CN1526765A - Composite bone tissue engineering rack material and its prepn - Google Patents
Composite bone tissue engineering rack material and its prepn Download PDFInfo
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- CN1526765A CN1526765A CNA031443087A CN03144308A CN1526765A CN 1526765 A CN1526765 A CN 1526765A CN A031443087 A CNA031443087 A CN A031443087A CN 03144308 A CN03144308 A CN 03144308A CN 1526765 A CN1526765 A CN 1526765A
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- collagen
- timbering material
- hydroxyapatite
- tissue engineering
- chondroitin sulfate
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Abstract
The composite bone tissue engineering rack material includes collagen, hydroxyapatite, chondroitin sulfate and bone morphogenetic protein; and has 3D porous structure of aperture 50-150 microns. The ratio between collagen and hydroxyapatite is 1 to 1-10, and the chondroitin sulfate is compounded with other components via cross-linking. The material may be constituted into tissue engineering artificial bone based on tissue engineering principle. The present invention has excellent physical, chemical and biological performance, is excellent bone tissue engineering rack material, and may be used in repairing bone defect caused by different reasons clinically.
Description
Technical field
The present invention relates to a kind of bone tissue engineering stent material, particularly collagen-hydroxyapatite (HA)-chondroitin sulfate (CS)-Delicious peptide (BMP) compound osseous tissue engineering timbering material and preparation method thereof.The application organizes engineering philosophy is that timbering material can make up tissue engineered artificial bone with this material, and it is damaged to be used to repair the bone that a variety of causes causes.
Background technology
The sickness rate that the bone that is caused by wound, tumour, infection and other cause of disease is damaged is higher, and it constitutes greatly threat to human beings'health.At present, the reparation that bone is damaged is mainly adopted from body bone, allogenic bone transplantation material and metal, macromolecular compound, natural or synthetic calcium phosphate material such as artificial bones such as coral, hydroxyapatite., immune response limited owing to donor, the reasons such as danger of disease carrying germ are arranged, these methods of treatment are very limited.Therefore, press for the graft materials that development has desirable biocompatibility and biological functionality.These material requirements have stable, good biological property, and are biodegradable when new osteogenesis is repaired, thereby really realize the reparation that bone is damaged.
In recent years, along with subject development such as cytobiology, Materials science, molecular biology, the damaged reparation of bone that forms of emerging organizational project subject provides new method and thinking.This method is that cell seeding is grown on the porous three-dimensional timbering material, bred, and the emiocytosis extracellular matrix also forms new osseous tissue and timbering material is degraded gradually, then it is implanted defect.Three-dimensional stent material, i.e. extracellular matrix surrogate is one of key of bone tissue engineer research, can it not only influences the biological behaviour and the culture efficiency of cell, and adapt to well, combine with body after determining to transplant and the effect of reparation.
The three-dimensional stent material that is used for bone tissue engineer should satisfy following requirement: (1) excellent biological compatibility and biological degradability, and its degraded product in vivo is harmless; (2) have osteoconductive, be beneficial to cell adhesion and propagation; (3) certain intensity is arranged, can be to external force resistance; (4) be easy to mouldingly, can be processed into different shape and size as required.At present, the timbering material that is used for bone tissue engineer mainly contains two classes, and a class is an inorganic materials, as bioactive ceramics; Another kind of is organic polymer material, as natural materialss such as synthetic materialss such as α-polyester, collagens.Studies show that these materials are all not ideal enough, the macromolecular material undercapacity, easy deformation when stressed can damage the cell of transplanting like this, and the biological ceramics degradation speed is slow, and is cracked easily after stressed.
Nature bone matrix mainly is made up of organic and inanimate matter.Comprise collegen filament and [amorphous in the organic matter.Wherein collegen filament account for 95% of organic matter, and most cells all are attached to the collagen surface growth in the osseous tissue, and collagen is also being kept the toughness of osseous tissue simultaneously, and the branch of the collegen filament under the scanning electron microscope in the visible ground substance of bone connects to netted.The natural collagen material has the effect of guiding and inducing cell growth, and we utilize the tissue leading regeneration collagen film (patent No.: 94118836.1) shown this biological function of collagenic material preparation.[amorphous is neutrality and acidic mucopolysaccharide, wherein based on basophilous chondroitin-4-suleate and 6-chondroitin sulfate.They can promote cell to attach, improve the intensity and the toughness of osseous tissue.The inorganic salt of bone are to be made of the sub-micro throw out that calcium phosphate forms, with inorganic salt HAP[Ca
10(PO
4)
6(OH)
2] very close, they are positioned at matrix, and the collegen filament material is arranged at surperficial and inner direction major axis along fiber.In osseous tissue, the crushing resistance and the elasticity of collagenous fiber are relatively poor, and HAP crystallization then fragility is bigger, and is easily cracked, but when both clocklike combined, osseous tissue promptly had unique intensity and toughness.Under physiological condition, osteoblastic differentiation and propagation are subjected to the adjusting of the various kinds of cell factor, comprise insulin-like growth factor (IGF), transforming growth factor-beta (TGF-β), Delicious peptide (BMP) etc., in the bone repair process, cytokine tells on by multiple factor synergy often.Wherein BMP is one group of protein with osteoinductive.In the structure of engineered artificial organs, no matter from bionic principle, still from the angle of functionalization, introducing polysaccharose substance and cell growth factor all has positive meaning.
Summary of the invention
The purpose of this invention is to provide a kind of new compound osseous tissue engineering timbering material and preparation method thereof.The present invention is a simulation natural bone tissue extracellular matrix components, in the tissue leading regeneration collagen film (patent No.: 94118836.1) on the patent basis, with beef tendon collagen, hydroxyapatite, chondroitin sulfate, Delicious peptide is raw material, makes up bone tissue engineering stent material.The application organizes engineering philosophy is that timbering material can make up tissue engineered artificial bone with this material.The present invention has good physics and chemistry and biology performance, is the bone tissue engineering stent material of excellent property.It is damaged to can be used for treating the bone that a variety of causes causes.
The present invention includes collagen, hydroxyapatite, chondroitin sulfate and Delicious peptide and constitute, have three-dimensional porous constitutional features, the aperture is the 50-150 micron.
The quality proportioning of described collagen and hydroxyapatite is 1: 1-10, it is by the freeze-drying moulding.
Described chondroitin sulfate is compound on the timbering material by crosslinked, contains 2-morpholino ethane sulfonic acid, carbodiimide, succinimide and chondroitin sulfate in the linking agent, and chondroitin sulfate cellulose content (mass ratio) is 0.2-2% in the linking agent.
Described Delicious peptide is a 0.35-0.5mg/mL timbering material volume, and it is attached on the timbering material by absorption.
The preparation of compound osseous tissue engineering timbering material comprises the steps:
1) be swelling solution with 0.3% propanedioic acid solution, the configuration solid content is the collagen swelling solution of 0.5-0.7%, takes by weighing collagen swelling solution and hydroxyapatite by proportioning, mixes, obtain the collagen-hydroxyapatite dispersion liquid,-20--60 ℃ of pre-freeze be 4h at least ,-30 ℃ of 15h ,-15 ℃ of 18h, 0 ℃ of 15h, 10 ℃ of 7h, the 20C4h lyophilize obtains uncrosslinked collagen-hydroxyapatite timbering material;
2) under the room temperature, the uncrosslinked collagen-hydroxyapatite timbering material (by the amount of 20ml/50mg collagen) for preparing is soaked in 30min in 40% ethanol that contains 50mmol/L 2-morpholino ethane sulfonic acid (MES); Then the amount of collagen-HA timbering material by 20ml/50mg collagen is immersed in the crosslinked fluid, crosslinked fluid contains 50mmol/L MES, 24mmol/L carbodiimide (EDC), 5mmol/L succinimide (NHS), the chondroitin sulfate of 0.2-2%; Crosslinked 4h under the room temperature; With the Na of support at 0.1mol/L
2HPO
4In clean 1h, in the NaCl of 1mol/L, clean 2h, among the NaCl of 2mol/L soaking and washing 1 round the clock, last, water cleans removes remaining crosslinked fluid and NaCl ,-30 ℃ of pre-freezes are 4h at least, secondary freeze-drying in the freeze drier ,-20 ℃ of 8h, 10 ℃ of 4h, 20 ℃ of 4h.
3) take by weighing people's Delicious peptide 2 of recombinating, be dissolved in glycine buffer, concentration is 1 μ g/ μ L, PH=6.5, leave standstill 30min under 0 ℃, the timbering material of sterilization is immersed in the above-mentioned reaction solution, leave standstill 60min under 0 ℃, take out timbering material, air-dry fast in Bechtop, aseptic sealing up for safekeeping.
Description of drawings
Fig. 1 scanning electron microscope of the present invention (SEM) photo.
The SEM photo of Fig. 2 scleroblast after external three weeks of cultivation on the timbering material of the present invention.
Embodiment
Embodiment 1
With 0.3% propanedioic acid solution is swelling solution, and the configuration solid content is 0.5% collagen swelling solution.Take by weighing solid content 0.5% collagen swelling solution 60g and hydroxyapatite 0.3g, mix, obtain collagen-HA dispersion liquid.-30 ℃ of pre-freezes of elder generation are 4h at least, again with-30 ℃ of 15h, and-15 ℃ of 18h, 0 ℃ of 15h, 10 ℃ of 7h, 20 ℃ of 4h lyophilizes obtain uncrosslinked collagen-HA timbering material.
Under the room temperature, uncrosslinked collagen-HA the timbering material of preparation is soaked in 30min in 40% ethanol that contains 120ml50mmol/L2-morpholino ethane sulfonic acid (MES), then collagen-HA timbering material is immersed in the (MES that contains 50mmol/L in the 120ml crosslinked fluid, the carbodiimide of 24mmol/L (EDC), the succinimide of 5mmol/L (NHS), 0.2% chondroitin sulfate) crosslinked 4h under the room temperature.With the Na of support at 0.1mol/L
2HPO
4In clean 1h, in the NaCl of 1mol/L, clean 2h, among the NaCl of 2mol/L soaking and washing 1 round the clock, last, water cleans removes remaining crosslinked fluid and NaCl ,-30 ℃ of pre-freezes are 4h at least, secondary freeze-drying in the freeze drier ,-20 ℃ of 8h, 10 ℃ of 4h, 20 ℃ of 4h.
Timbering material after crosslinked is cut into diameter 16mm, the disk of thick 3mm, take by weighing rhBMP-2 (people recombinate Delicious peptide 2), be dissolved in glycine buffer, concentration is 1 μ g/ μ L, leave standstill 30min under 0 ℃, the timbering material of sterilization is immersed in the 223 μ L rhBMP-2 solution, leave standstill 60min under 0 ℃.Take out timbering material, air-dry fast in Bechtop, aseptic sealing up for safekeeping.
Scanning electron microscopic observation (SEM):
After the timbering material surface gold-plating, carry out SEM with HITACHI S-3500N scanning electron microscope and observe.SEM observes as can be known, and timbering material is the porous three-dimensional structure, and the aperture on average is about 100um.(see figure 1)
The plantation of scleroblast on timbering material:
Get the 3-4 that the is in exponential phase of growth scleroblast for the marrow mesenchymal stem source, the trypsin solution digestion with 0.25% is blown and beaten, centrifugal, be suspended in the DMEM nutrient solution cell counting count board counting again, the trypan blue staining is surveyed the cytoactive rate, adjusts cell concn to 1.0 * 10 then
7Individual cells/ml.
Timbering material is placed 24 well culture plates, add each 0.1ml of scleroblast suspension respectively, add 1 milliliter of DMEM nutrient solution containing 10% foetal calf serum again, 37 ℃, 5%CO
2Following cultivation, nutrient solution of replacing in per 2 days.
In the 3rd week, get the tissue engineered artificial bone sample in the cultivation, in glutaraldehyde, fix 2 hours, fix 1 hour in the osmic acid, critical drying; The ion degree film; HITACHI S-3500N scanning electron microscope row SEM observes.
Scleroblast is well-grown in timbering material.The secretory cell epimatrix is deposited in cell peripheral, and a large amount of cells are by the extracellular matrix embedding, between cell and support, cell and iuntercellular formation cytodesma.(see figure 2)
Embodiment 2
With 0.3% propanedioic acid solution is swelling solution, and the configuration solid content is 0.5% collagen swelling solution.Take by weighing solid content 0.5% collagen swelling solution 60 grams and hydroxyapatite 1.2 grams, mix, obtain collagen-HA dispersion liquid.Elder generation's pre-freeze obtains uncrosslinked collagen-HA timbering material with lyophilize again.
Under the room temperature, uncrosslinked collagen-HA the timbering material of preparation is soaked in 30min in 40% ethanol that contains 120ml50mmol/L2-morpholino ethane sulfonic acid (MES), then collagen-HA timbering material is immersed in the (MES that contains 50mmol/L in the 120ml crosslinked fluid, the carbodiimide of 24mmol/L (EDC), the succinimide of 5mmol/L (NHS), 0.5% chondroitin sulfate) crosslinked 4h under the room temperature.With the Na of support at 0.1mol/L
2HPO
4The middle 1h that cleans cleans 2h in the NaCl of 1mol/L, clean 1 among the NaCl of 2mol/L round the clock, and last, water cleans crosslinked fluid and the NaCl that removes remnants, secondary freeze-drying in the freeze drier.
Timbering material after crosslinked is cut into diameter 16mm, and the disk of thick 3mm takes by weighing rhBMP-2, be dissolved in glycine buffer, concentration is 1 μ g/ μ L, leaves standstill 30min under 0 ℃, the timbering material of sterilization is immersed in the 223 μ L rhBMP-2 solution, leaves standstill 60min under 0 ℃.Take out timbering material, air-dry fast in Bechtop, aseptic sealing up for safekeeping.
Embodiment 3
With 0.3% propanedioic acid solution is swelling solution, and the configuration solid content is 0.7% collagen swelling solution.Taking by weighing solid content is 0.7% collagen swelling solution, 60 grams and hydroxyapatite 0.84 gram, mixes, and obtains collagen-HA dispersion liquid.Elder generation's pre-freeze obtains uncrosslinked collagen-HA timbering material with lyophilize again.
Under the room temperature, uncrosslinked collagen-HA the timbering material of preparation is soaked in 30min in 40% ethanol that contains 168ml50mmol/L2-morpholino ethane sulfonic acid (MES), then collagen-HA timbering material is immersed in the (MES that contains 50mmol/L in the 168ml crosslinked fluid, the carbodiimide of 24mmol/L (EDC), the succinimide of 5mmol/L (NHS), 0.2% chondroitin sulfate) crosslinked 4h under the room temperature.With the Na of support at 0.1mol/L
2HPO
4The middle 1h that cleans cleans 2h in the NaCl of 1mol/L, clean 1 among the NaCl of 2mol/L round the clock, and last, water cleans crosslinked fluid and the NaCl that removes remnants, secondary freeze-drying in the freeze drier.
Timbering material after crosslinked is cut into diameter 16mm, and the disk of thick 3mm takes by weighing rhBMP-2, be dissolved in glycine buffer, concentration is 1 μ g/ μ L, leaves standstill 30min under 0 ℃, the timbering material of sterilization is immersed in the 223 μ L rhBMP-2 solution, leaves standstill 60min under 0 ℃.Take out timbering material, air-dry fast in Bechtop, aseptic sealing up for safekeeping.
Claims (6)
1, a kind of compound osseous tissue engineering timbering material is characterized in that it comprises that collagen, hydroxyapatite, chondroitin sulfate and Delicious peptide constitute, and has three-dimensional porous constitutional features, and the aperture is the 50-150 micron.
2, compound osseous tissue engineering timbering material according to claim 1, the quality proportioning that it is characterized in that described collagen and hydroxyapatite is 1: 1-10.
3, compound osseous tissue engineering timbering material according to claim 1, it is characterized in that described chondroitin sulfate is compound on the collagen-hydroxyapatite matrix material by crosslinked, contain 2-morpholino ethane sulfonic acid, carbodiimide, succinimide and chondroitin sulfate in the linking agent, the chondroitin sulfate mass content is 0.2-2% in the linking agent.
4, compound osseous tissue engineering timbering material according to claim 1 is characterized in that described Delicious peptide is a 0.35-0.5mg/mL timbering material volume, and it is attached on the timbering material by absorption.
5, the preparation method of the described compound osseous tissue engineering timbering material of claim 1 is characterized in that it is to comprise the steps:
1) be swelling solution with 0.3% propanedioic acid solution, the configuration solid content is the collagen swelling solution of 0.5-0.7%, takes by weighing collagen swelling solution and hydroxyapatite by proportioning, mix, obtain the collagen-hydroxyapatite dispersion liquid, elder generation-20-60 ℃ of pre-freeze is again with lyophilize,-30 ℃ of 15h,-15 ℃ of 18h, 0 ℃ of 15h, 10 ℃ of 7h, 20 ℃ of 4h obtain uncrosslinked collagen-hydroxyapatite timbering material;
2) under the room temperature, the uncrosslinked collagen-hydroxyapatite timbering material of preparation is soaked in 30min in 40% ethanol that contains 50mmol/L2-morpholino ethane sulfonic acid (MES) by the amount of 20ml/50mg collagen; Then the amount of collagen-HA timbering material by 20ml/50mg collagen is immersed in the crosslinked fluid crosslinked 4h under the room temperature; With the Na of support at 0.1mol/L
2HPO
4In clean 1h, in the NaCl of 1mol/L, clean 2h, among the NaCl of 2mol/L soaking and washing 1 round the clock, last, water cleans removes remaining crosslinked fluid and NaCl, elder generation-20-60 ℃ of pre-freeze, secondary freeze-drying in the freeze drier ,-20 ℃ of 8h, 10 ℃ of 4h, 20 ℃ of 4h.
3) take by weighing people's Delicious peptide 2 of recombinating, be dissolved in glycine buffer, concentration is 1 μ g/ μ L, PH=6.5 leaves standstill 30min under 0 ℃, and the collagen-hydroxyapatite matrix material of sterilization is immersed in the reaction solution, leave standstill 60min under 0 ℃, take out timbering material, air-dry fast in Bechtop, aseptic sealing up for safekeeping.
6, the preparation method of compound osseous tissue engineering timbering material according to claim 1, it is characterized in that described crosslinked fluid contains 50mmol/L2-morpholino ethane sulfonic acid (MES), the carbodiimide of 24mmol/L (EDC), the succinimide of 5mmol/L (NHS), the chondroitin sulfate of 0.2-2%.
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Cited By (15)
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| CN100341588C (en) * | 2005-10-10 | 2007-10-10 | 胡庆柳 | Method for preparing porous collagen composite nano hydroox apatite artificial bone |
| EP1964583A1 (en) * | 2007-02-09 | 2008-09-03 | Royal College of Surgeons in Ireland | Process for producing a collagen/hydroxyapatite composite scaffold |
| GB2424223B (en) * | 2005-03-07 | 2010-11-10 | Univ Cambridge Tech | Biomaterial |
| CN102532585A (en) * | 2011-11-29 | 2012-07-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of collagen/hydroxyapatite composite bracket material cross-linked with chondroitin sulfate |
| CN101357242B (en) * | 2007-05-31 | 2012-08-29 | 生立生物科技股份有限公司 | Structure for modulating intraocular pressure |
| CN102886075A (en) * | 2012-09-19 | 2013-01-23 | 中南大学 | Human hard tissue repair material and preparation method thereof |
| CN101229394B (en) * | 2007-01-24 | 2013-05-22 | 张立海 | Titanium alloy-collagen-hydroxyl apatite bone repairing material and preparing method thereof |
| CN101229392B (en) * | 2007-01-24 | 2013-05-22 | 张立海 | Inversely crystallized collagen-hydroxyl apatite bone repairing material and preparing method thereof |
| CN103127556A (en) * | 2011-12-05 | 2013-06-05 | 贵州金玖生物技术有限公司 | Absorbable composite artificial bone |
| CN101229393B (en) * | 2007-01-24 | 2013-07-17 | 张立海 | Collagen-chitosan-hydroxyl apatite bone repairing material and preparing method thereof |
| CN104211982A (en) * | 2013-05-31 | 2014-12-17 | 嘉兴学院 | Manufacturing method of multi-aperture tissue engineering scaffold for cell growth |
| CN104922732A (en) * | 2014-03-19 | 2015-09-23 | 北京天新福医疗器材有限公司 | Preparation method of oral biofilm |
| CN107638594A (en) * | 2017-08-14 | 2018-01-30 | 中国科学院上海硅酸盐研究所 | Hydroxyapatite overlong nanowire/collagen composite multiporous biological support and its application |
| CN109954167A (en) * | 2019-02-28 | 2019-07-02 | 天新福（北京）医疗器材股份有限公司 | A kind of bone renovating material and its application |
| CN112432852A (en) * | 2020-11-19 | 2021-03-02 | 中国医学科学院生物医学工程研究所 | Method for testing mechanical properties of 3D printing tissue engineering auricular cartilage and support |
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2003
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB2424223B (en) * | 2005-03-07 | 2010-11-10 | Univ Cambridge Tech | Biomaterial |
| CN100341588C (en) * | 2005-10-10 | 2007-10-10 | 胡庆柳 | Method for preparing porous collagen composite nano hydroox apatite artificial bone |
| CN101229392B (en) * | 2007-01-24 | 2013-05-22 | 张立海 | Inversely crystallized collagen-hydroxyl apatite bone repairing material and preparing method thereof |
| CN101229393B (en) * | 2007-01-24 | 2013-07-17 | 张立海 | Collagen-chitosan-hydroxyl apatite bone repairing material and preparing method thereof |
| CN101229394B (en) * | 2007-01-24 | 2013-05-22 | 张立海 | Titanium alloy-collagen-hydroxyl apatite bone repairing material and preparing method thereof |
| EP1964583A1 (en) * | 2007-02-09 | 2008-09-03 | Royal College of Surgeons in Ireland | Process for producing a collagen/hydroxyapatite composite scaffold |
| WO2008096334A3 (en) * | 2007-02-09 | 2008-10-30 | Royal College Of Surgeons Ie | A collagen/hydroxyapatite composite scaffold, and process for the production thereof |
| EP2517738A1 (en) * | 2007-02-09 | 2012-10-31 | Royal College of Surgeons in Ireland | A collagen/hydroxyapatite scaffold, and process for the production thereof |
| CN101357242B (en) * | 2007-05-31 | 2012-08-29 | 生立生物科技股份有限公司 | Structure for modulating intraocular pressure |
| CN102532585A (en) * | 2011-11-29 | 2012-07-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of collagen/hydroxyapatite composite bracket material cross-linked with chondroitin sulfate |
| CN103127556A (en) * | 2011-12-05 | 2013-06-05 | 贵州金玖生物技术有限公司 | Absorbable composite artificial bone |
| CN102886075A (en) * | 2012-09-19 | 2013-01-23 | 中南大学 | Human hard tissue repair material and preparation method thereof |
| CN102886075B (en) * | 2012-09-19 | 2015-01-14 | 中南大学 | Human hard tissue repair material and preparation method thereof |
| CN104211982A (en) * | 2013-05-31 | 2014-12-17 | 嘉兴学院 | Manufacturing method of multi-aperture tissue engineering scaffold for cell growth |
| CN104922732A (en) * | 2014-03-19 | 2015-09-23 | 北京天新福医疗器材有限公司 | Preparation method of oral biofilm |
| CN107638594A (en) * | 2017-08-14 | 2018-01-30 | 中国科学院上海硅酸盐研究所 | Hydroxyapatite overlong nanowire/collagen composite multiporous biological support and its application |
| CN109954167A (en) * | 2019-02-28 | 2019-07-02 | 天新福（北京）医疗器材股份有限公司 | A kind of bone renovating material and its application |
| CN112432852A (en) * | 2020-11-19 | 2021-03-02 | 中国医学科学院生物医学工程研究所 | Method for testing mechanical properties of 3D printing tissue engineering auricular cartilage and support |
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