CN107137763A - A kind of study of vascularized tissue engineering bone and preparation method thereof - Google Patents
A kind of study of vascularized tissue engineering bone and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of study of vascularized tissue engineering bone and preparation method thereof, the study of vascularized tissue engineering bone is the support being made up of the coral hydroxyapatite of polylysine modification and is wrapped in being constituted into endothelial cellular membrane piece and Gegenbaur's cell diaphragm for the support internal layer successively.The present invention is with poly-D-lysine（PLL）Modify coral hydroxyapatite（CHA）The extraneous scaffold of acquisition, is conducive to blood vessel to creep and grows into, and final vascularization, and precondition is provided for tissue engineered bone inner cell long term survival, while reducing immunity of organism rejection and inflammatory reaction；Double cell patches are prepared from fat mesenchymal stem cell, without the endothelial cell of extra addition separate sources, cell derived is enriched, it is easy to which separation is obtained, and small to donor wound, without prejudice to Ethical Principles, fostering requirement is low.The study of vascularized tissue engineering bone preparation method of the present invention is simple, and mild condition, with good skeletonization and into vascular performance, meets the requirement of biological vivo applications, is had a good application prospect as a kind of novel vascular tissue engineered bone.
Description
Technical field
It is more particularly to a kind of by Gegenbaur's cell diaphragm and thin into blood vessel endothelium the present invention relates to field of biomedicine technology
Study of vascularized tissue engineering bone that after birth piece is built and preparation method thereof.
Background technology
The treatment of bulk Cranial defect caused by severe trauma, tumor resection, infection, congenital abnormality etc. is modern medicine
The problem and huge challenge faced, be always the mankind deepen continuously for centuries research and explore important topic.At present
Clinically conventional reparation means have autologous bone transplanting, allogenic bone transplantation and use artificial bone etc., but above method is present
Certain defect.Autologous bone transplanting is the goldstandard of generally acknowledged bone tissue reparation, but patient will be subjected to autograft operation
Wound, and for area it is limited, therefore, autologous bone transplanting can not be considered as the restorative procedure of preferable large area Cranial defect；Allosome
There is immunological rejection, transmission equivalent risk in bone collection, even jeopardize patients ' lives sometimes；Artificial bone implantation is easily caused
Foreign material repulsion reaction, infection etc..Therefore, it is necessary to find a kind of reparation means of new bulk Cranial defect.
In the case, the rise and development of organizational engineering, provides new possibility, to make up for the reparation of Cranial defect
The defect of current study on bone defect healing method brings hope.
In recent years, the research of bone tissue engineer has made great progress, using tissue engineering technique in small-sized mammalian
Bone tissue is built in vivo, and the method for the Cranial defect of repairing small-size has been mature on the whole, but a wide range of for large mammal
Or by the not good Cranial defect of area's blood supply, because graft early stage is without independent blood supply, nutrient permeation is not enough, and poroma is formed
The reason such as slow, skeletonization effect is still unstable.There are some researches show, it is resolved that and the pass of restriction Tissue Engineering Bone for Repair of Bone Defect curative effect
Key is the speed and degree of its vascularization in vivo.Sahota etc. be also considered as organizational project implant failure main reason is that
The sluggishness of vascularization.Therefore, effective blood supply how is set up, the tissue engineered bone of energy vascularization in time is built in vivo,
Shorten the important directions that bone healing time is current bone tissue engineer research, be also restriction tissue engineered bone larger scale clinical
The key of application.
The vascularization strategy of current tissue engineered bone mainly includes：The the designing and developing of support, using growth factor, in vivo bury
Plant, internal artery cell and overall culture systems etc..Although blood vessel of these methods when solving tissue construction to a certain degree
Change problem, but Shortcomings.Such as, in the manufacturing process of support, the pore size and interconnection of material internal are entered
Row modification, can be conducive to growing into for rete vasculosum, but there is inflammatory reaction, potential immunogenicity risk etc., and vascularization speed
Degree is undesirable；There are regulation and control when angiogenic growth factor is applied, half-life period is shorter in vivo for growth factor, heavy dose application
There is teratogenesis possible again, and some pathologic processes may be accelerated, it is such as angiomatous to occur；Internal heeling-in and internal artery cell
Second operation is both needed to, donor seletion is difficult, has potential transmission risk etc., limits clinical practice；Overall culture systems
For perfect " man-made organ ", but technical difficulty is high, realizes difficult.
In recent years, domestic and international researcher is built by vascular endothelial cell and the technical method of Gegenbaur's cell Combined culture
Go out study of vascularized tissue engineering bone, you can forming bone tissue again can vascularization simultaneously.But traditional " cell-scaffold " strategy is not
Foot part is：Seed cell is during plantation is to timbering material, and the cell for having 30-40% can not adhere to support and flow
Lose, cell attachment rate is low, utilization rate is poor；The interphase interaction of cell is few, it is difficult to form the due microenvironment of internal cell, cell
After scaffold complex implants, bon e formation always occurs in the periphery of support, influences the size and quality of skeletonization.In order to solve
Above mentioned problem, people work out cell patch technology.It is by vitro on cell carry out High Density Cultivation, make cell growth into
For multilayer and a large amount of extracellular matrixs are secreted, form the cell patch being made up of cell and extracellular matrix.Cell patch technology
Applied to mainly there is three aspect advantages in organizational project:First, the formation of diaphragm can reduce the stream of cell during tissue construction
Become estranged damage, improve cell utilization rate；2nd, cell patch has certain mechanical strength, can enter in the case of unsupported
Row tissue construction, avoids histocompatibility issues caused by timbering material, and easily operated, cheap；That the 3rd, enriches is thin
Extracellular matrix provides suitable growing environment for cell, and stores in growth course and activate growth factor, for the hair of tissue
Offer 26S Proteasome Structure and Function is educated to help.So, if Gegenbaur's cell diaphragm is combined into endothelial cell diaphragm, pass through suitable method structure
Build study of vascularized tissue engineering bone, it is contemplated that will be co-cultured compared with Gegenbaur's cell and endothelial cell and have more vascularization and skeletonization advantage.
Fat mesenchymal stem cell (adipose-derived stem cells, ADSCs) is present in adipose tissue
Adult stem cell.It is similar to mesenchymal stem cells MSCs, with into fat, into cartilage, skeletonization, sarcoblast and neural source
Many differentiation potentials of the differentiation such as cell, are a kind of preferable seed cells.Compared with mesenchymal stem cells MSCs, there is source wide
General, acquisition modes are simple, and fostering requirement is low, and amplification in vitro ability is strong, and generation time is short, the advantages of small to body wound, is expected to
As organizational project more preferably cell derived.At present, adipose-derived mescenchymal stem cell is widely used to regeneration
With reparation.Although the existing many researchs of the structure of study of vascularized tissue engineering bone, using adipose-derived Gegenbaur's cell and
Endothelial cell, preparation builds the strategy of study of vascularized tissue engineering bone with skeletonization and into double cell compound film sheets of vessel patency
But also few people are related to.
In addition, in addition to cell, the biocompatibility and biological degradability of timbering material be also influence vascularization because
Element.Timbering material must have good 3 D stereo loose structure, and such blood vessel can just creep and grow into, and final vascularization,
Precondition is provided for tissue engineered bone inner cell long term survival.
Current bone tissue engineering stent material includes inorganic material and the major class of organic material two.
Organic material substitutes field in hard tissue repair and is applied to bone earliest, and is widely used as bone renovating material, main
To include PLA (PLA), it is poly- acetic acid (PGA), polymeric polyglycolide-polylactide copolymer (PLGA), poly-epsilon-caprolactone (PCL), poly-
Acid anhydrides, polyphosphazene, poe etc..Studied in organic material it is more be polyhydroxy acid class (mainly including PLA, PGA,
PLGA).This family macromolecule polymer has obtained U.S. FDA approval due to its good biocompatibility, is widely used in doctor
Field.Wherein, PLGA is the high-molecular copolymer formed by poly- PLA and PGA, changes PLA and PGA ratio, be can adjust
PLGA mechanical strength and its degradation time in vivo.PLGA has good histocompatbility, has been approved by the FDA in the United States use
It is so far using one of most bone renovating material in clinic.But PLGA mechanical strengths are poor, catabolite is slightly acidic, easily
Cause internal inflammatory reaction, and because PLGA surface hydrophilicities are poor, activity functional groups are lacked in strand, its bioactivity
It is slightly worse, it is become relatively difficult with specific cells interaction.
Inorganic material for bone tissue engineering scaffold mainly include hydroxyapatite (HA), tricalcium phosphate (TCP) and
Other kinds of ceramic material etc..This kind of bioceramic material has good bioactivity and biocompatibility due to it, into
For wide variety of bone grafting substitute.Although it has good biocompatibility and certain degradability, higher chemistry are steady
The advantages of qualitative and stronger osteoacusis and osteoinductive.But this material have be difficult that moulding, intensity is not enough, fragility is big,
The low shortcoming of degradation rate.
The content of the invention
The purpose of the present invention is that the defect for overcoming above-mentioned prior art utilizes stem cell and cell patch there is provided one kind
Technique construction has good skeletonization and into vessel patency, and cell derived is abundant, it is easy to which separation is obtained, to donor wound
Small, fostering requirement is low, and effectively reduction produces the study of vascularized tissue engineering bone of the immune responses such as inflammation, rejection to host.
It is a further object of the present invention to provide the preparation method of above-mentioned study of vascularized tissue engineering bone.
The technical scheme taken for achieving the above object is：
A kind of study of vascularized tissue engineering bone, it is characterised in that it is the coral hydroxyapatite structure by polylysine modification
Into support and be wrapped in the (outer into endothelial cellular membrane piece (internal layer) and Gegenbaur's cell diaphragm of the rack surface successively
Layer) constitute.
It is above-mentioned to be used to prepare the endothelial cell into endothelial cellular membrane piece and for preparing Gegenbaur's cell diaphragm
Gegenbaur's cell derives from fat mesenchymal stem cell.
The preparation method of above-mentioned study of vascularized tissue engineering bone, it is characterised in that its technique includes：
1) preparation of the coral hydroxyapatite of polylysine modification
By coral hydroxyapatite mass concentration for 0.25%~1.25% Poly-L-Lysine Solution in 0~4 DEG C of temperature
15~24h is soaked under the conditions of degree, negative pressure leaching, puts -20 DEG C of refrigerator freezings and stay overnight afterwards, freeze-drying；
2) preparation of Gegenbaur's cell diaphragm
By the third generation or forth generation fat mesenchymal stem cell first with Trypsin Induced to most cell roundings, from
Bottom of bottle comes off, and then digestion is terminated with DMEM/F12 culture mediums, by 1 × 106~3 × 106Individual/cm2Density be seeded to culture dish
In, add on the DMEM/F12 culture mediums containing 10~12% hyclones and 1~1.5% dual anti-(penicillin, streptomysin), be placed in
37 DEG C, 5%CO224~48h is cultivated in constant incubator, is then within 10~14 days with the culture of Gegenbaur's cell induction broth again
Can；
3) into the preparation of endothelial cellular membrane piece
The third generation or forth generation fat mesenchymal stem cell are taken by 2 × 106~4 × 106Individual/cm2Density be inoculated in containing 10
24~48h is cultivated in~12% hyclone and 1~1.5% dual anti-DMEM/F12 culture mediums, it is then thin with blood vessel endothelium again
Born of the same parents' induction liquid is persistently cultivated 10~14 days；
4) structure of study of vascularized tissue engineering bone
By process 3) obtained by be wrapped in process 1 into endothelial cellular membrane piece) obtained by polylysine modification coral
Coral hydroxyapatite scaffold surface, parcel one enclose, then by process 2) obtained by Gegenbaur's cell diaphragm surround it is above-mentioned into blood vessel
Outside endothelial cell diaphragm, wrapping one is enclosed, and is formed volume layer sample complex, is fastened with silk thread.
Said process 1) in, concussion in every 3 hours shakes up soak.
Said process 1) in, the negative pressure leaching to have no any bubble from coral hydroxyapatite surface pore overflow i.e.
Can.
Said process 1) in, the freeze-drying refers to：By the coral soaked with Poly-L-Lysine Solution after freeze overnight
Coral hydroxyapatite is put into freeze drier, under the conditions of -42 DEG C~-47 DEG C of temperature, negative pressure 20-28Pa, after precooling 30min
Start to vacuumize, control 25~28Pa of vacuum, be freeze-dried 48~72h.
Said process 2) in, the Gegenbaur's cell induction broth composition is：DMEM/F12 170~180ml of culture medium, tire
Cow's serum 17~22ml, dual anti-1.7~2.7ml, 2~3ml of glutamine, 550~580ul of ascorbic acid, sodium β-glycerophosphate 2
~3ml, 15~25ul of dexamethasone.
Said process 2) in, during with induction broth culture, the next day change liquid culture.
Said process 3) in, the vascular endothelial cell induction liquid composition is：Endothelial Cell Growth Medium (EGM-2) 90
~110ml, 4.5~5.5ml of hyclone, 0.09~0.11ml of vitamin C, gentamicin-amphotericin B (GA-1000)
0.09~0.11ml, hydrocortisone (hydrocortisone) 0.036~0.044ml, hEGF (hEGF)
0.09~0.11ml, human fibroblastic growth factor (hFGF-B) 0.45~0.55ml, number growth factor of para-insulin
(IGF-I) 0.09~0.11ml, vascular endothelial growth factor (VEGF) 0.09~0.11ml.
Said process 3) in, induced with vascular endothelial cell in liquid incubation, liquid culture was changed every 2-3 days.
Said process 4) in, the coral hydroxyapatite support of the polylysine modification using it is preceding with containing 10~
The DMEM/F12 culture mediums of 12% hyclone and 1~1.5% dual anti-(penicillin, streptomysin) soak 12~24h.
Coral hydroxyapatite (coralline hydroxyapatite, CHA) is to react coral through hydrothermal exchange
The product arrived, the characteristics of remaining coral porous and high porosity dramatically increases former coral mechanical strength, and can lead to
The condition of control hydro-thermal conversion reaction is crossed to adjust the degradation rates of CHA in vivo.CHA not only overcomes the degraded of natural coral
Hurry up, it is fragile the shortcomings of, and prepare simple, there is a good osteoinductive and biocompatibility, and can with a variety of growths because
Son is combined or surface modification albumen equimolecular, is had a good application prospect in terms of repairing bone defect.
Poly-D-lysine (polylysine, PLL) is the polyvalent cation condensate being made up of lysine monomer, by multiple
Amino acid fragment is polymerized, and lysine can be decomposed into vivo and participates in body metabolism, without any side effects.It is commonly used for
The surface modification of bone tissue engineering stent material, main reason is that：1. lysine residue is positively charged in material surface, can increase
The adhesive capacity of strong negatively charged fibroblast etc.；2. amido, hydroxyl contained by it etc. can imitate extracellular matrix, so that
Improve the surface-active of material, improve cell adherence rate, promote cell growth, breeding；3. Subchondral drilling can be promoted；It is 4. safe,
Histocompatbility is good, and its analyte is essential amino acid；5. hydrophily is strong, beneficial to the adhesion between improvement cell and material
Deng.There is scholar's report PLL to handle cell culturing bracket, bone tissue engineering scaffold etc., cell adhesion forces and rate of vaccination can be increased,
It is a kind of good tissue engineering bracket material surface modification method.
The thought that the present invention is designed according to optimization of material, coral hydroxyapatite is modified with poly-D-lysine (PLL)
(CHA), so as to obtain good biocompatibility and the extraneous scaffold with 3 D stereo loose structure；It is dry thin using fat mesenchymal
Born of the same parents have the characteristic of multi-lineage potential, are induced differentiation, and preparing has osteogenic ability and double cell membranes into vessel patency
Piece；Stem cell and cell patch technology and bone tissue growth course are fully used for reference, the innovative vascularization group of compound structure is inquired into
The idea and method of Engineering Bone is knitted, skeletonization is finally constructed with the compound PLL/CHA of double cell patches and vascularization effect is preferable
Study of vascularized tissue engineering bone.The technical advantage of the study of vascularized tissue engineering bone of the present invention is embodied in the following aspects：
1st, the extraneous scaffold that coral hydroxyapatite (CHA) is obtained is modified with poly-D-lysine (PLL), is conducive to blood vessel to climb
Row is grown into, and final vascularization, precondition is provided for tissue engineered bone inner cell long term survival, while reducing immunity of organism
Rejection and inflammatory reaction；
2nd, double cell patches are prepared from fat mesenchymal stem cell, without the endothelial cell of extra addition separate sources,
Cell derived is enriched, it is easy to which separation is obtained, and small to donor wound, without prejudice to Ethical Principles, fostering requirement is low；
3rd, study of vascularized tissue engineering bone preparation method of the invention is simple, mild condition, with good skeletonization and into blood
Pipe performance, meets the requirement of biological vivo applications, is had a good application prospect as a kind of novel vascular tissue engineered bone.
4th, it is of the invention succeed in developing can be for preferable study of vascularized tissue engineering bone exploitation and bulk Cranial defect again
Raw treatment provides the proposition and experiment of experiment basis and theoretical foundation, especially double cell patches of fat stem cell induction, because
Operating technology is relatively easy, cost is relatively low, wound is small and skeletonization and vascularization effect preferably, make study of vascularized tissue engineering bone
Using with broader prospect, the need for meeting the various clinical sclerous tissues's defects of reparation.
Study of vascularized tissue engineering bone prepared by the present invention migrates to the nude mice dorsal sc of immune deficiency.It is postoperative to enter respectively
Row gross examination of skeletal muscle, SEM and histomorphometric analysis etc. detect the ability of its ectopic osteogenesis and the influence to revascularization.As a result table
It is bright：At the 8th week, filling collagen sample tissue, there is vascular distribution in experimental group brace aperture.There is different degrees of calcification within 12nd week
Visible a large amount of lumen of vessels in area, tissue.Masson dyeing is shown in：The 8th week nearly tissue sides of support color depth, and density is high, with new rubber
Based on fibrillation.Visible fiber different degrees of mineralising at the 12nd week, it is seen that a large amount of lumen of vessels, collagenous fibres are sparse.SEM is visible
(× 100) are filled in various degree by fibrous connective tissue in CHA holes.At 5000 times, as a result show：Fiber surface at the 12nd week
Great amount of hydroxy group apatite spline structure material is formed, extensively, mineralising is most ripe for area coverage.Coral hydroxyapatite support with it is double thin
The compound study of vascularized tissue engineering bone built of after birth piece has good skeletonization, into vascular performance.
Brief description of the drawings
Fig. 1 schemes for CHA (left side) and PLL/CHA (right side) SEM；
Fig. 2 is the pictorial diagram of Gegenbaur's cell diaphragm (left side) and endothelial cell diaphragm (right side)；
Fig. 3 is PLL/CHA supports and the compound study of vascularized tissue engineering bone pictorial diagram built of double cell patches；
Fig. 4 is that the double cell patch compounds of PLL/CHA/ are transplanted in nude mice by subcutaneous；
Fig. 5 is A groups transplanting 8 weeks (left sides) and (right side) HE dyeing (× 200) in 12 weeks；
Fig. 6 is the scanning electron microscope (SEM) photograph that A groups transplant 8 weeks (left sides) and 12 weeks (right side) (× 100).
Specific implementation method
The present invention is explained with example, it should be understood that example is to be used to illustrate rather than to this below
The limitation of invention.The scope of the present invention is determined with core content according to claims.
First, the preparation of the coral hydroxyapatite of polylysine modification and sign
(1) main agents
Coral hydroxyapatite (CHA) (China, Beijing meaning Hua Jian), poly-D-lysine (PLL) (U.S., Sigma).
(2) instrument and equipment
Supersonic wave cleaning machine (China, Kunshan instrument and equipment factory), (China, Shanghai is more public than bright instrument for vacuum freeze drier
Department), s-3400N SEM (Japan, HITACHI).
(3) experimental method
1. PLL soaks CHA
PLL is configured to 0.25%~1.25%w/v concentration with distilled water, through 0.22 μm of filter filtration sterilization.0~4
DEG C, 15~24h of CHA are soaked with the PLL solution prepared, concussion shakes up soak per 3h, PLL is fully contacted with CHA.
2. negative pressure leaching
The container for soaking CHA is placed in connection negative pressure leaching bottle, the sterile drying basin of negative pressure leaching machine, good seal joint
With drying basin lid mouthful, 20 DEG C of continuous negative pressure suction filtration 5h, until having no that any bubble overflows from CHA surface pores, take out immersion and hold
Device, puts -20 DEG C of refrigerator freezings and stays overnight.
3. it is freeze-dried
Soaking container is placed in freeze drier, preset -42 DEG C~-47 DEG C, negative pressure 20-28Pa, opened after precooling 30min
Beginning vacuumizes, 25~28Pa of vacuum.48h is freeze-dried, material is weighed and encapsulated, it is labelled, put -20 DEG C of low temperature refrigerators
Preserve stand-by.
4. characterize
Compound rest sample is taken, its surface observes the surface topography of support with SEM after ion sputtering instrument metal spraying plated film.
5. result
SEM results show that CHA and PLL/CHA are loose structure, and hole is mutually communicated, and pore diameter is 200-500 μ
m.Under high power lens, CHA surfaces are by the spherical hydroxyapatite crystal (diameter 500-4000nm) being made up of leaflet crystal, PLL/
Outside the hydroxyapatite crystal of CHA surfaces, also one layer PLL irregular crystalline solid.
2nd, using rabbit ADSCs (fat mesenchymal stem cell) inductions differentiation Gegenbaur's cell diaphragm is built respectively, into intravascular
Chrotoplast diaphragm
(1) main agents
Tetrazolium bromide (Alarmar Blue) (Shanghai, assist is holy), ascorbic acid (Ascrobic acid) (Beijing Suo Laibaosheng
Thing Science and Technology Ltd.), direct red 80 (Direct Red 80) (U.S. Sigma), DMEM/F12 culture mediums, phosphate-buffered
Liquid (PBS), hyclone (FBS) (U.S. Hyclone).
(4) instrument and equipment
Infinite M200Pro ELIASAs (Switzerland, Nano Quan), H-7650 transmission electron microscopes (Japan,
HITACHI), s-3400N SEM (Japan, HITACHI), petrographic microscope DM2500P (Germany, Leica), just
Put fluorescence microscope BX-511250CCD (Japan, Olympus), CO2 incubators (Heraeus companies, Germany).
(3) experimental method
1. the preparation of Gegenbaur's cell diaphragm
The good forth generation ADSCs (fat mesenchymal stem cell) of vegetative state is routinely used into 1% Trypsin Induced, extremely
Most cell roundings, come off from bottom of bottle, then terminate digestion with DMEM/F12 culture mediums.Cell density is adjusted to 1 × 106
~3 × 106Individual/cm2, be inoculated in advance with the coated culture dish of 1% gelatin, add contain 10~12% hyclones and 1~
The DMEM/F12 culture mediums of 1.5% dual anti-(penicillin, streptomysin), are placed in 37 DEG C, 5%CO2Cultivated in constant incubator, 24~
Be replaced by after 48h Gegenbaur's cell induction broth (DMEM/F12 170~180ml of culture medium, hyclone 17~22ml, it is dual anti-
(penicillin, streptomysin) 1.7~2.7ml, 2~3ml of glutamine, 550~580ul of ascorbic acid, sodium β-glycerophosphate 2~
3ml, 15~25ul of dexamethasone), the next day change liquid once, observe diaphragm forming process and formational situation, persistently cultivate 10~
14 days, the visible translucent milky film sample thing in ware bottom was light with cell scraper when having the tubercle that multiple whites differ in size in film
It is light to scrape, membranoid substance is separated with ware bottom, Gegenbaur's cell diaphragm can be obtained.
2. into the preparation of endothelial cellular membrane piece
The third generation or forth generation fat mesenchymal stem cell are taken by 2 × 106~4 × 106Individual/cm2Density be inoculated in containing 10
In the DMEM/F12 culture mediums of~12% hyclone and 1~1.5% dual anti-(penicillin, streptomysin) cultivate 24~48h, after more
Change into vascular endothelial cell induction liquid (Endothelial Cell Growth Medium (EGM-2) 90~110ml, 4.5~5.5ml of hyclone,
0.09~0.11ml of vitamin C, gentamicin-amphotericin B (GA-1000) 0.09~0.11ml, hydrocortisone
(hydrocortisone) 0.036~0.044ml, hEGF (hEGF) 0.09~0.11ml, human fibroblasts
Growth factor (hFGF-B) 0.45~0.55ml, number 0.09~0.11ml of growth factor (IGF-I) of para-insulin, blood vessel endothelium
Porcine HGF (VEGF) 0.09~0.11ml), the change of observed and recorded cellular morphology changed liquid every 2-3 days.Persistently cultivate 14
My god, endothelial cell diaphragm can be obtained.
3rd, the double cell patches of PLL/CHA/ build heterotopic transplantation experiment in study of vascularized tissue engineering bone and animal body
(1) main agents
Su Mian Xin II parenteral solutions (Jilin, Sheng Da), revive No. 3 parenteral solutions (Jilin, Sheng Da), Power DryHeto LL3000
Vacuum freeze drier (U.S., Thermo Fisher), remaining reagent is that analysis is pure.
(2) instrument and equipment
SHZ-D (III) circulating water types vavuum pump (China is given in Gongyi).
(3) experimental method
1. the double cell patch complexs of PLL/CHA/ build study of vascularized tissue engineering bone
PLL/CHA is cut into 0.5*0.5*2cm size cuboids, oxirane disinfection sterilizing, sterile packaged is standby.
Soaked using preceding with the DMEM/F12 culture mediums containing 10~12% hyclones and 1~1.5% dual anti-(penicillin, streptomysin)
12~24h.
Double cell patches (Gegenbaur's cell diaphragm and into endothelial cellular membrane piece) prepared by preceding method, are scraped with cell
Knife is careful to be scraped from culture dish bottom wall periphery, membranoid substance is separated with ware.
Support (coral hydroxyapatite of PLL modifications) inner surface, parcel one will be wrapped in into endothelial cellular membrane piece
Circle, then surrounds into Gegenbaur's cell diaphragm outside endothelial cellular membrane piece, and wrapping one is enclosed, and forms volume layer sample complex,
It is fastened with silk thread, is designated as complex A, is experimental group；Simple Gegenbaur's cell diaphragm and the wrapping of endothelial cell diaphragm are compound
The circle of support one builds complex and is designated as complex B and complex C respectively, is control group.It is stand-by after cultivating 3 days.
2. tested without the heterotopic transplantation of extraneous scaffold study of vascularized tissue engineering bone
By 36 nude mices, A groups, B groups, C groups are randomly divided into, the double cell patch complex nude mice by subcutaneous of PLL/CHA/ are implanted into
Detected within postoperative 8 weeks and 12 weeks (n=6/ groups/time point).Nude mice by subcutaneous is implanted into：Su Mian Xin liquid will be diluted according to 0.1ml-
0.2ml/20g intramuscular anesthesias, transverse incision is done in back, is subcutaneously implanted construction.Experimental group is complex A, and control group is
Complex B and diaphragm complex C.
3. characterize
Gross examination of skeletal muscle：Observation nude mice animation and wound healing situation and graft change after tissue engineered bone implantation；
Observation corium, surrounding tissue and graft relation before materials.
Histology：The graft of taking-up is divided into two parts, portion fixes 24h, SEM samples using 2.5% glutaraldehyde
Prepare, observe sample cross-section structure.A routine paraffin wax embedding, makes 5 μm of serial section, chooses 4, tissue block centre
Slice row HE is dyed, and carries out Microvessel Count according to report methods such as Weidner, and statistical is carried out to result with SPSS20.0
Analysis；4 slice row Masson dyeing are chosen, fiber mineralising situation is observed.
4. result
As a result show, after double cell patch complexs are combined with support, be brought into close contact.It is implanted into after nude mice by subcutaneous, otch is not
See inflammatory reaction.Postoperative 8th week, it is seen that perigraft tissue's adhesion, with capillary.12nd week, around graft
Tissue increases, and surface attachment thin vessels increase；Completely by faint yellow tissue filling in CHA holes, it is seen that capillary is stretched into.HE
Coloration result is visible：At the 8th week, it is full of in brace aperture in collagen sample tissue, tissue and is dispersed in the blood vessel that distributed quantity is not waited, can
Show cell, Gegenbaur's cell form.12nd week visible tissue inner cell quantity substantial increase, two groups of appearance of A, B are different degrees of
Calcification area, it is most obvious with A groups mineralising.C groups have no the blood vessel of visible mass efficient perfusion in mineralised zones, tissue.Masson contaminates
Color result is shown：It is full of in 8th week brace aperture by the collagenous fibres of inequality, fiber distribution is sparse, it is seen that vascularization.Three
Notable difference is had no between group.At the 12nd week, it is seen that the different degrees of mineralising of tissue fibers, wherein the most obvious with A groups.C groups can
See a large amount of lumen of vessels, collagenous fibres are sparse.SEM results show, in the 8th week visible three groups of CHA hole by fibrous connective tissue not
With degree filling.12nd week, three groups of CHA holes were filled completely by fibroid tissue completely, are tightly combined between tissue and support.
Microvessel Count result is shown：8th, 12 weeks when, tri- groups of capilary number group differences of A, B, C are statistically significant, at two
Between point component C not with the statistically significant (P of A, B group difference<0.05), illustrate C groups have into vessel patency apparently higher than other two
Group.At the 8th week, the also statistically significant (P of difference between A groups and B groups<0.05) A groups number of microvessels height of eye at the 8th week, is illustrated
In B groups.As a result show, the study of vascularized tissue engineering bone that the double cell patch complexs of PLL/CHA/ are built possess good skeletonization,
Into vascular performance.
Claims (11)
1. a kind of study of vascularized tissue engineering bone, it is characterised in that it is made up of the coral hydroxyapatite of polylysine modification
Support and be wrapped in being constituted into endothelial cellular membrane piece and Gegenbaur's cell diaphragm for the rack surface successively.
2. according to the study of vascularized tissue engineering bone described in claim 1, it is characterised in that described thin into blood vessel endothelium for preparing
The endothelial cell of after birth piece and derive from fat mesenchymal stem cell for the Gegenbaur's cell for preparing Gegenbaur's cell diaphragm.
3. a kind of preparation method of study of vascularized tissue engineering bone as claimed in claim 1 or 2, it is characterised in that its technique bag
Include：
1）The preparation of the coral hydroxyapatite of polylysine modification
By coral hydroxyapatite mass concentration for 0.25%~1.25% Poly-L-Lysine Solution in 0~4 DEG C of temperature conditions
15~24h of lower immersion, negative pressure leaching, puts -20 DEG C of refrigerator freezings and stays overnight afterwards, freeze-drying；
2）The preparation of Gegenbaur's cell diaphragm
The third generation or forth generation fat mesenchymal stem cell are first used into Trypsin Induced, then terminated with DMEM/F12 culture mediums
Digestion, by 1 × 106~3 × 106 Individual/cm2Density be seeded in culture dish, add containing 10~12% hyclones and 1~
On 1.5% dual anti-DMEM/F12 culture mediums, 37 DEG C, 5% CO are placed in2In constant incubator cultivate 24~48h, then again with into
Osteocyte induction broth culture 10~14 days；
3）Into the preparation of endothelial cellular membrane piece
The third generation or forth generation fat mesenchymal stem cell are taken by 2 × 106~4 × 106 Individual/cm2Density be inoculated in containing 10~
24~48h is cultivated in 12% hyclone and 1~1.5% dual anti-DMEM/F12 culture mediums, is then lured again with vascular endothelial cell
Drain is persistently cultivated 10~14 days；
4）The structure of study of vascularized tissue engineering bone
By process 3）Gained into endothelial cellular membrane piece is wrapped in process 1）The coral hydroxyl of the polylysine modification of gained
Base apatite rack surface, parcel one is enclosed, then by process 2）The Gegenbaur's cell diaphragm of gained surrounds above-mentioned into blood vessel endothelium
Outside cell patch, wrapping one is enclosed, and is formed volume layer sample complex, is fastened with silk thread.
4. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 1）In, concussion in every 3 hours shakes up
Soak.
5. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 1）In, the negative pressure leaching is not to
See that any bubble overflows from coral hydroxyapatite surface pore.
6. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 1）In, the freeze-drying is
Refer to：The coral hydroxyapatite soaked with Poly-L-Lysine Solution after freeze overnight is put into freeze drier, temperature-
42 DEG C~-47 DEG C, under the conditions of negative pressure 20-28Pa, start to vacuumize after precooling 30min, control 25~28Pa of vacuum, freezing is dry
Dry 48~72h.
7. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 2）In, the Gegenbaur's cell induction
Nutrient solution is constituted：DMEM/F12 170~180ml of culture medium, hyclone 17~22ml, dual anti-1.7~2.7ml, glutamy
2~3ml of amine, 550~580ul of ascorbic acid, 2~3ml of sodium β-glycerophosphate, 15~25ul of dexamethasone.
8. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 2）In, use induction broth culture
When, the next day change liquid culture.
9. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 3）In, the vascular endothelial cell
Induction liquid, which is constituted, is：90~110ml of Endothelial Cell Growth Medium, 4.5~5.5ml of hyclone, vitamin C 0.09~
0.11ml, gentamicin -0.09~0.11ml of amphotericin B, 0.036~0.044ml of hydrocortisone, human epidermal growth
0.09~0.11ml of the factor, 0.45~0.55ml of human fibroblastic growth factor, number growth factor 0.09 of para-insulin
~0.11ml, 0.09~0.11ml of vascular endothelial growth factor.
10. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 3）In, use vascular endothelial cell
Induce in liquid incubation, liquid culture was changed every 2-3 days.
11. according to the study of vascularized tissue engineering bone described in claim 3, it is characterised in that process 4）In, the poly-D-lysine
The coral hydroxyapatite support of modification is trained using preceding with the dual anti-DMEM/F12 containing 10~12% hyclones and 1~1.5%
Support base and soak 12~24h.
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Cited By (5)
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| CN107899079A (en) * | 2017-11-20 | 2018-04-13 | 华东交通大学 | Nano sheet-shaped hydroxyapatite/synthesized thin film and preparation method with brick mud structure |
| CN108578778A (en) * | 2018-05-17 | 2018-09-28 | 广东芙金干细胞再生医学有限公司 | A kind of cell for Ocular surface damage reparation plants piece and preparation method thereof |
| CN109112094A (en) * | 2018-08-10 | 2019-01-01 | 广东唯泰生物科技有限公司 | A kind of method that fat mesenchymal stem cell is induced to differentiate into vascular endothelial cell |
| CN110923201A (en) * | 2019-11-15 | 2020-03-27 | 广州医科大学附属口腔医院（广州医科大学羊城医院） | Method for enhancing osteogenesis capacity of human umbilical cord mesenchymal stem cells |
| CN113633824A (en) * | 2021-08-25 | 2021-11-12 | 宝鸡文理学院 | Hydroxyapatite coating based on polyether-ether-ketone and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107899079A (en) * | 2017-11-20 | 2018-04-13 | 华东交通大学 | Nano sheet-shaped hydroxyapatite/synthesized thin film and preparation method with brick mud structure |
| CN108578778A (en) * | 2018-05-17 | 2018-09-28 | 广东芙金干细胞再生医学有限公司 | A kind of cell for Ocular surface damage reparation plants piece and preparation method thereof |
| CN109112094A (en) * | 2018-08-10 | 2019-01-01 | 广东唯泰生物科技有限公司 | A kind of method that fat mesenchymal stem cell is induced to differentiate into vascular endothelial cell |
| CN110923201A (en) * | 2019-11-15 | 2020-03-27 | 广州医科大学附属口腔医院（广州医科大学羊城医院） | Method for enhancing osteogenesis capacity of human umbilical cord mesenchymal stem cells |
| CN110923201B (en) * | 2019-11-15 | 2024-04-02 | 广州医科大学附属口腔医院（广州医科大学羊城医院） | Method for enhancing osteogenic capacity of human umbilical mesenchymal stem cells |
| CN113633824A (en) * | 2021-08-25 | 2021-11-12 | 宝鸡文理学院 | Hydroxyapatite coating based on polyether-ether-ketone and preparation method thereof |
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