CN103990180A - Preparation method and application of deproteinated decalcification bone matrix implantable microcarrier - Google Patents
Preparation method and application of deproteinated decalcification bone matrix implantable microcarrier Download PDFInfo
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- CN103990180A CN103990180A CN201410225274.0A CN201410225274A CN103990180A CN 103990180 A CN103990180 A CN 103990180A CN 201410225274 A CN201410225274 A CN 201410225274A CN 103990180 A CN103990180 A CN 103990180A
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Abstract
The invention provides a preparation method of a deproteinated decalcification bone matrix implantable microcarrier and an application of the deproteinated decalcification bone matrix implantable microcarrier in preparation of tissue engineering materials. The preparation method comprises the following steps: carrying out cryogenic grinding on bovine cortex bones, sifting out bone particles with particle sizes of less than 200 micrometers, and carrying out degreasing, decalcification and deproteinization treatment on the bone particles. The deproteinated decalcification bone matrix implantable microcarrier obtained by using the preparation method is low in immunogenicity and high in cell adhesion rate. Cells cultivated by adopting the deproteinated decalcification bone matrix implantable microcarrier under a static cultivation condition are high in activity, stem cell phenotypes are kept perfectly, and as protein active ingredients in a decalcification bone matrix are removed, the deproteinated decalcification bone matrix particles do not cause change of bone components in target cells such as stem cells; and dynamic cultivation experiments of BMSCs (bone marrow mesenchymal stem cells) prove that the microcarrier particles are available for dynamic suspension cultivation conditions and can successfully form suspension at a stirring rate suitable for cell cultivation, compared with the cell adhesion rate of cytodex3, the cell adhesion rate of the microcarrier particles is higher, and the amplification efficiency of the microcarrier particles is similar to that of the cytodex3.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method and application of Deproteinization decalcified bone matrix embedded type microcarrier.
Background technology
The at present use of seed cell mainly be take autogenous cell as main, but its can amount to obtain very little, can not meet clinical use far away required, therefore need to increase in vitro, wait to reach the structure that the laggard step of required number completes tissue engineered bone.Aspect the amplification in vitro of seed cell, this year, the culture effect under dynamic suspension condition of culture was comparatively superior and extensively approved with microcarrier.
But, using comparatively widely at present, can be used as like product considers the microcarrier of standard substance and selects to be mainly Cytodex series of products, wherein comparatively extensive with Cytodex3 application, this product is the dextran granule containing Denatured protein pan coating of synthetic, has good cell adhesion and homogeneity; Its cell culture efficiency is high, and the seed cell that can increase in a large number is at short notice for the batch production of vaccine, coenzyme etc., or the seed cell such as a large amount of expanding stem cells collect and build for organizational project after digesting separation.But Cytodex series products is synthetic material, mostly be non-degradable material, cannot be directly used in in-vivo tissue repair process, the seed cell that causes cultivating amplification through microcarrier can not directly be take microcarrier transplantation carrier in body and be implanted.Abundant experimental results shows, extra cell dissociation separation process meeting destruction extracellular matrix, thus cause the forfeiture of cytoactive and the waste of cell number.In the process addressing the above problem, researcheres are attempted to improve cell utilization rate and effective percentage by methods such as improvement cell obtaining means, optimization microcarrier material and preparations, but mostly, because cytoactive reduces or the interior local acid accumulation of body is not improved and stops, cause the microcarrier material that can be directly used in transplanting in body still comparatively to lack.
In order to obtain the microcarrier granule that quality is unified, controllability is good (one of basic demand of microcarrier), in preparation of microcarriers process, synthetic material and the methods of adopting are carried out microcarrier processing more, this certainly will cause the poor or non-degradable of microcarrier material degradability in vivo, thereby can not be as transplantation carrier in the material implanted body that is applied to seed cell, cause seed cell after microcarrier is cultivated amplification, still needing digest separation to carry out the adherent process again of itself and stent graft material.Generally speaking, under existing preparation of microcarriers thinking, microcarrier granule prepared by employing synthetic material is using the transplantation carrier being difficult to as seed cell, and the seed cell of cultivating amplification gained by it will inevitably digest separation again.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application of Deproteinization decalcified bone matrix embedded type microcarrier, prepare the microcarrier that can be used for external dynamic suspension condition of culture a large amount of amplification seed cells.
For achieving the above object, the present invention adopts following technical scheme:
A kind of Deproteinization decalcified bone matrix embedded type microcarrier is in the application of preparing aspect tissue engineering material.
Described tissue engineering material is for repairing the tissue engineering material in lacuna shape tissue defect district.
Described tissue engineering material is for repairing the tissue engineering material of large segmental bone defect.
A kind of preparation method of Deproteinization decalcified bone matrix embedded type microcarrier, cattle cortical bone is carried out after freezing and pulverizing, sift out the osseous granules that particle diameter is less than 200um, and osseous granules is carried out to defat, decalcification and deproteinization and process, obtain Deproteinization decalcified bone matrix embedded type microcarrier.
Before described freezing and pulverizing by cattle cortical bone-80 ℃ preserve 24h more than.
Described defat detailed process is: in degreaser, add osseous granules also to continue to use distilled water flushing osseous granules after stirring 2-3h, then the osseous granules after rinsing is joined in degreaser, continue to stir 2-3h, wherein in every 3mL degreaser, add 0.8-1.2g osseous granules, degreaser is the mixture that methanol and chloroform volume ratio are 1:1.
Described decalcification is specially: osseous granules is joined in the hydrochloric acid of 0.5-0.6mol/L, continue to stir 8-12h, then osseous granules is taken out, then repeat said process 2-3 time; Wherein, in every 3mL hydrochloric acid, adding the quality of osseous granules is 0.8-1.2g.
Described deproteinization is processed and is specially: under room temperature, by the osseous granules after decalcification processing, be placed in successively the CaCl of 2-3mol/L
2solution stirring 16-24h, stirs 4h in the EDTA solution of 0.5mol/L, in the LiCl solution of 8mol/L, stirs 16-24h, then under the water-bath of 52 ℃, heats 4-6h, then uses 6mol/L urea cellulose solution extracting 36-48h, collecting granules after washing, and through frozen dried; Wherein, the CaCl of 2mol/L used
2the volume of the EDTA solution of solution, 0.5mol/L, the LiCl solution of 8mol/L, 6mol/L urea cellulose solution be the osseous granules quality after decalcification is processed 2-3 doubly.
With respect to prior art, the beneficial effect that the present invention has: the present invention carries out decalcification and deproteinization by cattle cortical bone and processes, the immunogenicity of gained Deproteinization decalcified bone matrix embedded type microcarrier is low, cell adhesion rate is high.Under static culture conditions, high, the stem Cell Phenotypic of the activity of its cultured cell keeps complete and owing to having removed the protein active composition in decalcified bone matrix (as BMPs etc.), such Deproteinization decalcified bone matrix granule will can not cause the object cell generation Osteoblast Differentiation such as stem cell in incubation; By the dynamic cultivation experiment to BMSCs (mesenchymal stem cells MSCs), confirm, this microcarrier granule can be used for dynamic suspension condition of culture, be suitable for can forming smoothly suspension under the mixing speed of cell culture, its cell adhesion rate is compared with cytodex3 is slightly high, amplification efficiency is similar, the culture period of 14 days finishes rear cultured cell to be identified, confirm BMSCs phenotype, versatility, the maintenance of multiplication capacity is satisfactory again; By preliminary test in animal body, confirm that this microcarrier can inject in the smooth perfect aspect of 16# injection needle, and can be better at ectopic osteogenesis and cell survival.
In addition, the present invention has carried out immunoreation and degradability in the body of laboratory animal (rat, new zealand rabbit), osteogenic detection equally to cattle Deproteinization decalcified bone matrix granule, and result all can reach tissue engineering tissue construction for the requirement of embedded type biomaterial; With gel mixing microcarrier-cell complexes, carrying out in the process of early immune reaction test in body, postoperative 7d is shown in that pettiness amount immunocyte infiltrates, and shows that its immunogenicity is low, good biocompatibility.
In the present invention, the preparation method of Deproteinization decalcified bone matrix embedded type microcarrier is simple, easy operating, and the immunogenicity of the microcarrier making is low, cell adhesion rate is high.
Accompanying drawing explanation
Fig. 1 is the surface scan Electronic Speculum picture of DpDBM microcarrier;
Fig. 2 is the cell adhesion low power scanning electron microscope picture of DpDBM microcarrier;
Fig. 3 is the cell adhesion high power scanning electron microscope picture of DpDBM microcarrier;
Fig. 4 is the surface scan Electronic Speculum picture of Cytodex3 microcarrier;
Fig. 5 is the cell adhesion low power scanning electron microscope picture of Cytodex3 microcarrier;
Fig. 6 is the cell adhesion high power scanning electron microscope picture of Cytodex3 microcarrier.
The specific embodiment
Below in conjunction with accompanying drawing, the present invention is described in detail.Solution in the present invention all refers to aqueous solution.
A preparation method for Deproteinization decalcified bone matrix embedded type microcarrier, comprises the following steps:
First cattle cortical bone is preserved more than 24h (freezing processing can reduce the immunogenicity of material) at-80 ℃, again cattle cortical bone is carried out after freezing and pulverizing, join in degreaser, continue to stir 2-3h, wherein in every 3mL degreaser, add 0.8-1.2g g osseous granules to sift out the osseous granules that particle diameter is less than 200um, and osseous granules is carried out to defat, in degreaser, add osseous granules also to continue to use distilled water flushing osseous granules after stirring 2-3h, then the osseous granules after rinsing, degreaser is the mixture of methanol, chloroform volume ratio 1:1, then carry out decalcification, being about to osseous granules joins in the hydrochloric acid of 0.5-0.6mol/L, continue to stir 8-12h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.5-0.6mol/L, continue to stir 8-12h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.5-0.6mol/L, continue to stir 12-24h, then osseous granules is taken out to the hydrochloric acid of putting into 0.5-0.6mol/L, continue to stir 12-24h, wherein in every 3mL hydrochloric acid, adding the quality of osseous granules is 0.8-1.2g, because decalcification is very fast while starting, so changed decalcifying Fluid once with every 12h in first 24 hours, then just osseous granules carries out deproteinization processing, filters out suitable size particles (diameter is less than the granule of 200um), obtains Deproteinization decalcified bone matrix embedded type microcarrier.
Wherein, deproteinization is processed the BMP leaching process that adopts standard, is specially under room temperature, by the osseous granules after decalcification processing, is placed in successively the CaCl of 2-3mol/L
2solution stirring 16-24h, stirs 4h in the EDTA solution of 0.5mol/L, in the LiCl solution of 8mol/L, stirs 16-24h, then under the water-bath of 52 ℃, heats 4-6h, then uses 6mol/L urea cellulose solution extracting 36-48h, collecting granules after washing, and through frozen dried; Wherein, the CaCl of 2mol/L used
2the volume of the EDTA solution of solution, 0.5mol/L, the LiCl solution of 8mol/L, 6mol/L urea cellulose solution be the osseous granules quality after decalcification is processed 2-3 doubly.
Embodiment 1
A preparation method for Deproteinization decalcified bone matrix embedded type microcarrier, comprises the following steps:
First cattle cortical bone is preserved more than 24h (freezing processing can reduce the immunogenicity of material) at-80 ℃, again cattle cortical bone is carried out after freezing and pulverizing, join in degreaser, continue to stir 2h, wherein in every 3mL degreaser, add 0.8g osseous granules to sift out the osseous granules that particle diameter is less than 200um, and osseous granules is carried out to defat, in degreaser, add osseous granules also to continue to use distilled water flushing osseous granules after stirring 3h, then the osseous granules after rinsing, degreaser is the mixture of methanol, chloroform volume ratio 1:1; Then carry out decalcification, being about to osseous granules joins in the hydrochloric acid of 0.6mol/L, continue to stir 12h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.5mol/L, continue to stir 8h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.5mol/L, continue to stir 24h, then osseous granules is taken out to the hydrochloric acid of putting into 0.6mol/L, continue to stir 12h, wherein in every 3mL hydrochloric acid, adding the quality of osseous granules is 1.2g, because decalcification is very fast while starting, so changed decalcifying Fluid once with every 12h in first 24 hours; Then osseous granules is carried out to deproteinization processing, filter out suitable size particles (diameter is less than the granule of 200um), obtain Deproteinization decalcified bone matrix embedded type microcarrier.
Wherein, deproteinization is processed the BMP leaching process that adopts standard, is specially under room temperature, by the osseous granules after decalcification processing, is placed in successively the CaCl of 2mol/L
2solution stirring 16h, stirs 4h in the EDTA solution of 0.5mol/L, in the LiCl solution of 8mol/L, stirs 24h, then under the water-bath of 52 ℃, heats 6h, then uses 6mol/L urea cellulose solution extracting 36h, collecting granules after washing, and through frozen dried; Wherein, the CaCl of 2mol/L used
2the volume of the EDTA solution of solution, 0.5mol/L, the LiCl solution of 8mol/L, 6mol/L urea cellulose solution is 3 times of osseous granules quality after decalcification is processed.
Embodiment 2
A preparation method for Deproteinization decalcified bone matrix embedded type microcarrier, comprises the following steps:
First cattle cortical bone is preserved more than 24h (freezing processing can reduce the immunogenicity of material) at-80 ℃, again cattle cortical bone is carried out after freezing and pulverizing, join in degreaser, continue to stir 3h, wherein in every 3mL degreaser, add 1.2g osseous granules to sift out the osseous granules that particle diameter is less than 200um, and osseous granules is carried out to defat, in degreaser, add osseous granules also to continue to use distilled water flushing osseous granules after stirring 2h, then the osseous granules after rinsing, degreaser is the mixture of methanol, chloroform volume ratio 1:1; Then carry out decalcification, being about to osseous granules joins in the hydrochloric acid of 0.5mol/L, continue to stir 8h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.6mol/L, continue to stir 12h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.6mol/L, continue to stir 12h, then osseous granules is taken out to the hydrochloric acid of putting into 0.5mol/L, continue to stir 12h, wherein in every 3mL hydrochloric acid, adding the quality of osseous granules is 0.8g, because decalcification is very fast while starting, so changed decalcifying Fluid once with every 12h in first 24 hours; Then osseous granules is carried out to deproteinization processing, filter out suitable size particles (diameter is less than the granule of 200um), obtain Deproteinization decalcified bone matrix embedded type microcarrier.
Wherein, deproteinization is processed the BMP leaching process that adopts standard, is specially under room temperature, by the osseous granules after decalcification processing, is placed in successively the CaCl of 3mol/L
2solution stirring 24h, stirs 4h in the EDTA solution of 0.5mol/L, in the LiCl solution of 8mol/L, stirs 16h, then under the water-bath of 52 ℃, heats 4h, then uses 6mol/L urea cellulose solution extracting 48h, collecting granules after washing, and through frozen dried; Wherein, the CaCl of 2mol/L used
2the volume of the EDTA solution of solution, 0.5mol/L, the LiCl solution of 8mol/L, 6mol/L urea cellulose solution is 3 times of osseous granules quality after decalcification is processed.
Embodiment 3
A preparation method for Deproteinization decalcified bone matrix embedded type microcarrier, comprises the following steps:
First cattle cortical bone is preserved more than 24h (freezing processing can reduce the immunogenicity of material) at-80 ℃, again cattle cortical bone is carried out after freezing and pulverizing, join in degreaser, continue to stir 3h, wherein in every 3mL degreaser, add 1g osseous granules to sift out the osseous granules that particle diameter is less than 200um, and osseous granules is carried out to defat, in degreaser, add osseous granules also to continue to use distilled water flushing osseous granules after stirring 2h, then the osseous granules after rinsing, degreaser is the mixture of methanol, chloroform volume ratio 1:1; Then carry out decalcification, be about to osseous granules and join in the hydrochloric acid of 0.5mol/L, continue to stir 10h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.6mol/L, continue to stir 11h, then osseous granules is taken out, after rinsing, put into the hydrochloric acid of 0.5mol/L, continue to stir 18h, wherein in every 3mL hydrochloric acid, adding the quality of osseous granules is 1g, because decalcification is very fast while starting, so changed decalcifying Fluid once with every 12h in first 24 hours; Then osseous granules is carried out to deproteinization processing, filter out suitable size particles (diameter is less than the granule of 200um), obtain Deproteinization decalcified bone matrix embedded type microcarrier.
Wherein, deproteinization is processed the BMP leaching process that adopts standard, is specially under room temperature, by the osseous granules after decalcification processing, is placed in successively the CaCl of 3mol/L
2solution stirring 20h, stirs 4h in the EDTA solution of 0.5mol/L, in the LiCl solution of 8mol/L, stirs 19h, then under the water-bath of 52 ℃, heats 5h, then uses 6mol/L urea cellulose solution extracting 40h, collecting granules after washing, and through frozen dried; Wherein, the CaCl of 2mol/L used
2the volume of the EDTA solution of solution, 0.5mol/L, the LiCl solution of 8mol/L, 6mol/L urea cellulose solution is 2 times of osseous granules quality after decalcification is processed.
The application of Deproteinization decalcified bone matrix embedded type microcarrier prepared by the present invention realizes by following experiment:
The microcarrier dynamic suspension of seed cell is cultivated (take BMSCs as example): adopt stirring type bioreactor dynamic suspension to cultivate the stem cell amount that is cultured in advance some, take and carry out expansion of stem cells with the similar inoculum concentration of Cytodex3 and culture environment (inoculation parameter is 3mg/mL; 1*105cell/mL; 40rpm/min).It is primary that BMSCs adopts 8w wistar rat marrow to rinse the BMSCs cultivating, be expanded to P3 and carry out microcarrier inoculation after generation, planting density and microcarrier density are as above-mentioned inoculation parameter, because forming contrast with CYTODEX3, therefore adopt the conventional inoculation parameter (recommended parameter of also using for microcarrier) of CYTODEX3.
Cultivate in gained microcarrier-cell complexes body and transplant:
1) utilize alginate jelly and microcarrier-mixing with cells
By mass fraction, be that 1-3% solution of sodium alginate joins 100mmol/L calcium chloride solution formation calcium alginate gel, first adopt solution of sodium alginate suspendible microcarrier-cell complexes, slowly add subsequently calcium chloride solution and form gel, prepare the gelatinous mixture that can inject by 20mL syringe needle, the mode that the tissue engineering material of structure can be injected by Wicresoft is for repairing lacuna shape tissue defect district.
2) due to Deproteinization decalcified bone matrix embedded type microcarrier granule of the present invention less (<200um), therefore in tissue engineering building process, can be used as the directly transplanting carrier of the highly active seed cell of carrying, with integral form (implant units) and large aperture material use in conjunction, thereby obtain the required mechanical characteristic of heavy burden district tissue substitute material, its advantage is: large aperture material monolithic nutrition supply rate is higher, but its effective area that can adhere to is corresponding deficiency, just solve this difficult problem with the use in conjunction of microcarrier-cell complexes, and also can be the use of microcarrier, large aperture material provides required mechanical property, thereby that widens implantable microcarrier utilizes approach and mode.
Deproteinization decalcified bone matrix embedded type microcarrier of the present invention can be used for building tissue engineering biological substitution material, is specially: the external amplification of the dynamic cultivation to seed cell (can simultaneously complete the steps such as required relevant induction); As materials such as the directly transplanting carrier of seed cell and gel, macropore supports, be united and applied in the reparation of large segmental bone defect in body.
Deproteinization decalcified bone matrix embedded type microcarrier of the present invention and artificial-synthetic copolymer are similar,, containing the active component of induction differentiation, therefore can not complete the rapid amplifying of seed cell and phenotype are maintained; For the part cultivation of noble cells, add corresponding inductivity culture fluid and can obtain maintaining of phenotype; Deproteinization decalcified bone matrix embedded type microcarrier of the present invention has good biocompatibility and degradation characteristic, immunologic rejection is little, therefore obtain good tissue repair effect after can be in being implanted into body, and by its cultured cells, its rate of increase is high, active good, has guaranteed equally the high efficiency of tissue repair.
Fig. 1-3 are the scanning electron microscope picture that DpDBM microcarrier and cell attach situation, Fig. 4-6 are and Cytodex3 microcarrier and cell attach the scanning electron microscope picture of situation, from Fig. 1-6, can find out with Cytodex3 microcarrier and compare, DpDBM microcarrier has been obtained similar cell culture expanding effect.
The present invention has carried out immunoreation and degradability in the body of laboratory animal (rat, new zealand rabbit), osteogenic detection equally to cattle Deproteinization decalcified bone matrix granule, and result all can reach tissue engineering tissue construction for the requirement of embedded type biomaterial; With gel mixing microcarrier-cell complexes, carrying out in the process of early immune reaction test in body, postoperative 7d is shown in that pettiness amount immunocyte infiltrates, and shows that its immunogenicity is low, good biocompatibility.
Claims (8)
1. a Deproteinization decalcified bone matrix embedded type microcarrier is in the application of preparing aspect tissue engineering material.
2. application according to claim 1, is characterized in that, described tissue engineering material is for repairing the tissue engineering material in lacuna shape tissue defect district.
3. application according to claim 1, is characterized in that, described tissue engineering material is for repairing the tissue engineering material of large segmental bone defect.
4. the preparation method of a Deproteinization decalcified bone matrix embedded type microcarrier, it is characterized in that, cattle cortical bone is carried out, after freezing and pulverizing, sifting out the osseous granules that particle diameter is less than 200um, and osseous granules is carried out to defat, decalcification and deproteinization and process, obtain Deproteinization decalcified bone matrix embedded type microcarrier.
5. the preparation method of a kind of Deproteinization decalcified bone matrix embedded type microcarrier according to claim 4, is characterized in that, before described freezing and pulverizing by cattle cortical bone-80 ℃ preserve 24h more than.
6. the preparation method of a kind of Deproteinization decalcified bone matrix embedded type microcarrier according to claim 4, it is characterized in that, described defat detailed process is: in degreaser, add osseous granules also to continue to use distilled water flushing osseous granules after stirring 2-3h, then the osseous granules after rinsing is joined in degreaser, continue to stir 2-3h, wherein in every 3mL degreaser, add 0.8-1.2g osseous granules, degreaser is the mixture that methanol and chloroform volume ratio are 1:1.
7. the preparation method of a kind of Deproteinization decalcified bone matrix embedded type microcarrier according to claim 4, it is characterized in that, described decalcification is specially: osseous granules is joined in the hydrochloric acid of 0.5-0.6mol/L, continue to stir 8-12h, then osseous granules is taken out, then repeat said process 2-3 time; Wherein, in every 3mL hydrochloric acid, adding the quality of osseous granules is 0.8-1.2g.
8. a kind of Deproteinization decalcified bone matrix according to claim 4 is prepared the preparation method of embedded type microcarrier, it is characterized in that, described deproteinization is processed and is specially: under room temperature, by the osseous granules after decalcification processing, be placed in successively the CaCl of 2-3mol/L
2solution stirring 16-24h, stirs 4h in the EDTA solution of 0.5mol/L, in the LiCl solution of 8mol/L, stirs 16-24h, then under the water-bath of 52 ℃, heats 4-6h, then uses 6mol/L urea cellulose solution extracting 36-48h, collecting granules after washing, and through frozen dried; Wherein, the CaCl of 2mol/L used
2the volume of the EDTA solution of solution, 0.5mol/L, the LiCl solution of 8mol/L, 6mol/L urea cellulose solution be the osseous granules quality after decalcification is processed 2-3 doubly.
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| CN109320970A (en) * | 2018-10-09 | 2019-02-12 | 山西宾大干细胞生物科技有限公司 | A kind of temperature-sensitive hydrogel and the preparation method and application thereof for repair of cartilage |
| CN112023112A (en) * | 2020-09-22 | 2020-12-04 | 天津中津生物发展有限公司 | Bone hemostatic material with osteogenesis inducing activity and preparation method thereof |
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