CN103948969A - Method for constructing tissue engineered cartilage by using PCL kernel-containing material - Google Patents
Method for constructing tissue engineered cartilage by using PCL kernel-containing material Download PDFInfo
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- CN103948969A CN103948969A CN201410163412.7A CN201410163412A CN103948969A CN 103948969 A CN103948969 A CN 103948969A CN 201410163412 A CN201410163412 A CN 201410163412A CN 103948969 A CN103948969 A CN 103948969A
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Abstract
The invention relates to a method for constructing tissue engineered cartilage by using a PCL kernel-containing material. The method comprises the steps of a, making a PLC kernel; b, making a PLC kernel-containing PGA/PLA compound ear bracket; c, making a PGA/PLA compound ear bracket; d, culturing cartilage cells; e, constructing cartilage auricle bracket; and f, nude mouse subcutaneous replantation of in-vitro constructed auricle: respectively carrying out nude mouse subcutaneous implantation on the cartilage cells and an ear bracket compound after culturing in vitro for a week. According to the method, the tissue engineered cartilage can be constructed by using the PLC kernel-containing material, so that enough strength is provided to resist the tension caused by the subcutaneous implantation and then the original complicated and precise shape is maintained.
Description
Technical field
The present invention relates to biological tissue field, relates in particular to a kind of method that application band PCL inner nuclear material builds tissue engineering bone/cartilage.
Background technology
Microtia (microtia) is that the body surface that sickness rate is the highest after harelip is in congenital malformation, and traditional treatment mainly be take autologous costal cartilage grafting as main, and Dui Gong causes in district very big damage.Theoretically, tissue engineering technique is at least obviously better than traditional treatment pattern aspect following three: (1) is little to patient trauma, after the cell amplification that can take a morsel, builds bulk cartilage; (2) cartilage building has function, is structure and reconstruction truly; (3) can be accurately moulding, more meet clinical practice demand.
Yet the cartilage of external structure of the prior art is because mechanical property is not enough, is difficult to overcome subcutaneous tension force after implanting in animal body and maintains original form.Although titanium net supporter auxiliary transplantation can solve form in nude mouse and maintain problem, in large animal body, can cause foreign body reaction and disturb regenerating bone or cartilage.
In view of above-mentioned defect, creator of the present invention has obtained this creation finally through long research and practice.
Summary of the invention
The object of the present invention is to provide a kind of method of application band PCL inner nuclear material structure tissue engineering bone/cartilage, in order to overcome above-mentioned technological deficiency.
For achieving the above object, the invention provides a kind of method that application band PCL inner nuclear material builds tissue engineering bone/cartilage, detailed process is: step a, makes PLC kernel; PCL kernel is the latticed porous support that PCL granular materials is made with heat fusing deposition rapid forming technology, latticed PCL is cut into and is applicable to size, be placed in the pressurization of auricle mould fixing, heating pressurization, until being cooled in pressure process after room temperature, take out into the PCL kernel of auricle type, prune excess stock;
Step b, makes band PCL kernel PGA/PLA composite ear support;
Step b1, tears long fibre to make sub-thread PGA non-woven cotton, and PGA non-woven cotton is layered in special-purpose rectangle box uniformly, is made into rectangular small pieces, and every agreement that contracts a film or TV play to an actor or actress 150mg, needs 2 altogether;
Step b2, is placed in auricle mould by above-mentioned small pieces and with dehydrated alcohol, soaks fullly, and pressurization is fixing, after absolute ethanol volatilizes, and auricle form preliminarily stabilised;
Step b3, drip the PLA dichloromethane solution of 0.3%-3%, and it is moulding in mould, to continue pressurization, first weighs before dripping PLA solution at every turn, when material is about 158-165mg, no longer drip PLA solution, finally obtain two auricle form PGA/PLA compound rests;
Step b4, is superimposed on the latticed PCL of above-mentioned auricle form between above-mentioned two auricle form PGA supports, is placed in the pressurization of auricle mould and fixes, and heating pressurization is cooling, makes micro-PCL melting and upper and lower two-layer PGA/PLA compound rest close adhesion;
Step b5, takes out nuclear scaffold in the band prepare, prunes unnecessary material, then drips and in mould, pressurize after the PLA dichloromethane solution of a 0.3%-3% mouldingly, finally forms nuclear scaffold in the band with auricle form true to nature;
Step c, makes PGA/PLA composite ear support;
B is identical with above-mentioned steps, and long fibre is torn and made sub-thread PGA non-woven cotton, is made into rectangular small pieces, and every agreement that contracts a film or TV play to an actor or actress 300mg drips 1%PLA, when material is about 330mg, has prepared, and is trimmed to auricle shape standby;
Steps d, cultured chondrocytes;
Steps d 1, cuts the one-sided auricular cartilage of new zealand white rabbit under aseptic condition, add 0.25% trypsin of 3 times of volumes, be placed in 37 ℃ of water bath with thermostatic control agitator digestion 30min, then use phosphate buffer (PBS) to rinse 3 times, sedimentation cell, removes trypsin;
Steps d 2, adds 0.15%II Collagenase Type, with cultured chondrocytes liquid, is made into respective concentration, and 0.22 micron of pin type filter filters, and uses immediately, is placed in digestion 6～8h in 37 ℃ of water bath with thermostatic control agitators:
Steps d 3, until most of cartilage piece digested after, the 100 microns of cell strainer filterings for Digestive system that obtain, 1500r/min, centrifugal 5min, the cell of precipitation is with resuspended containing 10% hyclone and 1% 3 anti-height sugar DMEM, and gained cell is counted with Trypan Blue, and check chondrocyte vigor, under inverted microscope, after hematimeter counting, adjust cell density to 1x10
4/ cm
2concentration be seeded on 100mm culture dish, at 37 ℃, 5%CO
2, under saturated humidity condition, with cultured chondrocytes liquid, cultivate amplification, until chondrocyte, grow to when 70%-80% merges and go down to posterity;
Step e, auricular branch framework is built cartilage;
By auricle support, with after 75% ethanol disinfection, PBS rinses 3 times, and cultured chondrocytes liquid will prop up and be placed on preculture in culture fluid after rinsing 3 times; The first generation (P1) rabbit ear chondrocyte is collected with 0.25% trypsinization, with resuspended to concentration 100 * 10 containing 10%FBS and 1% 3 anti-DMEM culture fluid
6/ ml, equivalent is seeded on two pack support materials uniformly;
Step f, the nude mice by subcutaneous Hui Zhi of external structure auricle, carries out respectively nude mice by subcutaneous implantation by chondrocyte-ear supporter complex In vitro culture after 1 week.
Further, in above-mentioned steps f, during implantation, adopt chloral hydrate intraperitoneal injection of anesthesia nude mice, at the about 2cm of buttocks lateral dissection skin, with ophthalmology curved scissors, at subcutaneous cephalad, isolate lacuna; By cell material complex with PBS rinsing 3 times to remove serum composition, little drag hook exposes the lacuna of separating, ophthalmic tweezers is grasping tissue edge gently, tissue is sent in subcutaneous space, the ear supporter concave face skin that reclines, fixes position, with 5-0 nylon line suture otch; With 1mm syringe, by skin, thrust, suction air, makes skin and sarcolemma be close to cell material complex, after 8 weeks, draws materials.
Further, in above-mentioned steps e, chondrocyte equivalent is seeded on two pack support materials uniformly, at 37 ℃, 5%CO
2, under saturated humidity condition, cultivate after 4 hours, add cultured chondrocytes liquid, i.e. 10% hyclone, 1% 3 anti-DMEM culture medium, continues to cultivate, and changes every other day liquid.
Further, in above-mentioned steps d1, cut the one-sided auricular cartilage of new zealand white rabbit under aseptic condition, the careful perichondrium of stripper surface, is cut into the piece of tissue of 1mm * 1mm * 1mm size, and chloromycetin flushing liquor rinses 3 times.
Further, in above-mentioned steps b3, while dripping PLA solution, PLA content accounts for the 5%-10% of total PGA/PLA support.
Further, in above-mentioned steps b4, under 55-70 ℃ of high temperature, heat, within 1 hour, being placed under pressure device 200N-500N, to be forced into mould thoroughly cooling.
Further, in above-mentioned steps a, mesh spacing 1-3mm, floor height 0.1mm.
Further, in above-mentioned steps a, at 55-70 ℃, heat after about 5-10 minute, pressurization rapidly, pressure is 200N-500N.
Beneficial effect of the present invention is compared with prior art: the present invention applies band PCL inner nuclear material can build tissue engineering bone/cartilage, thereby have enough subcutaneous the brought tension force of intensity antagonism implantation, maintains the accurate shape of original complexity.
The present invention intends the netted PCL kernel that adopts and has significant advantage building aspect the specific form cartilages such as auricle: (1) this support can be prepared by 3 D-printing, mesh size, and the key parameters such as grid thickness all can accuracy controlling; (2) this support can be accurately moulding by mould under the condition of conventional heating (approximately 60 ℃) pressurization, and after cool to room temperature or body temperature, form remains unchanged, and is obviously conducive to auricle form and accurately controls and maintain; (3) under the prerequisite of parcel PGA fiber, heating pressurization is moulding, in PCL, endorse and the adhesion of PGA fibre compact, and mesh part also can be filled by PGA, and the phenomenon of kernel and peripheral material disengaging can not occur.
In addition, the cell of inoculation guides the PCL mesh of growing into inner through PGA, through In vitro culture, after PGA degraded, formed chondroid tissue not only fully covers PCL surface, and its mesh of also fully growing into is inner, form anchoring structure, further prevented that peripheral organization and inner nuclear material from departing from.
The present invention constructs has accurate form and not in time and the organizational project auricular cartilage of distortion transform to be explored new thinking and method for organizational project is pleasant to clinical practice.
The specific embodiment
Below, to the present invention is above-mentioned, be described in more detail with other technical characterictic and advantage.
The detailed process that the present invention application builds the method for tissue engineering bone/cartilage with PCL inner nuclear material is,
Step a, makes PLC kernel; PCL kernel is the latticed porous support that PCL granular materials is made with heat fusing deposition rapid forming technology, in the present embodiment, and mesh spacing 2mm, floor height 0.1mm.Latticed PCL is cut into and is applicable to size, be placed in the pressurization of auricle mould and fix.At 55-70 ℃, heat after about 5-10 minute, pressurization rapidly, pressure is 200N-500N; Until being cooled to after room temperature in pressure process, take out into the PCL kernel of auricle type, prune excess stock.
Step b, makes band PCL kernel PGA/PLA composite ear support;
Step b1, tears long fibre to make sub-thread PGA non-woven cotton, and PGA non-woven cotton is layered in special-purpose rectangle box uniformly, is made into rectangular small pieces, and every agreement that contracts a film or TV play to an actor or actress 150mg, needs 2 altogether;
Step b2, is placed in auricle mould by above-mentioned small pieces and with dehydrated alcohol, soaks fullly, and pressurization is fixing, after absolute ethanol volatilizes, and auricle form preliminarily stabilised;
Step b3, drip the PLA dichloromethane solution of 0.3%-3%, and it is moulding in mould, to continue pressurization, first weighs before dripping PLA solution at every turn, when material is about 158-165mg, no longer drip PLA solution, finally obtain two auricle form PGA/PLA compound rests; While dripping PLA solution, PLA content accounts for the 5%-10% of total PGA/PLA support; In general, do not use the very few PGA fiber that easily makes of PLA or PLA too fluffy and be unfavorable for moulding; But PLA content too much easily causes again material too fine and close, causes PGA fiber to be difficult to fully fill PGL mesh, can affect in addition the porosity, hydrophilic of PGA/PLA material etc.; General PLA content can not cause obvious impact to the biocompatibility of material while being less than 20%.
Step b4, the latticed PCL of above-mentioned auricle form is superimposed between above-mentioned two auricle form PGA supports, be placed in the pressurization of auricle mould fixing, under 55-70 ℃ of high temperature, heat, within 1 hour, being placed under pressure device 200N-500N, to be forced into mould thoroughly cooling, makes micro-PCL melting and upper and lower two-layer PGA/PLA compound rest close adhesion;
Step b5, takes out nuclear scaffold in the band prepare, prunes unnecessary material, then drips and in mould, pressurize after the PLA dichloromethane solution of a 0.3%-3% mouldingly, finally forms nuclear scaffold in the band with auricle form true to nature; In like manner can also produce the support of other forms, as circle, triangle, square, trachea shape etc.
Step c, makes PGA/PLA composite ear support;
B is identical with above-mentioned steps, and long fibre is torn and made sub-thread PGA non-woven cotton, is made into rectangular small pieces, and every agreement that contracts a film or TV play to an actor or actress 300mg drips 1%PLA, when material is about 330mg, has prepared, and is trimmed to auricle shape standby.
Steps d, cultured chondrocytes;
Steps d 1, cuts the one-sided auricular cartilage of new zealand white rabbit under aseptic condition, the careful perichondrium of stripper surface is cut into the piece of tissue of 1mm * 1mm * 1mm size, and chloromycetin flushing liquor rinses 3 times; 0.25% trypsin that adds 3 times of volumes, is placed in 37 ℃ of water bath with thermostatic control agitator digestion 30min, then uses phosphate buffer (PBS) to rinse 3 times, and sedimentation cell, removes trypsin;
Steps d 2, adds 0.15%II Collagenase Type, and NB4, is made into respective concentration with cultured chondrocytes liquid, and 0.22 micron of pin type filter filters, and uses immediately, is placed in digestion 6～8h in 37 ℃ of water bath with thermostatic control agitators;
Steps d 3, until most of cartilage piece digested after, the 100 microns of cell strainer filterings for Digestive system that obtain, 1500r/min, centrifugal 5min, the cell of precipitation is with resuspended containing 10% hyclone and 1% 3 anti-height sugar DMEM, and gained cell is counted with Trypan Blue, and check chondrocyte vigor, under inverted microscope, after hematimeter counting, adjust cell density to 1x10
4/ cm
2concentration be seeded on 100mm culture dish, at 37 ℃, 5%CO
2, under saturated humidity condition, with cultured chondrocytes liquid, cultivate amplification, until chondrocyte, grow to when 70%-80% merges and go down to posterity.
Step e, auricular branch framework is built cartilage;
By auricle support, with after 75% ethanol disinfection, PBS rinses 3 times, and cultured chondrocytes liquid will prop up and be placed on preculture in culture fluid after rinsing 3 times; The first generation (P1) rabbit ear chondrocyte is collected with 0.25% trypsinization, with resuspended to concentration 100 * 10 containing 10%FBS and 1% 3 anti-DMEM culture fluid
6/ ml, equivalent is seeded on two pack support materials uniformly.At 37 ℃, 5%CO2, cultivates after 4 hours under saturated humidity condition, adds cultured chondrocytes liquid, i.e. 10% hyclone, and 1% 3 anti-DMEM culture medium, continues to cultivate, and changes every other day liquid.
Step f, the nude mice by subcutaneous Hui Zhi of external structure auricle;
Chondrocyte-ear supporter complex In vitro culture is carried out respectively to nude mice by subcutaneous implantation after 1 week; During implantation, adopt chloral hydrate intraperitoneal injection of anesthesia nude mice, at the about 2cm of buttocks lateral dissection skin, with ophthalmology curved scissors, at subcutaneous cephalad, isolate lacuna; By cell material complex with PBS rinsing 3 times to remove serum composition, little drag hook exposes the lacuna of separating, ophthalmic tweezers is grasping tissue edge gently, tissue is sent in subcutaneous space, the ear supporter concave face skin that reclines, fixes position, with 5-0 nylon line suture otch; With 1mm syringe, by skin, thrust, suction air, makes skin and sarcolemma be close to cell material complex, after 8 weeks, draws materials.
The present invention is in vitro in building process, and peripheral PGA material is fully degraded, and the secreted autologous cartilaginous tissue of chondrocyte being vaccinated thereon substitutes, the foreign body reaction that therefore can effectively avoid PGA support to cause; External structure is formed is chondroid tissue, but not simple cell material complex, so performance is more stable, is also more conducive to operation; External structure technology platform is conducive to utilize from now on stem cell constructing cartilage, because stem cell must experience In vitro culture, realizes ripe cartilage directed differentiation.
Interpretation of result:
Chondrocyte and material
General 12～24 hours of primary chondrocyte is adherent, and drawout completely after 48 hours, starts propagation.When chondrocyte is just adherent, be still circular, ovalize, triangle and polygon after stretching gradually, nucleus be circle or oval.After going down to posterity, chondrocyte increases acceleration, and somatoblast is common, is polygon mostly, refractivity is strong, within general about 5 to 7 days, can reach more than 90% fusion state, immunofluorescence dyeing nucleus PI lining dyes for redness, expresses and has the cell pulp of II Collagen Type VI for green.
In vitro culture is used in 1 week and is inverted the demonstration of aberration microscope Continuous Observation, and the chondrocyte of experimental group and matched group stably sticks on PGA material fiber, and division growth, gradually extracellular matrix secretion.Biocompatibility detection display experimental group and matched group DNA detection by quantitative indifference, result has statistical significance, shows that PCL kernel does not affect the adhesion of chondrocyte on material.Scanning electron microscope shows, the cell of two groups of materials is drawout form abundant extracellular matrix completely all, on the visible PCL kernel of experimental group tangent plane, also has cell adhesion and secretes substrate.
PCL-PGA/PLA composite ear support of the present invention, the auricle form cartilage based on this ear supporter external structure succeeds, and can long term maintenance form after implanting in nude mouse.The auricle in the past building based on PGA/PLA auricular branch cannot maintain form in nude mouse; With titanium net supporter auxiliary transplantation, contribute to form to maintain, but may cause foreign body reaction in large animal body.Therefore, the present invention adopts PCL support as kernel, peripheral parcel PGA fiber, then through PLA embedding, form PCL-PGA/PLA composite ear support; At present, based on this stent applications animal cartilage cells in vitro, build auricle form cartilage and succeed, and can long term maintenance form after implanting in nude mouse.
By extending Time in Vitro, improve external structure cartilage quality, solve and utilize cell material complex at immune animal internal regeneration cartilaginous tissue.
The PLA embedding PGA that adopts 10%～20% content, not only increases the mechanical property of timbering material but also delay its degradation rate, realizes in vitro and builds product form long sustaining; Adopt medical pure titanium inner support net auxiliary auricle form that maintains when subcutaneous implantation; Subcutaneous by implanting after the auricle removal inner support of above-mentioned construct in vitro, form can continue to maintain again.
PCL inner nuclear material contributes to maintain the accurate form of auricle in nude mouse in building process, and good biocompatibility, can form the PCL cartilage complex of consolidation.
The foregoing is only preferred embodiment of the present invention, is only illustrative for invention, and nonrestrictive.Those skilled in the art is understood, and in the spirit and scope that limit, can carry out many changes to it in invention claim, revise, and even equivalence, but all will fall within the scope of protection of the present invention.
Claims (8)
1. application builds a method for tissue engineering bone/cartilage with PCL inner nuclear material, it is characterized in that, this detailed process is:
Step a, makes PLC kernel; PCL kernel is the latticed porous support that PCL granular materials is made with heat fusing deposition rapid forming technology, latticed PCL is cut into and is applicable to size, be placed in the pressurization of auricle mould fixing, heating pressurization, until being cooled in pressure process after room temperature, take out into the PCL kernel of auricle type, prune excess stock;
Step b, makes band PCL kernel PGA/PLA composite ear support;
Step b1, tears long fibre to make sub-thread PGA non-woven cotton, and PGA non-woven cotton is layered in special-purpose rectangle box uniformly, is made into rectangular small pieces, and every agreement that contracts a film or TV play to an actor or actress 150mg, needs 2 altogether;
Step b2, is placed in auricle mould by above-mentioned small pieces and with dehydrated alcohol, soaks fullly, and pressurization is fixing, after absolute ethanol volatilizes, and auricle form preliminarily stabilised;
Step b3, drip the PLA dichloromethane solution of 0.3%-3%, and it is moulding in mould, to continue pressurization, first weighs before dripping PLA solution at every turn, when material is about 158-165mg, no longer drip PLA solution, finally obtain two auricle form PGA/PLA compound rests;
Step b4, is superimposed on the latticed PCL of above-mentioned auricle form between above-mentioned two auricle form PGA supports, is placed in the pressurization of auricle mould and fixes, and heating pressurization is cooling, makes micro-PCL melting and upper and lower two-layer PGA/PLA compound rest close adhesion;
Step b5, takes out nuclear scaffold in the band prepare, prunes unnecessary material, then drips and in mould, pressurize after the PLA dichloromethane solution of a 0.3%-3% mouldingly, finally forms nuclear scaffold in the band with auricle form true to nature;
Step c, makes PGA/PLA composite ear support;
B is identical with above-mentioned steps, and long fibre is torn and made sub-thread PGA non-woven cotton, is made into rectangular small pieces, and every agreement that contracts a film or TV play to an actor or actress 300mg drips 1%PLA, when material is about 330mg, has prepared, and is trimmed to auricle shape standby;
Steps d, cultured chondrocytes;
Steps d 1, cuts the one-sided auricular cartilage of new zealand white rabbit under aseptic condition, add 0.25% trypsin of 3 times of volumes, be placed in 37 ℃ of water bath with thermostatic control agitator digestion 30min, then use phosphate buffer (PBS) to rinse 3 times, sedimentation cell, removes trypsin;
Steps d 2, adds 0.15%II Collagenase Type, with cultured chondrocytes liquid, is made into respective concentration, and 0.22 micron of pin type filter filters, and uses immediately, is placed in digestion 6～8h in 37 ℃ of water bath with thermostatic control agitators;
Steps d 3, until most of cartilage piece digested after, the 100 microns of cell strainer filterings for Digestive system that obtain, 1500r/min, centrifugal 5min, the cell of precipitation is with resuspended containing 10% hyclone and 1% 3 anti-height sugar DMEM, and gained cell is counted with Trypan Blue, and check chondrocyte vigor, under inverted microscope, after hematimeter counting, adjust cell density to 1x10
4/ cm
2concentration be seeded on 100mm culture dish, at 37 ℃, 5%CO
2, under saturated humidity condition, with cultured chondrocytes liquid, cultivate amplification, until chondrocyte, grow to when 70%-80% merges and go down to posterity;
Step e, auricular branch framework is built cartilage;
By auricle support, with after 75% ethanol disinfection, PBS rinses 3 times, and cultured chondrocytes liquid will prop up and be placed on preculture in culture fluid after rinsing 3 times; The first generation (P1) rabbit ear chondrocyte is collected with 0.25% trypsinization, with resuspended to concentration 100 * 10 containing 10%FBS and 1% 3 anti-DMEM culture fluid
6/ ml, equivalent is seeded on two pack support materials uniformly;
Step f, the nude mice by subcutaneous Hui Zhi of external structure auricle, carries out respectively nude mice by subcutaneous implantation by chondrocyte-ear supporter complex In vitro culture after 1 week.
2. application band PCL inner nuclear material according to claim 1 builds the method for tissue engineering bone/cartilage, it is characterized in that, in above-mentioned steps f, during implantation, adopt chloral hydrate intraperitoneal injection of anesthesia nude mice, at the about 2cm of buttocks lateral dissection skin, with ophthalmology curved scissors, at subcutaneous cephalad, isolate lacuna; By cell material complex with PBS rinsing 3 times to remove serum composition, little drag hook exposes the lacuna of separating, ophthalmic tweezers is grasping tissue edge gently, tissue is sent in subcutaneous space, the ear supporter concave face skin that reclines, fixes position, with 5-0 nylon line suture otch; With 1mm syringe, by skin, thrust, suction air, makes skin and sarcolemma be close to cell material complex, after 8 weeks, draws materials.
3. application band PCL inner nuclear material according to claim 1 and 2 builds the method for tissue engineering bone/cartilage, it is characterized in that, in above-mentioned steps e, chondrocyte equivalent is seeded on two pack support materials uniformly, and at 37 ℃, 5%CO
2, under saturated humidity condition, cultivate after 4 hours, add cultured chondrocytes liquid, i.e. 10% hyclone, 1% 3 anti-DMEM culture medium, continues to cultivate, and changes every other day liquid.
4. application band PCL inner nuclear material according to claim 3 builds the method for tissue engineering bone/cartilage, it is characterized in that, in above-mentioned steps d1, under aseptic condition, cut the one-sided auricular cartilage of new zealand white rabbit, the perichondrium of careful stripper surface, be cut into the piece of tissue of 1mm * 1mm * 1mm size, chloromycetin flushing liquor rinses 3 times.
5. application band PCL inner nuclear material according to claim 3 builds the method for tissue engineering bone/cartilage, it is characterized in that, in above-mentioned steps b3, while dripping PLA solution, PLA content accounts for the 5%-10% of total PGA/PLA support.
6. application band PCL inner nuclear material according to claim 5 builds the method for tissue engineering bone/cartilage, it is characterized in that, in above-mentioned steps b4, under 55-70 ℃ of high temperature, heats, and within 1 hour, being placed under pressure device 200N-500N, to be forced into mould thoroughly cooling.
7. application band PCL inner nuclear material according to claim 5 builds the method for tissue engineering bone/cartilage, it is characterized in that, and in above-mentioned steps a, mesh spacing 1-3mm, floor height 0.1mm.
8. application band PCL inner nuclear material according to claim 7 builds the method for tissue engineering bone/cartilage, it is characterized in that, in above-mentioned steps a, at 55-70 ℃, heat after about 5-10 minute, and pressurization rapidly, pressure is 200N-500N.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105854085A (en) * | 2016-04-25 | 2016-08-17 | 上海国睿生命科技有限公司 | Method for constructing tissue engineered cartilages in vivo |
| CN110327495A (en) * | 2019-07-02 | 2019-10-15 | 上海国睿生命科技有限公司 | Organizational project auricle form compound rest and preparation method thereof |
| CN112843334A (en) * | 2021-01-13 | 2021-05-28 | 东华大学 | Bionic trachea constructed by three-dimensional printing composite aerogel and preparation method thereof |
| CN113730651A (en) * | 2020-12-21 | 2021-12-03 | 刘永兴 | Biological material for promoting bone regeneration, preparation method and application |
| CN113925048A (en) * | 2021-10-13 | 2022-01-14 | 北京大学 | Animal specimen ear and animal specimen and manufacturing process thereof |
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2014
- 2014-04-18 CN CN201410163412.7A patent/CN103948969A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105854085A (en) * | 2016-04-25 | 2016-08-17 | 上海国睿生命科技有限公司 | Method for constructing tissue engineered cartilages in vivo |
| CN110327495A (en) * | 2019-07-02 | 2019-10-15 | 上海国睿生命科技有限公司 | Organizational project auricle form compound rest and preparation method thereof |
| CN110327495B (en) * | 2019-07-02 | 2021-11-30 | 上海国睿生命科技有限公司 | Tissue engineering auricle form composite scaffold and preparation method thereof |
| CN113730651A (en) * | 2020-12-21 | 2021-12-03 | 刘永兴 | Biological material for promoting bone regeneration, preparation method and application |
| CN112843334A (en) * | 2021-01-13 | 2021-05-28 | 东华大学 | Bionic trachea constructed by three-dimensional printing composite aerogel and preparation method thereof |
| CN112843334B (en) * | 2021-01-13 | 2022-07-08 | 东华大学 | Bionic trachea constructed by three-dimensional printing composite aerogel and preparation method thereof |
| CN113925048A (en) * | 2021-10-13 | 2022-01-14 | 北京大学 | Animal specimen ear and animal specimen and manufacturing process thereof |
| CN115581811A (en) * | 2022-11-03 | 2023-01-10 | 上海交通大学医学院附属第九人民医院 | Autologous tissue engineering living cell meibomian plate substitute and preparation method and application thereof |
| CN115581811B (en) * | 2022-11-03 | 2024-04-02 | 上海交通大学医学院附属第九人民医院 | Autologous tissue engineering living cell meibomian substitute and preparation method and application thereof |
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