matrix cut with orthopaedics carry out pulverization process, with the stainless steel steel of 80 mesh screen out particle diameter 0.2cm with
Under particle, with bone matrix particle of the stainless steel steel of the 30 mesh sieve screening particle diameter between 0.2~0.5cm, sterile storage after collection
Deposit, 4 DEG C save backup.
Step 6: the structure of tissue engineered bone
Standby mesenchymal stem cells MSCs condensate and bone or decalcifed bone matrix will be handled well in such a way
It is combined the tissue engineered bone for being configured to Bone Defect Repari regeneration.2 mesenchymal stem cells MSCs condensates are laid in bottom
Bone or decalcifed bone matrix material, are uniformly laid on mesenchymal stem cells MSCs condensate by layer；Again by between 2 marrow
Mesenchymal stem cells condensate is tiled in bone material, and bone or decalcifed bone matrix material are uniformly laid between marrow by continuation
On mesenchymal stem cells condensate；Finally 2 mesenchymal stem cells MSCs condensates are tiled on material；Three confluent monolayer cells are formed to gather
The finished product of zoarium two layers of bone matrix particle of parcel.
Rat calvarial limit defect repair is carried out using the present embodiment methods described, repair process and effect are shown in Fig. 1 respectively
And Fig. 2.Figure 1A gives second operation figure after rat 2cm skull limit defect model succeeds 2 months, diaphragm condensate（Marrow
Mescenchymal stem cell condensate）It is laid on the bottom（Figure 1B）, the 2 months autogenous bone materials that freeze prepared are placed in diaphragm condensate
On（Fig. 1 C）, diaphragm condensate is laid on the bottom（Fig. 1 D）；Draw materials within the 4th, 8,12 week after reparation and CT is detected, 4 weeks
Begin with new bone formation afterwards, there are a large amount of bon e formations at 8 weeks, defect repair is basically completed after 12 weeks（Fig. 2）.
Embodiment 2：Rabbit cranial bone limit defect repair
The present embodiment is repaired to its skull limit defect using rabbit as examination biology, specifically includes following steps.
Step 1: the preparation of nutrient solution
The present embodiment has used 4 kinds of nutrient solutions, respectively nutrient solution I, nutrient solution II, nutrient solution III and nutrient solution IV.Its
In, nutrient solution I, nutrient solution II are the same as embodiment 1.
Nutrient solution III is by FBS, DMEM nutrient solution, vitamin C, 8- isopentene groups naringenin, licochalcone A and excessive sheep
Leaves of pulse plants glycosides forms.Wherein, FBS volume fraction is 10%, and ascorbic concentration is 100mg/L, 8- isopentene group naringenins it is dense
Spend for 10-5Mol/L, the concentration of licochalcone A is 10-6Mol/L, the concentration of icariin is 10-6mol/L。
Nutrient solution IV is by FBS, DMEM nutrient solution, β-phosphoglycerol sodium, dexamethasone, vitamin C, 8- isopentene group mandarin oranges
Tangerine element, licochalcone A and icariin composition；Wherein, FBS volume fraction is for the concentration of 10%, β-phosphoglycerol sodium
10mmol/L, the concentration of dexamethasone is 20nM, and ascorbic concentration is for the concentration of 50 μ g/ml, 8- isopentene group naringenins
10-6Mol/L, the concentration of licochalcone A is 5 × 10-6Mol/L, the concentration of icariin is 10-5mol/L。
Step 2: the separation and culture of mesenchymal stem cells MSCs
3 monthly age screech owl white rabbits are chosen, take femur bone marrow 20ml, marrow is obtained through centrifugation and blows and beats into list repeatedly with syringe
Stratum nucleare suspension；Strainer filtering and counting with 150 mesh, it is 1 × 10 to adjust cell concentration with nutrient solution I6Individual/mL, is inoculated in
75mL blake bottles, nutrient solution are nutrient solution II.In 37 DEG C, 5%CO2, cultivate 48h under the conditions of moisture-saturated, abandon nutrient solution II, use
Concentration is 0.1mol/L phosphate buffer（PBS）The nutrient solution II for washing twice and more renewing.Culture is changed per 72h later
Liquid II 1 times, when cell confluency reaches 75%, carry out passage and expand culture, take P3 to be used as structure medulla mesenchyma for cell
The polymeric material of stem cells.
Step 3: the single treatment of bone matrix
3 monthly age screech owl white rabbits are taken to carry out skull diameter 2cm limit defect modelings（With taking the rabbit of marrow in step 2 one by one
It is corresponding, tested for autologous stem cell transplantation）.The osteocomma taken out at skull modeling is handled, with containing penicillin and strepto-
Element without Ca2+、Mg2+PBS skull osteocomma, discard skull osteocomma remaining blood, connective tissue, bone particle etc., will collect
Skull osteocomma（Bone tissue material）Carry out 3~5 times to rinse and screen repeatedly, by the bone piece of acquisition（Skull osteocomma）It is placed in sterile
In 5mL centrifuge tubes, sealed membrane sealing is placed in -80 DEG C of refrigerators and carries out stored frozen.
Step 4: mesenchymal stem cells MSCs condensate is built
The mesenchymal stem cells MSCs in the P3 generations of step 2 culture is taken, contains 1.0 × 10 per 1mL nutrient solutions III7It is individual thin
Born of the same parents, it is prepared into mesenchymal stem cells MSCs suspension.By mesenchymal stem cells MSCs suspension using 2mL volume be inoculated in aperture as
In the orifice plates of 3.5cm six, 4.5-5.5% CO are statically placed in2, cultivate in 37 ± 1 DEG C of incubators；Nutrient solution III 1 was changed every 24 hours
It is secondary, co-culture 10 days, the visible milky film spline structure of naked eyes（Diaphragm or mesenchymal stem cells MSCs Cell-aggregates）, diaphragm exists
When six orifice plate rims have slight tilting, now mesenchymal stem cells MSCs Cell-aggregates are formed.Nutrient solution IV is changed, continues to train
Support 3 days.Mesenchymal stem cells MSCs Cell-aggregates are cleaned with physiological saline, are gently torn with tweezers from ware bottom, physiological saline
Rinsing 3 times, physiological saline rinse each 5min, are used in 8 hours and complete to transplant.
Step 5: the preparation of bone matrix particle
After 2 months, the bone matrix of the single treatment frozen is taken out, 37 DEG C of water-baths are thawed, by bone matrix under aseptic condition
Cut with orthopaedics and carry out pulverization process, the particle below particle diameter 0.2cm is screened out with the stainless steel steel of 80 mesh, with the stainless steel of 30 mesh
Bone matrix particle of the steel sieve screening particle diameter between 0.2~0.5cm, sterile storage after collection, 4 DEG C save backup.
Step 6: the structure of tissue engineered bone
Standby mesenchymal stem cells MSCs condensate will be handled well to carry out in such a way with bone matrix granular materials
Combination structure：2 mesenchymal stem cells MSCs condensates are laid in bottom, bone matrix granular materials is uniformly laid in bone
On bone marrow-drived mesenchymal stem condensate, then 2 mesenchymal stem cells MSCs condensates are tiled on bone matrix particle, continued equal
Even is laid in bone matrix granular materials on mesenchymal stem cells MSCs condensate, finally by 2 mesenchymal stem cells MSCs
Condensate tiles on material；Form the reparation that three layers of mesenchymal stem cells MSCs condensate wrap up two layers of bone matrix granular materials
Product.
Rabbit cranial bone limit defect repair is carried out using the present embodiment methods described, by building rabbit cranial bone limit defect
（Diameter=2cm defect）Model, simulation clinical treatment once open cranium, -80 DEG C of Preservation in sterile condition of skull of taking-up.By step 2 institute
Separation, culture mesenchymal stem cells MSCs are stated, builds volume Cell-aggregates diaphragm sample active material（Mesenchymal stem cells MSCs gathers
It is fit）See Fig. 3 A.After 2 months, transplanting bone defect healing is carried out with the tissue engineered bone of structure, sees Fig. 3 B.From Fig. 3 C, structure
The organizational project diostosis built, which is transplanted to, very strong ectopic bone formation under rabbit skin.Treat that tissue engineered bone carries out Cranial defect and repaiied
After multiple 12 weeks, CT surface sweepings identification bone defect healing is completed and effect is preferable, sees Fig. 3 D.This example demonstrates that the tissue work of structure
Journey bone has ectopic bone formation, and the defect repair time is short, there is preferable Bone Defect Repari function.
Embodiment 3：Pig skull limit defect repair,
The present embodiment is repaired to its skull limit defect using pig as examination biology, specifically includes following steps.
Step 1: the preparation of nutrient solution
The present embodiment has used 4 kinds of nutrient solutions, respectively nutrient solution I, nutrient solution II, nutrient solution III and nutrient solution IV.Its
In, nutrient solution I, nutrient solution II are the same as embodiment 1 and embodiment 2.
Nutrient solution III is by FBS, DMEM nutrient solution, vitamin C, 8- isopentene groups naringenin, licochalcone A and excessive sheep
Leaves of pulse plants glycosides forms.Wherein, FBS volume fraction is 10%, and ascorbic concentration is 100mg/L, 8- isopentene group naringenins it is dense
Spend for 10-6Mol/L, the concentration of licochalcone A is 10-5Mol/L, the concentration of icariin is 10-5mol/L。
Nutrient solution IV is by FBS, DMEM nutrient solution, β-phosphoglycerol sodium, dexamethasone, vitamin C, 8- isopentene group mandarin oranges
Tangerine element, licochalcone A and icariin composition；Wherein, FBS volume fraction is for the concentration of 10%, β-phosphoglycerol sodium
20mmol/L, the concentration of dexamethasone is 20nM, and ascorbic concentration is for the concentration of 50 μ g/ml, 8- isopentene group naringenins
10-6Mol/L, the concentration of licochalcone A is 5 × 10-5Mol/L, the concentration of icariin is 10-5mol/L。
Step 2: the separation and culture of mesenchymal stem cells MSCs
1 age long informal voucher pig takes femur bone marrow 50ml, and separating liquid centrifuges and blows and beats into monokaryon layer repeatedly with syringe
Suspension；Strainer filtering and counting with 150 mesh, it is 1 × 10 to adjust cell concentration with nutrient solution I6Individual/mL, is inoculated in 75mL
Blake bottle, nutrient solution are nutrient solution II.In 37 DEG C, 5%CO2, cultivate 48h under the conditions of moisture-saturated, abandon nutrient solution II, use concentration
For 0.1mol/L phosphate buffer（PBS）The nutrient solution II for washing twice and more renewing.Nutrient solution II is changed per 72h later
1 time, when cell confluency reaches 75%, pass on expanding and cultivate, take P3 to be used as structure medulla mesenchyma for cell and do carefully
The material of born of the same parents' Cell-aggregates.
Step 3: the single treatment of bone matrix
1 age, long informal voucher pig carried out skull diameter 5cm limit defect modelings（A pair of the pig 1 with taking marrow in step 2
Should, tested for autologous stem cell transplantation）, the osteocomma taken out at skull modeling is handled, with containing penicillin and streptomysin
Without Ca2+、Mg2+PBS skull osteocomma, skull osteocomma remaining blood, connective tissue, bone particle etc. are discarded, by collection
Skull osteocomma（Bone tissue material）Carry out 3 times to rinse and screen repeatedly, by the bone piece of acquisition（Skull osteocomma）It is placed in sterile 50mL
In centrifuge tube, sealed membrane sealing is placed in -80 DEG C of refrigerators and carries out stored frozen.
Step 4: mesenchymal stem cells MSCs condensate is built
The mesenchymal stem cells MSCs in the P3 generations of step 2 culture is taken, contains 5 × 10 per 1mL nutrient solutions III6Individual cell,
It is prepared into mesenchymal stem cells MSCs suspension.Mesenchymal stem cells MSCs suspension is inoculated in into aperture using 2mL volume to train as 5cm
Support in ware, be statically placed in 5% CO2, cultivate in 37 DEG C of incubators；Nutrient solution III was changed every 24 hours once, was co-cultured 10 days, naked eyes
It can be seen that milky film spline structure（Diaphragm or mesenchymal stem cells MSCs Cell-aggregates）, diaphragm has in six orifice plate rims slightly to be stuck up
When rising, now mesenchymal stem cells MSCs Cell-aggregates are formed.Nutrient solution IV is changed, continues culture 3 days.It is clear with physiological saline
Mesenchymal stem cells MSCs Cell-aggregates are washed, are gently torn with tweezers from ware bottom, physiological saline rinses 3 times, and is suspended in life
Manage standby in salt solution.
Step 5: the preparation of bone matrix particle
After 3 months, the bone material of -80 DEG C of preservations is taken out（Bone matrix）, 37 DEG C of water-baths are thawed, by bone matrix under aseptic condition
Pulverization process is carried out, the particle below particle diameter 0.2cm is screened out with the stainless steel steel of 80 mesh, screening is sieved with the stainless steel steel of 30 mesh
Bone matrix particle of the particle diameter between 0.2~0.5cm, sterile storage after collection, 4 DEG C save backup.
Step 6: the structure of tissue engineered bone
Standby mesenchymal stem cells MSCs condensate and bone matrix particle timbering material will be handled well in such a way
It is combined structure：2 mesenchymal stem cells MSCs condensates are laid in bottom, uniformly bone matrix granular materials tiled
Tiled on mesenchymal stem cells MSCs condensate, then by 2 mesenchymal stem cells MSCs condensates on bone matrix particle, after
It is continuous that uniformly bone matrix granular materials is laid on mesenchymal stem cells MSCs condensate, finally 2 medulla mesenchymas are done
Cell-aggregates tile on material；Three layers of mesenchymal stem cells MSCs condensates two layers of bone matrix granular materials of parcel of formation
Repair product.
Pig skull limit defect repair is carried out using the present embodiment methods described.First, pig skull limit defect is built, directly
Footpath=5cm, thickness=3cm defect model, simulation clinical treatment once open cranium, -80 DEG C of Preservation in sterile condition after the bone processing of taking-up, together
When take marrow and be separately cultured mescenchymal stem cell and build Cell-aggregates diaphragm sample active material（See Fig. 4 a, Fig. 4 b, scheme
4c）.After 3 months, transplanting bone defect healing is carried out with the tissue engineered bone of structure, sees Fig. 4 d.Treat that tissue engineered bone carries out bone and lacked
After damage is repaired 12 weeks, CT surface sweepings identification bone defect healing is completed and effect is preferable, sees Fig. 4 e and Fig. 4 f.This example demonstrates that structure
The tissue engineered bone built has preferable physiology bone regeneration capability, and the defect repair time is short, there is preferable Bone Defect Repari function.
Further narration has been done to the present invention above in conjunction with embodiment, but the present invention is not limited to above-mentioned embodiment,
In one skilled in the relevant art's possessed knowledge, it can also be made on the premise of present inventive concept is not departed from
Various change.
Claims (5)
1. a kind of Cranial defect Regeneration and Repair tissue engineered bone, it is characterised in that the tissue engineered bone is by being filled between three layers of marrow
The composite layered structure that matter stem cell condensate and two layers of bone matrix particle are formed；The composite layered structure is from the bottom to top
Bottom mesenchymal stem cells MSCs condensate is followed successively by, it is evenly laid out in the bone base on bottom mesenchymal stem cells MSCs condensate
Matter particle, tile intermediate layer mesenchymal stem cells MSCs condensate on bone matrix particle, evenly laid out between the marrow of intermediate layer
Bone matrix particle on mesenchymal stem cells condensate, and the upper strata mesenchymal stem cells MSCs being laid on bone matrix particle gather
It is fit；
Wherein, the mesenchymal stem cells MSCs condensate includes mesenchymal stem cells MSCs and by mesenchymal stem cells MSCs
Secretion and be distributed in the extracellular matrix around mesenchymal stem cells MSCs.
2. Cranial defect Regeneration and Repair tissue engineered bone according to claim 1, it is characterised in that filled between every layer of marrow
Matter stem cell condensate includes two mesenchymal stem cells MSCs condensates, and every mesenchymal stem cells MSCs condensate has
6~10 layers of mesenchymal stem cells MSCs layer.
3. Cranial defect Regeneration and Repair tissue engineered bone according to claim 1, it is characterised in that the bone matrix particle is
The bone matrix particle prepared from autologous bone.
4. Cranial defect Regeneration and Repair tissue engineered bone according to claim 3, it is characterised in that the bone matrix particle
Particle diameter is 0.2~0.5cm.
5. building the method for the Cranial defect Regeneration and Repair tissue engineered bone as any one of Claims 1-4, its feature exists
In comprising the following steps：
Mesenchymal stem cells MSCs is separately cultured
Marrow blood sample is filtered, it is 5.0 × 10 to adjust mesenchymal stem cells MSCs concentration with nutrient solution I5~1.0 × 106Individual/mL,
And be inoculated in blake bottle, 12~18h is stood, culture medium I is replaced by nutrient solution II after mesenchymal stem cells MSCs is adherent；
In 37 ± 1 DEG C, 4.5~5.5%CO2, 48~72h is cultivated under the conditions of moisture-saturated, abandon nutrient solution II, use 0.1mol/L
The culture medium II that PBS is washed twice and more renewed, later every 72~96h change nutrient solution II 1 times, treat that cell confluency reaches
When 70%~85%, carry out passage and expand culture to P3 for cell；
The nutrient solution I is DMEM nutrient solutions；Nutrient solution II is the DMEM nutrient solutions containing FBS, and FBS in DMEM nutrient solutions
Volume fraction is 10%；
(2) the polymeric structure of mesenchymal stem cells MSCs
By P3 for mesenchymal stem cells MSCs, cell concentration is configured to as 5.0 × 10 with nutrient solution III5~1.0 × 107Individual/mL's
Mesenchymal stem cells MSCs suspension, 2mL~8mL mesenchymal stem cells MSCs suspensions are taken to be inoculated in a diameter of 3cm~10cm cultures
In ware, 37 ± 1 DEG C, 4.5~5.5%CO are statically placed in2Cultivated in incubator；Nutrient solution III was changed every 22~26 hours once, training
Support 7~10 days and formed to mesenchymal stem cells MSCs condensate, nutrient solution III is replaced by nutrient solution IV, continue culture 3 days；With
Physiological saline clean mesenchymal stem cells MSCs condensate, from culture dish take out mesenchymal stem cells MSCs condensate and with give birth to
Manage saline rinse 3 times, it is standby；
The nutrient solution III is by FBS, DMEM nutrient solution, vitamin C, 8- isopentene groups naringenin, licochalcone A and barrenwort
Glycosides forms；Wherein, FBS volume fraction is 10%, and ascorbic concentration is 50~100mg/L, 8- isopentene group naringenins
Concentration is 10-5~10-8Mol/L, the concentration of licochalcone A is 5 × 10-4~5 × 10-6Mol/L, the concentration of icariin are
10-5~10-7mol/L；
The nutrient solution IV is by FBS, DMEM nutrient solution, β-phosphoglycerol sodium, dexamethasone, vitamin C, 8- isopentene group citruses
Element, licochalcone A and icariin composition；Wherein, FBS volume fraction is that the concentration of 10%, β-phosphoglycerol sodium is 10
~20mmol/L, the concentration of dexamethasone is 5~20nM, and ascorbic concentration is 50~100 μ g/ml, 8- isopentene group citruses
The concentration of element is 10-5~10-8Mol/L, the concentration of licochalcone A is 5 × 10-4~5 × 10-6Mol/L, icariin it is dense
Spend for 10-5~10-7mol/L；
(3) bone matrix particle preparation
Pretreated autologous bone or decalcified bone matrix are placed in -80 ± 5 DEG C of degree refrigerators and carry out stored frozen；37 DEG C of water-bath solutions
Freeze, aseptically crush autologous bone or decalcified bone matrix, the bone matrix that screening grain diameter is 0.2~0.5cm is sterile
Storage, 2~8 DEG C save backup；
(4) structure of tissue engineered bone
2 bottom mesenchymal stem cells MSCs condensates are tiled, are uniformly laid in autologous bone or decalcifed bone matrix
On bottom mesenchymal stem cells MSCs condensate；2 intermediate layer mesenchymal stem cells MSCs condensates are laid in autologous bone again
Or on decalcifed bone matrix, autologous bone or decalcifed bone matrix are uniformly laid in intermediate layer medulla mesenchyma and done by continuation
On Cell-aggregates；2 bottom mesenchymal stem cells MSCs condensates are finally laid in autologous bone or decalcifed bone matrix
On, form required tissue engineered bone.
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| CN106890364A (en) * | 2015-12-17 | 2017-06-27 | 西安组织工程与再生医学研究所 | A kind of preparation method for being engineered periodontium |
| CN106890363B (en) * | 2015-12-17 | 2021-09-10 | 西安组织工程与再生医学研究所 | Preparation method of engineered dental pulp |
| CN108704164A (en) * | 2018-05-17 | 2018-10-26 | 广东芙金干细胞再生医学有限公司 | A kind of injection cell auxiliary autologous fat transplantation object and preparation method thereof |
| CN108939151B (en) * | 2018-08-01 | 2019-11-05 | 北京大学 | Application of the nanoporous micro rack in regeneration and restoration |
| CN110055215A (en) * | 2019-05-06 | 2019-07-26 | 南京鼓楼医院 | A kind of high Osteoblast Differentiation ability human mesenchymal stem cell and preparation method thereof |
| CN112375742B (en) * | 2020-11-18 | 2023-09-29 | 中山大学附属第八医院（深圳福田） | Method for improving bone formation capacity of bone marrow mesenchymal stem cells and application |
| CN113679885A (en) * | 2021-08-19 | 2021-11-23 | 中国人民解放军联勤保障部队第九八八医院 | Method for repairing and reconstructing craniomaxillofacial surface based on 3D printing construction |
| CN113711993A (en) * | 2021-09-01 | 2021-11-30 | 杭州越波生物科技有限公司 | Method for quantitatively evaluating bone regeneration repair capacity of rat skull after bone defect by utilizing Micro-CT |
| CN114940967A (en) * | 2022-05-09 | 2022-08-26 | 中山大学 | Breast milk extracellular vesicle and application thereof in preparation of bone repair material |
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Denomination of invention: A tissue engineering bone for bone defect regeneration and repair and its construction method and Application Effective date of registration: 20200915 Granted publication date: 20171208 Pledgee: Xianyang financing guarantee Limited by Share Ltd. Pledgor: GUANGDONG FUJIN STEM CELL REGENERATION MEDICINE Co.,Ltd. Registration number: Y2020990001113 |
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