CN105854085A - Method for constructing tissue engineered cartilages in vivo - Google Patents
Method for constructing tissue engineered cartilages in vivo Download PDFInfo
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- CN105854085A CN105854085A CN201610261335.8A CN201610261335A CN105854085A CN 105854085 A CN105854085 A CN 105854085A CN 201610261335 A CN201610261335 A CN 201610261335A CN 105854085 A CN105854085 A CN 105854085A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, e.g. meniscus
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- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
The invention discloses a method for constructing tissue engineered cartilages in vivo, which comprises the following steps: carrying out multiplication culture on a great deal of auricular cartilage cells in vitro; preparing a stratified cartilage cell membrane; preparing a PLC (three-dimensional printing) inner core with a special form; preparing a sandwich mode of the stratified cartilage cell membrane and a material; and performing an operation for implanting a compound containing the membrane and the inner core into animal muscle. According to the method, a three-dimensional printed material is used as an inner core scaffold, and the material is coated by the cell membrane to prevent the scaffold material from being in direct contact with in vivo tissue, thereby reducing the inflammatory reaction caused by the scaffold material in vivo and increasing the success ratio of cartilage regeneration in vivo. In addition, through three-dimensional printing, inner core materials with various special forms can be manufactured, and tissue engineered cartilages corresponding to the inner core materials are constructed; and meanwhile, by adjusting the height of a printing layer, the density of grids and the mode of wiring, due to different superimposed layers of the membrane, the constructed cartilages have different mechanical strength which can reach a relatively high level.
Description
Technical field
The present invention relates to biological tissue field, particularly relate to a kind of application cladding chondrocyte diaphragm technology and combine
PCL inner nuclear material builds the method for the tissue engineering bone/cartilage with specific form in vivo.
Background technology
At present, in big animal construct in vitro large area and there is the tissue engineering bone/cartilage of certain mechanical strength become
A great problem and some demand clinically cannot be met, such as: the reparation of long section trachea defect.In recent years
The rise of tissue engineering technique and fast development are provided for new approaches, and it uses autogenous cell and material
Material support prebuild goes out to have the organization engineered cartilage of given shape.But existing structure tissue engineering bone/cartilage
Method owing to the most moulding, there are problems in the aspect such as mechanical strength and immunoreation, so not yet
Make a breakthrough.
Existing research is pointed out, aging chondrocyte there will be under the cultivation conditions of " cell accumulation " and divides
Change, again express chondrocyte characteristic phenotypic and secrete distinctive cartilage epimatrix.Studies have reported that simultaneously,
Utilize merely the external cladding of chondrocyte to cultivate and can build lamellar cartilage.The cartilage group built due to the method
Knit without timbering material, be the inflammatory reaction that autologous composition can avoid acellular factor to cause in vivo,
So being a kind of to be applicable to the construction method of regenerating bone or cartilage in big animal body.But, consequent cartilage is also
Without given shape, mechanical strength is poor simultaneously, tends not to meet the requirement of application.
But, there is now many biomaterials and have at morphology Control, the aspect such as mechanical strength and immunoreation
Good characteristic.Such as, pla-pcl (PCL) is through the one of ring-opening polymerisation by 6-caprolactone
New polymers, it not only has the features such as excellent biocompatibility, degradability and easy processing, simultaneously
It is the most moulding, and good mechanical properties, can by adjust PCL 3 D-printing floor height, mesh-density and
The approach such as mode that take the needle make its mechanical strength reach greater level.Superposed with cladding chondrocyte diaphragm by it
Sandwich pattern formed there is the tissue engineering bone/cartilage of specific form, can effective morphology Control solution mechanics
The problem of the aspect such as intensity and immunoreation.
Summary of the invention
A kind of method that it is an object of the invention to provide construct in vitro tissue engineering bone/cartilage, above-mentioned in order to overcome
Technological deficiency.
For achieving the above object, the present invention provides a kind of method of construct in vitro tissue engineering bone/cartilage, specifically walks
Rapid as follows:
S1, a large amount of amplification cultivation of Ear cartilage cells in vitro:
Cut auricular cartilage under S11, aseptic condition, add 0.25% trypsin of 3 times of volumes, put
After 37 DEG C of water bath with thermostatic control agitators digest 30min, rinse 3 times with 10mM phosphate buffer, heavy
Shallow lake cartilage block, removes trypsin；
S12, addition 0.15% II Collagenase Type, be made into respective concentration with cultured chondrocytes liquid, and 0.22
Micron pin type filter filters, and is placed in 37 DEG C of water bath with thermostatic control agitators digested 6～8h, until major part is soft
After bone piece is digested, it is thus achieved that Digestive system；
S13, by the Digestive system of gained with 100 micrometer cell strainer filterings, 1500r/min, centrifugal 5min,
The cell of the precipitation high sugar DMEM weight resisted containing 10% hyclone and 1% 3
After Xuan, count with Trypan Blue, and check chondrocyte vigor；
S14, count with hematimeter under inverted microscope after adjust cell density to 1x104/cm2
Concentration be seeded on 100mm culture dish, at 37 DEG C, 5%CO2, under the conditions of saturated humidity, thin with cartilage
Born of the same parents' culture fluid cultivates amplification, passes on when chondrocyte growth to 70%-80% merges；
S2, the preparation of cladding chondrocyte diaphragm:
S21, leave and take 3-4 culture dish when primary cell is inoculated with 3.0 × 104/cm2Cell density is inoculated
Chondrocyte, ware based on the cell of these culture dishs, with cartilaginous tissue culture fluid at 37 DEG C, 5%CO2,
Cultivate under the conditions of saturated humidity, change liquid after 3 days first, the next day of later, change liquid, until it is thin to build cladding cartilage
After birth sheet；
S22, remaining primary cell grow to pass on after 70-80% merges, the cell chondrocyte after passing on
Culture fluid is cultivated, until reaching P3 for chondrocyte；
S23, by P3 for cell dissociation, centrifugal, resuspended, with 8 × 105/cm2Concentration is inoculated in be left and taken before
Basic ware on, with cartilaginous tissue culture fluid cultivate, change liquid every other day；After cultivating 4 weeks, take off in culture dish
Play the cladding chondrocyte diaphragm of a diameter 100mm；
S3, make there is PCL kernel and the inner support of specific form:
S31, PCL granular materials is combined three-dimensional computer printing technique with heat fusing deposition rapid forming technology
The stock support with specific form made, obtains PCL kernel；Can be by adjusting the three-dimensional of inner nuclear material
Print floor height, mesh-density, the mechanical strength of the approach change kernels such as the mode that takes the needle.
S32, PCL granular materials is combined three-dimensional computer printing technique with heat fusing deposition rapid forming technology
The support with specific form of the atresia made, obtains inner support；
The preparation of the sandwich pattern complex of S4, cladding chondrocyte diaphragm and inner nuclear material:
Use cladding chondrocyte diaphragm, inner nuclear material, the pattern of cladding chondrocyte diaphragm make have special
Diaphragm/the material composite of form, and at periphery suture, three layers are fixed；
The operation of S5, complex implantation animal muscle:
When diaphragm/inner nuclear material complex is implanted to animal muscle environment, first isolate one piece of band blood supply
Thin layer fascia tissue parcel inner support, then by attached thereto for the complex of external structure.
Further, in above-mentioned steps S1.3 and step S2.2, described cultured chondrocytes formula of liquid is:
The hyclone of 10% is added on the basis of DMEM in high glucose culture fluid (Hyclone, Thermo, U.S.A)
(Hyclone, Thermo, U.S.A), Antibiotic solution (Hyclone, Thermo,
U.S.A) 1ml/100ml, and 2ng/mlbFGF.
Further, in above-mentioned steps S2.1 and step S2.3, described cartilaginous tissue culture fluid formula is:
Formula at DMEM in high glucose is: on the basis of DMEM in high glucose culture fluid (Hyclone, Thermo, U.S.A)
Add the hyclone (Hyclone, Thermo, U.S.A) of 10%, Antibiotic solution
(Hyclone, Thermo, U.S.A) 1ml/100ml, and 10ng/ml TGF-β 1.
Further, in above-mentioned steps S1.1, under aseptic condition, cut auricular cartilage, carefully peel off surface
Perichondrium, is cut into the piece of tissue of 1mm × 1mm × 1mm size, and chloromycetin flushing liquor rinses 3 times.
Further, in above-mentioned steps S2.3, by P3 for cell with 8 × 105/ cm2 concentration is inoculated in height
On the primary cell that density is cultivated, cladding cartilage can be constructed after cultivating 4 weeks with cartilaginous tissue culture fluid thin
After birth sheet.
Further, in above-mentioned steps S3, PCL kernel mesh spacing 2mm, the floor height 0.1mm of preparation,
Kernel concrete shape can be controlled by adjusting the setting that three-dimensional computer prints, changes the layer of kernel grid simultaneously
Height, mesh spacing, take the needle mode.PCL inner support tube is the pore-free material support of 3 D-printing, is used as body
As the inner support of tissue engineering bone/cartilage during interior transplanting.
Further, in above-mentioned steps S5, with chlore-ammonia ketone (5mg/kg) and Su Mian Xin when complex is implanted
(0.05-1ml/kg) intramuscular injection anesthesia, tracheal intubation is fixed.Row cervical region median incision cuts throat skin, solves
Cut the thin layer fascia tissue parcel inner support of one piece of band blood supply, then by the specific form that has of external structure
Complex with PBS rinse 3 times to remove serum composition, the most attached thereto.Dissect homonymy platysma, will
Muscle invests composite surface, then with 6-0 absorbable suture involutory muscle both side edges complex wrapped up into
In, carefully after hemostasis, layer-by-layer suture closes otch, draws materials after 8 weeks.Postoperative treat recovery from anesthesia, pull out trachea
Intubate, after situation is steady, send receptacle close observation, intramuscular injection penicillin in postoperative three days back to.
In another preference, described biodegradable inner nuclear material is except available polyglycolic acid (PGA)
Outside, also include the degradation material that pharmaceutically acceptable, biocompatibility is good, be selected from lower group:
Polylactic acid (PLA), PLGA, poly butyric, condensing model, poly-phosphazo, polyamino acid, false poly-amino
Acid, poe, polyester urethane, Merlon, Polyethylene Glycol, poly(ethylene oxide), poly-P-Dioxane
Ketone, collagen, gelatin, hyaluronic acid, ammonia polyose of candy, chitosan, chitin, alginate, de-cell
Substrate, and the various combinations etc. of these compositions.
The beneficial effects of the present invention is compared with prior art: the cartilage of present invention application band PCL kernel
Cladding cell patch can build the tissue engineering bone/cartilage with specific form in vivo, both can reduce support material
The inflammatory reaction that material causes in vivo, can effectively mould the tissue engineering bone/cartilage of construct in vitro simultaneously
Shape.It addition, which increase the biomechanical strength building cartilage, it is sufficient to meet trachea support function and resist
Various stress after trachea defect prothesis.
The present invention intends the PCL kernel that uses and has a significant advantage at the cartilage building the specific form such as tubulose: (1)
PCL plays support effect as kernel, and timbering material can be avoided directly to contact with in-vivo tissue, reduces support
The inflammatory reaction that material causes in vivo, improves the success rate of internal regenerating bone or cartilage.(2) cartilage is in vivo
During maturation, PCL material can constantly be degraded, and hard material long-term existence can be avoided may to cause not
Good reaction；(3) this support can be accurate by mould under conditions of conventional heating (about 60 DEG C) pressurizes
Moulding, after being cooled to room temperature or body temperature, form remains unchanged, it is clear that be conducive to form accurately control and maintain；
(4) this support can be prepared by 3 D-printing, mesh size, and web thickness, take the needle the key parameters such as mode
All can accuracy controlling.
The cartilaginous tissue that the cladding chondrocyte diaphragm technology that the present invention intends using builds in vitro is without support
Material, the inflammatory reaction that acellular factor can be avoided to cause in vivo, improves the success of internal regenerating bone or cartilage
Rate.Additionally, can be to Primary chondrocyte, the most autologous soft by a large amount of for chondrocyte amplification cultivation in this technology
The demand of osseous tissue substantially reduces.
What the present invention constructed have determines the tissue engineering bone/cartilage of form and enough biomechanical strengths, with full
Various demands to tissue engineering bone/cartilage the most clinically, this is that tissue engineering trachea etc. converts spy to clinical practice
The thinking of Suo Xin and method.
Accompanying drawing explanation
Fig. 1 is complex (diaphragm+kernel) preparation process；
In figure, A, B are that the front of tubulose PCL kernel is seen and side sight；C is the cladding of In vitro culture surrounding
Chondrocyte diaphragm；D, E, F are that the front of complex is seen, and side is seen and antapical view.
Detailed description of the invention
In order to make objects and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is carried out
Further describe.Should be appreciated that specific embodiment described herein only in order to explain the present invention,
It is not intended to limit the present invention.
Chondroblast
In the present invention, the source of chondroblast is not particularly limited, and can be that the cartilage of human or animal is thin
Born of the same parents, can derive from the various cartilages such as articular cartilage, costicartilage, Ear cartilage, nasal septal cartilage, tracheal cartilages
Tissue；Can also be other cell types of human or animal with cartilage differentiation potential, can derive from bone marrow,
Fat, muscle, skin etc. are organized.A kind of preferred source is soft from Ear cartilage or the joint of human or animal
Bone.
The method separating acquisition chondrocyte is the accepted method that document is repeatedly reported.A kind of preferably method is
Aseptic under general anesthesia or local anaesthesia cut cartilaginous tissue, after phosphate buffer (PBS) cleans, add 3 times of cartilages
The collagenase solution (concentration typically at 0.5-3mg/ml, is prepared with PBS or culture fluid) of volume, 37 DEG C of perseverances
Temperature vibration digestion 4-20 hour (depending on the source and digestion progress extent of cartilaginous tissue), is collected by filtration soft
Osteocyte suspension, centrifugal, washing, Trypan Blue, counted under microscope, Primary chondrocyte vigor one
As should be more than 80%.The method that stem cell induces differentiation into chondrocyte is also the side of this area routine
Method.
The cultivation of chondrocyte, propagating method and culture fluid are also well known in the art.A kind of preferably side
Method is to be cultivated in CO2 incubator by chondrocyte.Suitably culture fluid includes (but being not limited to):
1) F-12 culture medium or DMEM culture medium+5%-20% hyclone；2) F-12 culture medium or DMEM culture medium
+ 5%-20% autologous (or allosome) human serum；3) F-12/DMEM culture medium (1:1)+2%-20% hyclone or
Human serum.The one particularly preferred chondrocyte of class is the chondrocyte in the primary-the 3 generation of Isolation and culture.
Chondrocyte function and vigor now are all good, have the strongest Subchondral drilling ability, through Tri-labeling method
Learning dyeing proof has II expression of collagen, RT-PCR and in situ hybridization detection proof to have II Collagen Type VI and egg
The expression of white polysaccharide (aggrecan) mRNA.
Biodegradable inner nuclear material
The material that can be used for building organization engineered cartilage of the present invention is medically acceptable biodegradable
Material, including (but being not limited to):
(a) degradability synthesis macromolecular material, such as polylactic acid (PLA), polyglycolic acid (PGA), PLGA,
Poly butyric (PHB), condensing model (polyanhydrides), poly-phosphazo (polyphosphazenes),
Polyamino acid (polyamino acid), false polyamino acid (pesudo-polyamino acid), poly-ortho acid
Ester (polyorthoesters), polyester urethane (polyesterurethane), Merlon
(polycarbonate), Polyethylene Glycol, hyaluronic acid, poly-para-dioxanone (polydioxanone)
Deng；
(b) natural degradable material, such as collagen (collagen), gelatin (gelatin), ammonia polyose of candy
(glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginic acid
Salt and various acellular matrixes etc.；
C the copolymer of () above-mentioned material or compound material, especially macromolecular material are answered with natural material
Condensation material, and the composite of solid material and syringeability material.
The most medically acceptable Biodegradable material is solid material or solid, liquid composite wood
Material, such as polylactic acid (PLA), polyglycolic acid (PGA), collagen etc..When material is solid type material,
The size and shape that directly prefabricated one-tenth can be needed, it is also possible to by area of computer aided and rapid shaping
The model of technology customization carries out accurate plasticity to material.
Embodiment
S1, sheep Ear cartilage cell separation and cultivation；
S1.1, the aseptic credulous osteocomma cutting goat about 2cm × 2cm size, shredded into 1mm3Cartilage
Block, adds 0.25% trypsin of 3 times of volumes, is placed in 37 DEG C after repeatedly rinsing with chloromycetin solution
Water bath with thermostatic control agitator digestion 30min, to remove the fibrous connective tissue outside cartilaginous tissue, then uses phosphoric acid
Salt buffer (PBS) rinses 3 times, precipitates cartilage block, removes trypsin；
S1.2, addition 0.15% II Collagenase Type, be made into respective concentration with cultured chondrocytes liquid, and 0.22
Micron pin type filter filters, and uses immediately, is placed in 37 DEG C of water bath with thermostatic control agitators digested 6～8h；
S1.3 is until after major part cartilage block is digested, it is thus achieved that Digestive system by 100 micrometer cell filter screen mistakes
Filter, 1500r/min, centrifugal 5min, the cell of precipitation reaches with containing 10% hyclone and 1% 3 anti-high sugar
Er Baike MEM is resuspended, and gained cell counts with Trypan Blue, and checks chondrocyte
Vigor, with adjustment cell density after hematimeter counting to 1x10 under inverted microscope4/cm2Dense
Degree is seeded on 100mm culture dish, at 37 DEG C, 5%CO2, under the conditions of saturated humidity, trains with chondrocyte
Nutrient solution cultivates amplification, carries out passing on (figure one A, B) when chondrocyte growth to 70%-80% merges；
S2, the preparation of cladding chondrocyte diaphragm
S2.1, leave and take 3-4 culture dish (100mm) when primary cell is inoculated with 3.0 × 104/cm2Carefully
Born of the same parents' density inoculation chondrocyte, ware based on the cell of these culture dishs, with cartilaginous tissue culture fluid
37 DEG C, 5%CO2, cultivate under the conditions of saturated humidity, change liquid after 3 days first, the next day of later, change liquid, until
Build cladding chondrocyte diaphragm；
Remaining primary cell of S2.2 grows after nearly 70-80% merges and passes on, the cell chondrocyte after passing on
Culture fluid is cultivated, until reaching P3 for chondrocyte；
S2.3, by P3 for cell dissociation, centrifugal, resuspended, with 8 × 105/cm2Concentration is inoculated in be stayed before
On the basic ware taken, cultivate with cartilaginous tissue culture fluid, change liquid every other day；After cultivating 4 weeks, in culture dish
Uncover the cladding chondrocyte diaphragm (C in Fig. 1) of a diameter 100mm；
S3, making tubulose PCL kernel and inner support tube；PCL kernel is that PCL granular materials is with hot melt thaw collapse
Long-pending rapid shaping technique combines the latticed porous tubular scaffolds that three-dimensional computer printing technique is made, can be by adjusting
The 3 D-printing floor height of whole inner nuclear material, mesh-density, the mechanical strength of the approach change kernels such as the mode that takes the needle.
PCL inner support tube is the tubular bracket of atresia prepared by same method.
The preparation of the sandwich pattern complex of S4, cladding chondrocyte diaphragm and tubular material；Use cladding
Chondrocyte diaphragm, tubular material, the pattern of cladding chondrocyte diaphragm make tubular film/Material cladding
Thing, and at periphery suture by three layers fixing (D in Fig. 1, E, F)；
S5, using PCL pipe as inner support tube, tubular film/PCL complex is implanted to the other flesh of sheep trachea
Operation in meat；When tubular film/PCL inner nuclear material complex is implanted to animal neck muscle environment,
First isolate the thin layer fascia tissue parcel inner support tube of one piece of band blood supply, then the tubulose of external structure is combined
Thing is attached thereto.Draw materials after 8 weeks and carry out coherent detection assessment.
Result shows, has substantially formed tubulose cartilage seen from sight, and its surfaces externally and internally is porcelain white, all
Matter maturation cartilage.The dyeing display of histology HE, the cartilaginous tissue of formation is uniformly distributed lacuna spline structure,
And obvious inflammatory cell infiltration does not occur.Mechanical strength testing result proves, it can reach relatively flood
Flat.
The above is only the preferred embodiment of the present invention, it is noted that common for the art
For technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications,
These improvements and modifications also should be regarded as protection scope of the present invention.
Claims (7)
1. the method for a construct in vitro tissue engineering bone/cartilage, it is characterised in that comprise the steps:
S1, a large amount of amplification cultivation of Ear cartilage cells in vitro:
Cut auricular cartilage under S11, aseptic condition, add 0.25% trypsin of 3 times of volumes, put
After 37 DEG C of water bath with thermostatic control agitators digest 30min, rinse 3 times with 10mM phosphate buffer, heavy
Shallow lake cartilage block, removes trypsin；
S12, addition 0.15% II Collagenase Type, be made into respective concentration with cultured chondrocytes liquid, and 0.22
Micron pin type filter filters, and is placed in 37 DEG C of water bath with thermostatic control agitators digested 6～8h, until major part is soft
After bone piece is digested, it is thus achieved that Digestive system；
S13, by the Digestive system of gained with 100 micrometer cell strainer filterings, 1500r/min, centrifugal 5min,
The cell of the precipitation high sugar DMEM weight resisted containing 10% hyclone and 1% 3
After Xuan, count with Trypan Blue, and check chondrocyte vigor；
S14, count with hematimeter under inverted microscope after adjust cell density to 1x104/cm2
Concentration be seeded on 100mm culture dish, at 37 DEG C, 5%CO2, under the conditions of saturated humidity, thin with cartilage
Born of the same parents' culture fluid cultivates amplification, passes on when chondrocyte growth to 70%-80% merges；
S2, the preparation of cladding chondrocyte diaphragm:
S21, leave and take 3-4 culture dish when primary cell is inoculated with 3.0 × 104/cm2Cell density is inoculated
Chondrocyte, with cartilaginous tissue culture fluid at 37 DEG C, 5%CO2, cultivate under the conditions of saturated humidity, after 3 days
Change liquid first, the next day of later, change liquid, until building cladding chondrocyte diaphragm；
S22, remaining primary cell grow to pass on after 70-80% merges, the cell chondrocyte after passing on
Culture fluid is cultivated, until reaching P3 for chondrocyte；
S23, P3 is digested for chondrocyte, centrifugal, resuspended, with 8 × 105/cm2Before concentration is inoculated in
On the basic ware left and taken, cultivate with cartilaginous tissue culture fluid, change liquid every other day；After cultivating 4 weeks, in culture dish
In uncover the cladding chondrocyte diaphragm of a diameter 100mm；
S3, make there is PCL kernel and the inner support tube of specific form:
S31, PCL granular materials is combined three-dimensional computer printing technique with heat fusing deposition rapid forming technology
The stock support with specific form made, obtains PCL kernel；
S32, PCL granular materials is combined three-dimensional computer printing technique with heat fusing deposition rapid forming technology
Make the support with specific form of atresia, obtain inner support tube；
The preparation of the sandwich pattern complex of S4, cladding chondrocyte diaphragm and inner nuclear material:
Use cladding chondrocyte diaphragm, inner nuclear material, the pattern of cladding chondrocyte diaphragm make have special
Diaphragm/the material composite of form, and at periphery suture, three layers are fixed；
The operation of S5, complex implantation animal muscle:
When the diaphragm/inner nuclear material complex with specific form is implanted to animal muscle environment, first divide
Separate out the thin layer fascia tissue parcel inner support of one piece of band blood supply, then the complex of external structure is invested it
On.
The method of a kind of construct in vitro tissue engineering bone/cartilage the most according to claim 1, its feature exists
In, described cultured chondrocytes formula of liquid is: add 10% on the basis of DMEM in high glucose culture fluid
Hyclone, Antibiotic solution 1ml/100ml, and 2ng/mlbFGF.
The method of a kind of construct in vitro tissue engineering bone/cartilage the most according to claim 1, its feature exists
In, described cartilaginous tissue culture fluid formula is: add 10% on the basis of DMEM in high glucose culture fluid
Hyclone, Antibiotic solution1ml/100ml, and 10ng/ml TGF-β 1.
The method of a kind of construct in vitro tissue engineering bone/cartilage the most according to claim 1, its feature exists
In, in above-mentioned steps S1.1, under aseptic condition, cut auricular cartilage, carefully peel off the perichondrium on surface,
Being cut into the piece of tissue of 1mm × 1mm × 1mm size, chloromycetin flushing liquor rinses 3 times.
The method of a kind of construct in vitro tissue engineering bone/cartilage the most according to claim 1, its feature exists
In, in above-mentioned steps S2.3, by P3 for cell with 8 × 105/cm2Concentration is inoculated in High Density Cultivation
On primary cell, cladding chondrocyte diaphragm after cultivating 4 weeks with cartilaginous tissue culture fluid, can be constructed.
The method of a kind of construct in vitro tissue engineering bone/cartilage the most according to claim 1, its feature exists
In, in above-mentioned steps S3, the mesh spacing 2mm of the PCL kernel of preparation, floor height 0.1mm.
The method of a kind of construct in vitro tissue engineering bone/cartilage the most according to claim 1, its feature exists
In, in described step S5, the chlore-ammonia ketone of 5mg/kg and 0.05-1ml/kg when complex is implanted
After Su Mian Xin intramuscular injection anesthesia, fixed by tracheal intubation.
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