CN102921045A - Nano-hydroxyapatite/chitosan/chondroitin sulfuric acid composite stent - Google Patents
Nano-hydroxyapatite/chitosan/chondroitin sulfuric acid composite stent Download PDFInfo
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- CN102921045A CN102921045A CN2012104306623A CN201210430662A CN102921045A CN 102921045 A CN102921045 A CN 102921045A CN 2012104306623 A CN2012104306623 A CN 2012104306623A CN 201210430662 A CN201210430662 A CN 201210430662A CN 102921045 A CN102921045 A CN 102921045A
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Abstract
The invention discloses a nano-hydroxyapatite/chitosan/chondroitin sulfuric acid composite stent with high biological response and a preparation method of the nano-hydroxyapatite/chitosan/chondroitin sulfuric acid composite stent. The preparation method comprises the steps of: using chitosan/chondroitin sulfuric acid as an organic matrix and using a calcium-phosphor salt solution as an inorganic phase precursor; fully uniformly mixing a chitosan/chondroitin sulfuric acid acetic acid solution with a soluble calcium-phosphor salt solution, crosslinking with a crosslinking agent, carrying out injection molding and forming, carrying out in-situ biomimetic mineralization crystallization in alkaline liquor, freezing and drying to obtain the nano-hydroxyapatite/chitosan/chondroitin sulfuric acid composite stent with high biological response. The preparation method can be used for simulating a composition and a structure of natural bone on the aspect of material preparation, is mild in preparation condition, and is simple to operate; and the prepared material can be randomly shaped and re-machined according to the use demand.
Description
Technical field
The invention belongs to artificial bone repair materials technical field, be specifically related to a kind of high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest and preparation method thereof.
Background technology
Body bone tissue is the natural mineralising biomaterial with high-precision structure that is composited by the nanometer hydroxyapatite of weak degree of crystallinity and collagen assembling.In a broad sense, can be regarded as the double-phase composite or the Organic/Inorganic Composite Nanomaterials that contain nanocrystal in the matrix, these research and development for bone renovating material provide inspiration and inspiration, and the nanometer hydroxyapatite bone-repairing composite material arises at the historic moment.So-called nano composite material refers to that dispersed phase size at least less than the composite of 100nm, because the interfacial area between decentralized photo and the matrix is large, can more fully combines the performance of decentralized photo and matrix, thereby have good combination property.Usually the method that prepare the nanometer hydroxyapatite bone-repairing composite material mainly is after the nanometer hydroxyapatite with matrix material and synthetic passes through simple physical mechanical or chemical mode mix homogeneously, the mould that reinjects, lyophilization molding.Hydroxyapatite disperses inhomogeneously in the composite of simple composite preparation, and agglomeration easily occurs, and a little less than the interface binding power, so that the mechanical properties decrease of material, microstructure is unordered.The compound technology of preparing of original position is the emerging research field that development in recent years is got up, adopting the compound situ reaction of original position to prepare inorganic/organic system molecular composite material is a kind of more promising method for preparing organic/inorganic composite material, it can well reduce the interface energy at organic/inorganic interface, realizes the Uniform Dispersion of inorganic particulate on organic substrate
This research adopts the in-situ compositing preparation take chitosan/chondroitin sulfate as organic matrix, and homodisperse nano-HAP is the composite of inorganic phase, has realized that molecular level is compound, has guaranteed the mechanical property of material.
Chitosan is natural biomaterial, and is positively charged, and the main component-glycosaminoglycans in its structure and the extracellular matrix is very similar, and its catabolite has no side effect to human body, has good biological degradability and biocompatibility.Chondroitin sulfate is a kind of straight chain glycosaminoglycans with negative charge, is the main component in the extracellular matrix, and it can promote cell to attach, and improves intensity and the toughness of osseous tissue, so have fabulous biological activity.Chitosan, the amino of chondroitin sulfate, carboxyl, carbonyl group plays the current potential complementation for the crystallization of nanometer hydroxyapatite, binding and chelation, the guiding nanometer hydroxyapatite carries out in-situ crystallization at organic matrix, nanometer hydroxyapatite and the chitosan of in-situ crystallization have been caused, chondroitin sulfate has certain interaction, reduction hydroxyapatite presoma and chitosan have been played, the interface bonding energy of chondroitin sulfate storeroom, so that hydroxyapatite forms with matrix at organic matrix interactional Uniform Dispersion nano-particle is arranged, thereby realized the in-situ crystallization of nanometer hydroxyapatite on organic matrix.
Summary of the invention
The object of the present invention is to provide a kind of high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest and preparation method thereof, develop according to the compound preparation of original position and biomimetic mineralization principle that a kind of degree of crystallinity is controlled, the height Uniform Dispersion, and and form the nanometer hydroxyapatite compound rest that effective stable keys closes between the organic matrix, thereby at the bionical structure of 26S Proteasome Structure and Function realization to natural bone.The preparation process condition of the method is gentle, and easy and simple to handle, cost compare is low, and preparing resulting timbering material can according to the specific requirement of using position and shape, process moulding.
For achieving the above object, the present invention adopts following technical scheme:
The preparation method of a kind of high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest, take chitosan/chondroitin sulfate as organic matrix, calcium-microcosmic salt solution is the inorganic phase presoma; With chitosan/chondroitin sulfate acetic acid solution and soluble calcium salt and the abundant mix homogeneously of phosphate solution, crosslinked through cross-linking agent again, casting, the crystallization of alkali liquor situ biomimetic mineralization, lyophilization obtains high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest.
The concrete steps of described preparation method are as follows:
Step 1: preparation (1) preparation 1～2mol/L calcium salt soln and 0.6～1.2mol/L phosphate solution of support presoma mixed liquor; (2) with chitosan powder and chondroitin sulfate powder dissolution in volume fraction is 1～2% acetic acid solution, stir and make each component mix homogeneously; (3) material of above-mentioned (1), (2) is mixed to get the mixture of variable concentrations with different proportionings, calcium in the system-phosphorus mol ratio is 1.67:1, lasting stirring makes its abundant mix homogeneously, then adds cross-linking agent and carries out crosslinkedly, obtains support presoma mixed liquor;
Step 2: the molding (1) of the nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest mixed liquor that step 1 is prepared injects mould, move to 4 ℃ of refrigerators and leave standstill 4～6 h, put into subsequently the freezing at least 12h of ultra cold storage freezer, then change extremely fully dehydration of lyophilization in the freezer dryer over to; (2) drying sample with step 2 (1) gained places alkali liquor to soak, and then repeatedly embathe to neutrality with deionized water, carry out lyophilization after ultra cold storage freezer is freezing and obtain high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest.
The described calcium salt of step 1 (1) is lime nitrate or calcium chloride; Phosphate is a kind of in sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, the dipotassium hydrogen phosphate.
The concentration of chitosan is 10～20mg/mL in the final mixed liquor of step 1 (3), the concentration of chondroitin sulfate is 2～10mg/mL, it is 8～20mg/mL that the theoretical HAP of formation of the Ca/P precursor solution that adds measures, and the organic matrix component is 1:0.4～1 with the theoretical mass ratio that forms HAP.
The described mixed system of step 1 (3) crosslinked 4～6h under room temperature, cross-linking agent are 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy-succinamide (NHS); The concentration of cross-linking agent EDC is 2～5mg/mL, and the mol ratio of EDC and NHS is 1.5～5:1.
Freezing temperature range is-5 ℃～-80 ℃ in the described ultra cold storage freezer of step 2 (1).
The described alkali liquor of step 2 (2) is that mass fraction is that ethanol/deionized water solution or the mass fraction of 5～10%NaOH is ethanol/deionized water solution of 5～10%KOH; The volume ratio of ethanol and deionized water is 1～3:1 in the described alkali liquor.
The described drying sample of step 2 (2) placed the alkali liquor soak at room temperature at least 8 hours.
Chondroitin sulfate is chondroitin sulfate A or C.
Remarkable advantage of the present invention is: chitosan is a kind of natural cationic polysaccharide that contains a large amount of amino, chondroitin sulfate is a kind of anion glycosaminoglycans, chondroitin sulfate and chitosan are cross-linked to form the support organic matrix, on microstructure and physicochemical property, such as aspects such as surface charge, hydrophilic and hydrophobics, there are many similaritys with biomacromolecule.The groups such as the carboxyl that contains in the organic matrix, hydroxyl, sulfonic group, amido and amide groups not only can promote adhesion and the growth of cell, and are the nucleation sites of inorganic phase hydroxyapatite.The present invention utilizes the regular distribution on organic matrix of these groups just, the nucleation and crystallization of original position induced nano hydroxyapatite, thus prepare nano-particle high degree of dispersion, the controlled high biological response nano-composite scaffolds of degree of crystallinity.
The support of preparation gained has following advantage with respect to other compound rests:
(1) select the combination of chitosan, chondroitin sulfate and hydroxyapatite component, the biocompatibility of these three kinds of components, biological activity are all better, and the proportioning of three kinds of components can be adjusted;
(2) develop according to the compound preparation of original position and biomimetic mineralization principle that a kind of degree of crystallinity is controlled, the height Uniform Dispersion, and and the nano-composite scaffolds that the effective stable keys of formation closes between the organic matrix, because the Uniform Dispersion of nano-particle on the compound rest, the mechanical property of compound rest is improved;
(3) adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of compound support frame material, the result shows that this material cell compatibility is good;
(4) preparation process condition of this high biological response nano-composite scaffolds is gentle, and easy and simple to handle, cost is lower.
Description of drawings
Fig. 1-2 is the scanning electron microscope diagram of high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest.
Fig. 3 is the X-ray diffractogram of high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest.
Fig. 4-the 7th, the phase contrast microscope figure of high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest and the external compound cultivation of Osteoblasts of Newborn Mouse (the compound incubation time difference of Fig. 4-7: 4h, 12h, 72h, 96h; Because support is light tight, shows as shade under phase contrast microscope).
The specific embodiment
Embodiment 1
1) preparation 2mol/L lime nitrate saline solution, the 1.2mol/L potassium dihydrogen phosphate; The mass ratio of chitosan, chondroitin sulfate is 10:1;
2) chitosan/chondroitin sulfate with described quality proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of lime nitrate saline solution in above-mentioned chitosan/chondroitin sulfate mixed liquor, potassium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.4 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 2mg/mL, and the EDC/NHS mol ratio is 1.5:1, ambient cross-linking 6h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 4 h, puts into subsequently-10 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 5%NaOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-10 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 2
1) preparation 2mol/L calcium nitrate solution, the potassium dihydrogen phosphate of 0.6mol/L; The mass ratio of chitosan, chondroitin sulfate is 5:1;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium nitrate solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, potassium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.5 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 2.5mg/mL, and the EDC/NHS mol ratio is 2:1, ambient cross-linking 5h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 4h, puts into subsequently-20 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 5%NaOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-20 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 3
1) preparation 1mol/L calcium nitrate solution, the 1.2mol/L disodium phosphate soln; The mass ratio of chitosan, chondroitin sulfate is 10:3;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium nitrate solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, disodium phosphate soln stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.6 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 3mg/mL, and the EDC/NHS mol ratio is 2.5:1, ambient cross-linking 5h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 6h, puts into subsequently-80 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 5%NaOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-80 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
1) preparation 2mol/L calcium nitrate solution, the 0.6mol/L disodium phosphate soln; The mass ratio of chitosan, chondroitin sulfate is 5:2;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 2% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium nitrate solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, disodium phosphate soln stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.7 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 4mg/mL, and the EDC/NHS mol ratio is 3:1, ambient cross-linking 6h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 4 h, puts into subsequently-10 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 5%KOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-10 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 5
1) preparation 1mol/L calcium chloride solution, the 1.2mol/L potassium dihydrogen phosphate; The mass ratio of chitosan, chondroitin sulfate is 2:1;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 2% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium chloride solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, potassium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.8 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 5mg/mL, and the EDC/NHS mol ratio is 3:1, ambient cross-linking 5h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 6 h, puts into subsequently-20 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 5%KOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-20 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 6
1) preparation 2mol/L calcium chloride solution, the potassium dihydrogen phosphate of 0.6mol/L; The mass ratio of chitosan, chondroitin sulfate is 5:3;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium chloride solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, potassium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.9 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 4mg/mL, and the EDC/NHS mol ratio is 1.5:1, ambient cross-linking 4h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 5 h, puts into subsequently-10 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 5%KOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-10 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 7
1) preparation 2mol/L calcium nitrate solution, the 1.2mol/L sodium dihydrogen phosphate; The mass ratio of chitosan, chondroitin sulfate is 10:7;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium nitrate solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, sodium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:1 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 4mg/mL, and the EDC/NHS mol ratio is 2:1, ambient cross-linking 6h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 4 h, puts into subsequently-20 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 10%NaOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-20 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 8
1) preparation 2mol/L calcium nitrate solution, the 0.6mol/L sodium dihydrogen phosphate; The mass ratio of chitosan, chondroitin sulfate is 5:4;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium nitrate solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, sodium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.6 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 5mg/mL, and the EDC/NHS mol ratio is 1.5:1, ambient cross-linking 5h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 6 h, puts into subsequently-20 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 10%NaOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-20 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
Embodiment 9
1) preparation 1mol/L calcium chloride solution, the 1.2mol/L sodium dihydrogen phosphate; The mass ratio of chitosan, chondroitin sulfate is 4:1;
2) chitosan/chondroitin sulfate with described proportioning is dissolved in 1% acetic acid solution, stirs;
3) add lentamente a certain amount of calcium chloride solution in the chitosan of mix homogeneously/chondroitin sulfate mixed liquor, sodium dihydrogen phosphate stirs 1h; N (Ca wherein
2+): n (PO
4 3-)=1.67:1, organic component is 1:0.7 with the theoretical mass ratio that forms HAP;
4) add cross-linking agent EDC/NHS, the concentration of EDC is 5mg/mL, and the EDC/NHS mol ratio is 2:1, ambient cross-linking 6h; Final mixed liquor injects mould, moves to 4 ℃ of refrigerators and leaves standstill 5 h, puts into subsequently-10 ℃ of freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
5) drying sample is placed the ethanol of 10%KOH/deionized water soak at room temperature 10h, repeatedly embathe to neutrality with deionized water again, carry out lyophilization after-10 ℃ of ultra cold storage freezers are freezing and obtain product.
Adopt Osteoblasts of Newborn Mouse to set up the in-vitro evaluation model of timbering material, investigate the Cytological Characteristics of the synthetic nano-hydroxyapatite/chitosan of original position composite bionic/chondroitin sulfate compound rest.
This preparation method can be so that the original position compound rest be preformed into various shapes according to actual needs; Can observe support by SEM figure and form the interconnected pore structure that mutually runs through preferably, equally distributed tiny nano-particle is arranged on the support hole wall.By the XRD figure of support spectrum, can find that the diffraction maximum of the inorganic crystal thing in the support is consistent with the characteristic peak of HAP, and its diffraction maximum exists broadening, diffusing phenomenon, the nanometer hydroxyapatite of degree of crystallinity a little less than original position has formed on the support is described.Osteoblast and timbering material are united cultivation, can find out from phase contrast microscope figure, behind the adding cell suspension, cell is in the random distribution of edge of materials, cell is the trend of fusiformis, polygon tiling and oriented material end trend behind 12 h, can observe cell behind 72 h and constantly breed.Along with the prolongation of incubation time and the propagation of cell, can find that edge of materials assembling increasing cell, osteoblast is keeping good growth conditions always in the associating incubation, illustrates that the cell compatibility of compound rest is good.
The above only is better embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (10)
1. the preparation method of one kind high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest, it is characterized in that: take chitosan/chondroitin sulfate as organic matrix, calcium-microcosmic salt solution is the inorganic phase presoma; With chitosan/chondroitin sulfate acetic acid solution and soluble calcium salt and the abundant mix homogeneously of phosphate solution, crosslinked through cross-linking agent again, casting, the crystallization of alkali liquor situ biomimetic mineralization, lyophilization obtains high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest.
2. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 1/chondroitin sulfate compound rest, it is characterized in that: the concrete steps of described preparation method are as follows:
Step 1: the preparation of support presoma mixed liquor
(1) preparation 1～2mol/L calcium salt soln and 0.6～1.2mol/L phosphate solution;
(2) with chitosan powder and chondroitin sulfate powder dissolution in volume fraction is 1～2% acetic acid solution, stir and make each component mix homogeneously;
(3) material of above-mentioned (1), (2) is mixed to get the mixture of variable concentrations with different proportionings, calcium in the system-phosphorus mol ratio is 1.67:1, lasting stirring makes its abundant mix homogeneously, then adds cross-linking agent and carries out crosslinkedly, obtains support presoma mixed liquor;
Step 2: the molding of nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest
(1) mixed liquor that step 1 is prepared injects mould, moves to 4 ℃ of refrigerators and leaves standstill 4～6 h, puts into subsequently the freezing at least 12h of ultra cold storage freezer, then changes extremely fully dehydration of lyophilization in the freezer dryer over to;
(2) drying sample with step 2 (1) gained places alkali liquor to soak, and then repeatedly embathe to neutrality with deionized water, carry out lyophilization after ultra cold storage freezer is freezing and obtain high biological response nano-hydroxyapatite/chitosan/chondroitin sulfate compound rest.
3. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest, it is characterized in that: the described calcium salt of step 1 (1) is lime nitrate or calcium chloride; Phosphate is a kind of in sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, the dipotassium hydrogen phosphate.
4. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest, it is characterized in that: the concentration of chitosan is 10～20mg/mL in the final mixed liquor of step 1 (3), the concentration of chondroitin sulfate is 2～10mg/mL, it is 8～20mg/mL that the theoretical HAP of formation of the Ca/P precursor solution that adds measures, and the organic matrix component is 1:0.4～1 with the theoretical mass ratio that forms HAP.
5. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest is characterized in that: the described mixed system of step 1 (3) crosslinked 4～6h under room temperature, and cross-linking agent is EDC/NHS; The concentration of cross-linking agent EDC is 2～5mg/mL, and the mol ratio of EDC and NHS is 1.5～5:1.
6. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest, it is characterized in that: freezing temperature range is-5 ℃～-80 ℃ in the described ultra cold storage freezer of step 2 (1).
7. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest is characterized in that: the described alkali liquor of step 2 (2) is that mass fraction is that ethanol/deionized water solution or the mass fraction of 5～10%NaOH is ethanol/deionized water solution of 5～10%KOH; The volume ratio of ethanol and deionized water is 1～3:1 in the described alkali liquor.
8. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest, it is characterized in that: the described drying sample of step 2 (2) placed the alkali liquor soak at room temperature at least 8 hours.
9. the preparation method of high biological response nano-hydroxyapatite/chitosan according to claim 2/chondroitin sulfate compound rest, it is characterized in that: chondroitin sulfate is chondroitin sulfate A or C.
10. the high biological response nano-hydroxyapatite/chitosan that makes of the method for claim 1/chondroitin sulfate compound rest.
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| CN103143059A (en) * | 2013-03-01 | 2013-06-12 | 福州大学 | Nano-composite bone defect repair support with multistage pore diameter structure |
| CN103272282A (en) * | 2013-05-15 | 2013-09-04 | 福州大学 | Nanometer hydroxyapatite/chitosan/fish skin collagen composite scaffold |
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| CN111139594A (en) * | 2020-03-11 | 2020-05-12 | 昆明理工大学 | Preparation method of composite porous material for bone repair |
| CN117887134A (en) * | 2024-03-14 | 2024-04-16 | 四川大学 | Hydrogel biomimetic mineralization stent and preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103143059A (en) * | 2013-03-01 | 2013-06-12 | 福州大学 | Nano-composite bone defect repair support with multistage pore diameter structure |
| CN103272282A (en) * | 2013-05-15 | 2013-09-04 | 福州大学 | Nanometer hydroxyapatite/chitosan/fish skin collagen composite scaffold |
| CN103495210A (en) * | 2013-09-26 | 2014-01-08 | 福州大学 | Chitosan-hydroxylapatite in-situ loaded icariin composite microspheres |
| CN103495210B (en) * | 2013-09-26 | 2015-03-04 | 福州大学 | Chitosan-hydroxylapatite in-situ loaded icariin composite microspheres |
| CN108478880A (en) * | 2018-04-10 | 2018-09-04 | 暨南大学 | A kind of nano-hydroxyapatite/chitosan porous compound support frame material and its bionical dialysis mineralising preparation method and application |
| CN111139594A (en) * | 2020-03-11 | 2020-05-12 | 昆明理工大学 | Preparation method of composite porous material for bone repair |
| CN117887134A (en) * | 2024-03-14 | 2024-04-16 | 四川大学 | Hydrogel biomimetic mineralization stent and preparation method and application thereof |
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