CN102488925A - Injectable articular cartilage tissue repair material and its preparation method - Google Patents
Injectable articular cartilage tissue repair material and its preparation method Download PDFInfo
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- CN102488925A CN102488925A CN2011104516858A CN201110451685A CN102488925A CN 102488925 A CN102488925 A CN 102488925A CN 2011104516858 A CN2011104516858 A CN 2011104516858A CN 201110451685 A CN201110451685 A CN 201110451685A CN 102488925 A CN102488925 A CN 102488925A
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Abstract
The invention discloses an injectable articular cartilage tissue repair material and is characterized in that starting materials of the composite material contain the following components of: by weight, 100 parts of I-type collagen and 10-50 parts of hyaluronic acid, wherein oxidisability of hyaluronic acid is 20-60%. A monocyte layer in bone marrow fluid in vivo is embedded in the repair material. The thickness of the composite material is 1-5mm. Hyaluronic acid is oxidized into oxidized hyaluronic acid by the use of sodium periodate. The collagen solution, 5*PBS and a buffer are mixed at the volume ratio of 7:2:1 and the pH is adjusted to neutral. After dissolving oxidized hyaluronic acid by the use of ultrapure water, the dissolved oxidized hyaluronic acid is added into the neutral collagen solution and the mixed solution is uniformly stirred and stands in a refrigerator of 4 DEG C. The separated and extracted monocyte layer in the bone marrow fluid by a density gradient centrifugation method is uniformly mixed with the above collagen-oxidized hyaluronic acid solution. The mixed liquor is putted into a constant temperature incubator of 37 DEG C for standing or injection to the organism articular cartilage defects so as to form the composite material.
Description
Technical field
The present invention relates to a kind of injectable articular cartilage tissue repair materials and preparation method thereof, belong to the preparation field of tissue renovation material.
Technical background
Articular cartilage defect is a kind of common disease, but does not all have very effective treatment means clinically always, and the cartilaginous tissue cell content low, can not move, also lack blood vessel, lymph and nerve etc., self-repairing capability is very limited.The means of application organizes engineering make up cartilage tissue engineering rack comes cartilage defect is repaired, and will be a kind of very potential clinical treatment mode.
Collagen is the important component of animal somatic cell epimatrix, has excellent biological compatibility, biological degradability and poor antigen property, has become one of most widely used material in the biomedical sector.Hyaluronic acid is as a kind of composition of natural cartilage extracellular matrix; Can be specifically and the CD44 receptors bind; Promote the interaction between cell and the material also to regulate cell behavior, the secretion of maintenance, cell proliferation and the extracellular matrix polysaccharide of chondrocyte phenotype etc. is had important effect, but hyaluronic acid is as a kind of polysaccharide; Be water miscible, stripping from timbering material easily.
Add or utilize bioactive substance to prepare that timbering material is increasing to be applied in the tissue repair, to reach better tissue repair effect.Hydrogel is as a kind of injectable support; Can be used for filling the damaged of Any shape and size, and the wound of bringing is less, contains a large amount of moisture; The material that can make the material that is dissolved in wherein and low relative molecular mass is from infiltration diffusion therebetween; To support the exchange of nutrient substance and products of cellular metabolism, in addition, its particular structural characteristic makes seed cell can be evenly distributed in three-dimensional stent material inside; For seed cell provides bionical growing environment, it is a kind of advantageous tissue renovation material.
The patent of at present more existing cartilage tissue engineered materials, Chinese patent 03137617.7 discloses a kind of novel cartilage tissue engineered with syringeability hydrogel support.Be that monomer with initiator, aided initiating, deionized water, biocompatible polymer is hybridly prepared into homogeneous solution in proportion mutually, in water bath with thermostatic control, react, obtain can be used for cartilage tissue engineered syringeability hydrogel.But chemical reagent such as initiator, aided initiating have been selected for use in the disclosed hydrogel support of this patent.Chinese patent 02113378.6 discloses can be transplanted to intravital bone material, and it uses molecular weight to be the L-lactic acid polymer of 8-20 ten thousand and the calcium polyphosphate fiber of average length 5-15mm, adopts founding granule leaching to be composited.It is 5 * 10 that Chinese patent 200810150135.0 discloses a kind of hydroxypropyl emthylcellulose and concentration of 10%～15% of containing
6The cartilage tissue engineering bracket material for injections of cartilage seed cell.If select for use from the seed cell of body chondrocyte as cartilage tissue engineering rack; Owing to can not obtain chondrocyte immediately and disposable extracted amount is little; Need go down to posterity and cultivate the seed cell that just can obtain capacity several times, so this seed cell is used very inconvenience in clinical.
Summary of the invention
The objective of the invention is to the deficiency of prior art and a kind of injectable articular cartilage tissue repair materials and preparation method thereof is provided, be characterized in that this repair materials selects the good raw material of biocompatibility for use, the initiator that does not include in the chemical gel in the component to be added etc.; Biocompatibility and biological degradability are good, biologically active, and the fluid property of hydrogel predecessor is good; Be easy to injection; Can be self-moulding, the seed cell of selecting for use is easy to extract, and is convenient to clinical practice.
The object of the invention has following technical measures to realize that wherein said raw material umber is parts by weight except that specified otherwise.
The initiation material of injectable articular cartilage tissue repair materials is composed of the following components:
100 parts of type i collagens,
10～50 parts of hyaluronic acids are preferably 12～45 parts, are preferably 20～40 parts again, are preferably 30～40 parts again;
Wherein, hyaluronic oxidizability is 20%～60%; Be embedded with the mononuclear cell layer in the bone marrow fluid in the organism in the said repair materials, the thickness of composite is 1～5mm.
The method for preparing of injectable articular cartilage tissue repair materials may further comprise the steps:
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of step (2), add the sodium periodate solution of step (3), at room temperature lucifuge stirs, and reaction 5～10h, reactant liquor be with deionized water dialysis 60～96h, and lyophilization obtains oxidizability and be 20%～60% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 20%～60% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 3.15～15.75mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains is regulated pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of step (4) in the neutral collagen mixed solution of step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 6～20h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction organism bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 10～25min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to organism articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel, has good self-moulding ability.
Injectable articular cartilage tissue repair materials is used for the damaged of articular cartilage tissue, organizational project and clinical medicine domain.
Performance test:
1. test water content, the degradation property of repair materials with weight method;
2. with the degree of cross linking between hyaluronic effective content and collagen and the hyaluronic acid in the chemical method test compound material;
3. seed cell is embedded in experiment in the external and body in the material, observes the growth conditions of seed cell with laser confocal microscope;
4. detect the propagation situation and the substrate secretion of seed cell with fluorescence spectrophotometry and ultraviolet spectrophotometry;
5. detect the secretion of cartilage cell epimatrix and the formation of characteristic cartilage lacuna with histological stain and Immunohistochemical Staining.
The result shows: repair materials has very high water content, and is imitative like cartilaginous tissue in the organism, good degradation property, and hyaluronic acid effectively is fixed in the repair materials; Dependence test and observation to seed cell show that the raw material components in this composite has the effect of good one-tenth chondrocyte induction to seed cell, and the secretion of distinctive cartilage lacuna formation, cartilage cell epimatrix is arranged.
The present invention has the following advantages:
1, biological activity bionic material collagen can induce the stem cell in the mononuclear cell layer to become the cartilage differentiation in articular cartilage place original position with hyaluronic acid, the secretion cartilage cell epimatrix, thus make up cartilaginous tissue to reach the reparation purpose;
2, have syringeability, can form gel in damaged in-situ injection, the wound that organism is caused is little, avoids causing the secondary wound;
3, become gelling performance good, do not need organic solvent;
4, have good biological safety and biocompatibility, no cytotoxicity;
5, histological stain and immunohistochemical staining show, this hydrogel and seed cell can form soft in external compound cultivation
Osteoid tissue has the extracellular matrix secretion of cartilaginous tissue;
6, be convenient to clinical manipulation, method for preparing is simple to operate, and is environmentally friendly.
The specific embodiment
Below through embodiment the present invention is carried out concrete description; Be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; Can not be interpreted as the restriction to protection domain of the present invention, the person skilled in the art in this field can make some nonessential improvement and adjustment to the present invention according to the content of the invention described above.
It is 100 parts that embodiment 1 adopts type i collagen, and the oxidation hyaluronic acid is 10 parts; The hyaluronic oxidizability of oxidation is 20%, and the thickness of composite is 3mm.
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of 20ml step (2), add the sodium periodate solution of 5ml step (3), at room temperature lucifuge stirs, and reaction 5h, reactant liquor be with the deionized water 60h that dialyses, and lyophilization obtains oxidizability and be 20% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 20% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 3.15mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of 2ml step (4) in the neutral collagen mixed solution of 10ml step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 18h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction Japan large ear rabbit bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 15min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to Japan large ear rabbit articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel.
It is 100 parts that embodiment 2 adopts type i collagen, and the oxidation hyaluronic acid is 20 parts; The hyaluronic oxidizability of oxidation is 20%, and the thickness of composite is 3mm.
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of 20ml step (2), add the sodium periodate solution of 5ml step (3), at room temperature lucifuge stirs, and reaction 5h, reactant liquor be with the deionized water 60h that dialyses, and lyophilization obtains oxidizability and be 20% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 20% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 6.3mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of 2ml step (4) in the neutral collagen mixed solution of 10ml step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 10h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction Japan large ear rabbit bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 15min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to Japan large ear rabbit articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel.
It is 100 parts that embodiment 3 adopts type i collagen, and the oxidation hyaluronic acid is 20 parts; The hyaluronic oxidizability of oxidation is 40%, and the thickness of composite is 3mm.
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of 20ml step (2), add the sodium periodate solution of 10ml step (3), at room temperature lucifuge stirs, and reaction 8h, reactant liquor be with the deionized water 72h that dialyses, and lyophilization obtains oxidizability and be 40% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 40% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 6.3mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of 2ml step (4) in the neutral collagen mixed solution of 10ml step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 12h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction Japan large ear rabbit bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 15min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to Japan large ear rabbit articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel.
It is 100 parts that embodiment 4 adopts type i collagen, and the oxidation hyaluronic acid is 50 parts; The hyaluronic oxidizability of oxidation is 40%, and the thickness of composite is 3mm.
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of 20ml step (2), add the sodium periodate solution of 10ml step (3), at room temperature lucifuge stirs, and reaction 8h, reactant liquor be with the deionized water 72h that dialyses, and lyophilization obtains oxidizability and be 40% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 40% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 15.75mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of 2ml step (4) in the neutral collagen mixed solution of 10ml step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 12h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction Japan large ear rabbit bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 15min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to Japan large ear rabbit articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel.
It is 100 parts that embodiment 5 adopts type i collagen, and the oxidation hyaluronic acid is 10 parts; The hyaluronic oxidizability of oxidation is 60%, and the thickness of composite is 3mm.
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of 20ml step (2), add the sodium periodate solution of 15ml step (3), at room temperature lucifuge stirs, and reaction 10h, reactant liquor be with the deionized water 96h that dialyses, and lyophilization obtains oxidizability and be 60% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 60% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 3.15mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of 2ml step (4) in the neutral collagen mixed solution of 10ml step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 6h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction Japan large ear rabbit bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 15min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to Japan large ear rabbit articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel.
It is 100 parts that embodiment 6 adopts type i collagen, and the oxidation hyaluronic acid is 50 parts; The hyaluronic oxidizability of oxidation is 60%, and the thickness of composite is 3mm.
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of 20ml step (2), add the sodium periodate solution of 15ml step (3), at room temperature lucifuge stirs, and reaction 10h, reactant liquor be with the deionized water 96h that dialyses, and lyophilization obtains oxidizability and be 60% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 60% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 15.75mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of 2ml step (4) in the neutral collagen mixed solution of 10ml step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 6h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction Japan large ear rabbit bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 15min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to Japan large ear rabbit articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel.
Claims (3)
1. an injectable articular cartilage tissue repair materials is characterized in that the initiation material of this repair materials is composed of the following components, is by weight:
100 parts of type i collagens,
10～50 parts of hyaluronic acids;
Wherein, hyaluronic oxidizability is 20%～60%; Be embedded with the mononuclear cell layer in the bone marrow fluid in the organism in the said repair materials, the thickness of composite is 1～5mm.
2. according to the method for preparing of the said injectable articular cartilage tissue repair materials of claim 1, it is characterized in that this method may further comprise the steps:
(1) in ice-water bath, collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, slowly stir, be mixed with the collagen solution that concentration is 9mg/ml, place 4 ℃ of refrigerators of temperature to leave standstill;
(2) hyaluronic acid powder is dissolved in the ultra-pure water, stirs and make it abundant dissolving, be mixed with the hyaluronic acid solution that concentration is 10mg/ml;
(3) sodium metaperiodate powder lucifuge is dissolved in the ultra-pure water, stirs and be mixed with the sodium periodate solution that concentration is 20mM;
(4) in the hyaluronic acid solution of step (2), add the sodium periodate solution of step (3), at room temperature lucifuge stirs, and reaction 5～10h, reactant liquor be with deionized water dialysis 60～96h, and lyophilization obtains oxidizability and be 20%～60% oxidation hyaluronic acid;
(5) oxidizability that step (4) is obtained is 20%～60% oxidation hyaluronic acid, with ultra-pure water dissolving, stirs and is made into the oxidation hyaluronic acid solution that concentration is 3.15～15.75mg/ml, and this solution is subsequent use after with 0.22um filter filtration sterilization;
(6) under ice-water bath, add 5 * PBS solution and buffer in the collagen solution of step (1), slowly stir, the collagen mixed solution that obtains transfers pH to neutral, and wherein, 5 * PBS solution concentration is 0.05M, and the composition of buffer is NaOH-NaHCO
3-Hepes, the concentration of NaOH is 50mM, NaHCO
3Concentration be 260mM, the concentration of Hepes is 200mM, buffer and 5 * PBS solution are all used behind the 0.22um filter filtration sterilization subsequent use; Collagen solution, 5 * PBS solution and the blended volume ratio of buffer are 7: 2: 1;
(7) under ice-water bath, add the oxidation hyaluronic acid solution of step (4) in the neutral collagen mixed solution of step (6), stir, put into 4 ℃ of refrigerators of temperature and leave standstill 6～20h;
(8) with the cell of whole mononuclear cell layer in the density separation and Extraction organism bone marrow fluid, processing density is 1 * 10
8The cell suspension of individual/ml takes out collagen and hyaluronic mixed liquor of oxidation and said cell suspension mix homogeneously that step (7) obtains from 4 ℃ of refrigerators of temperature;
(9) sterile working is all attend at superclean bench in above-mentioned steps (5), (6), (7), (8);
(10) mixed liquor that step (8) is obtained is put into 37 ℃ of constant incubators of temperature and is left standstill 10～25min, obtains composite.
(11) mixed liquor that step (8) is obtained is expelled to organism articular cartilage tissue defect, utilizes the physiological temp of bio-tissue to make it original position formation hydrogel, has good self-moulding ability.
3. according to the purposes of the said injectable articular cartilage tissue repair materials of claim 1, it is characterized in that this articular cartilage tissue repair materials is used for the damaged of articular cartilage tissue, organizational project and clinical medicine domain.
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Application publication date: 20120613 |