CN106310378B - A kind of preparation method of imitation human bone material - Google Patents
A kind of preparation method of imitation human bone material Download PDFInfo
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- CN106310378B CN106310378B CN201610812243.4A CN201610812243A CN106310378B CN 106310378 B CN106310378 B CN 106310378B CN 201610812243 A CN201610812243 A CN 201610812243A CN 106310378 B CN106310378 B CN 106310378B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The present invention relates to a kind of preparation methods of imitation human bone material, belong to biomedical material technical applications.The porous material adds distilled water to be uniformly mixing to obtain aqueous gelatin solution using gelatin as raw material, is formed the Solution extrusion by mold, prepares the porous support combined with regular clear opening.Then the bracket is crosslinked in glutaraldehyde solution, the mineralising in SBF solution obtains the isinglass multi-hole bracket of the inorganic salts containing calcium and phosphorus.The isinglass multi-hole bracket has regular clear opening, and pore-size distribution is at 300-450 μm.Porosity is within the scope of 50%-95%；The isinglass multi-hole bracket axial compression strength reaches 12-14MPa, and lateral compression strength reaches 7-9 MPa.Present invention process is simple, convenient for operation, stable yield；The bracket cytotoxicity that this method is prepared is qualified, and skeletonization works well, has a good application prospect.
Description
Technical field
The present invention relates to a kind of preparation methods of imitation human bone material, belong to bio-medical material Technology application field.
Background technique
It is well known that natural bone tissue is mainly by with HAP inorganic phase as main component and organic based on collagen
Phase composition.So the material of imitated people's bone also should using both materials as standard, choose as far as possible with people's bone constituent structure
Similar material.In bone holder material, hydroxyapatite is combined with natural macromolecular material and prepares Organic-inorganic composite
Material, can be in conjunction with the advantages of natural macromolecular material and hydroxyapatite mineral.In natural macromolecular material, collagen is bone group
The main organic matter knitted, and gelatin is the natural products that collagen is obtained through partial denaturation or chemical treatment hydrolysis.It has
Excellent physical and chemical performance, such as hydrophily it is strong, be capable of forming gel, side chain radical because reactivity is high, convenient for chemical modification.
Due to these advantages, gelatin is widely used in various fields.Meanwhile it has good biocompatibility and degradability and valence
Lattice are cheap, from a wealth of sources, so can be used as tissue engineering bracket.And hydroxyapatite no matter the application in os osseum or cartilage
Good biocompatibility is all embodied, and one of the main constituent as osteocyte epimatrix, had good
Osteoacusis and synosteosis performance and high mechanical strength.However, the fracture toughness of artificial synthesized hydroxyapatite is lower, brittleness
Larger, toughness is insufficient and poor mechanical property, is restricted its application aspect in terms of load-bearing bone.It is asked to solve these
It inscribes, in recent years the special valuing researches of people and the various hydroxyapatite composite materials of exploitation, to improve hydroxylapatite biology material
The mechanical property and biology performance of material.
To biomineralization studies have shown that during entire biomineralization, interfacial molecular recognition process is played to Guan Chong
The effect wanted.Under certain conditions, for organic substrate by being self-assembly of template, which is providing structure for inorganic mineral
While frame, by with inorganic ions on interface electrostatic matching, geometric similarity and spatial chemistry is complementary etc. controls mine
The nucleation and growth of object, to control the structure and performance of biogenic mineral material.Crystal can be effectively controlled in mineralization method
Pattern and structure.
From the perspective of bionics, polymer prepared by polymers modulate biomimetic mineralization-hydroxyapatite composite wood
Material is similar to natural bone tissue on ingredient and structure.The selection for having crossed material by discussion before is needed organic gelatin
It is combined with hydroxyapatite.And synthesizing hydroxylapatite can be guided by organic substrate such as collagen by discussing,
Moreover gelatin materials are can to replace organic macromolecule matrix of the collagen as biomineralization.
Current natural macromolecular material/hydroxyapatite composite material preparation is typically all by hydroxyapatite powder
It is mixed with the aqueous solution of high molecular polymer, since such powder is not readily dissolved in water, which results in most lifes
Object activity hydroxy apatite has all been embedded in the inside of polymer, and being cultivated or be implanted into vitro defect can not be with
Cell effectively contacts.And polymer mostly has certain viscosity, and mixing physically is easy to cause hydroxylapatite powder
The reunion at end, has influenced the structure of bone bracket.And in the environment of close to physiological condition, high temperature is avoided or using organic
Solvent, controlled in aqueous solution by polymer inorganic mineral bionical deposition be material science in terms of an important research
Field.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of imitation human bone material, specifically includes the following steps:
(1) Ca (OH) that mass percent concentration is 0.1% ~ 0.3% is prepared2Solution exists in the ratio of 200g/L ~ 400g/L
Ca(OH)2Gelatin particle is added in solution, it is 1%-6% that then mass percent concentration, which is added, in the ratio of 100ml/L ~ 150ml/L
Glycerine, after gelatin particle is completely soluble in water, colloidal sol is placed on stand at room temperature to viscosity increase when pour into extruding hold
Cooling is stood in device, obtains Sol A；
(2) Sol A is subjected to extrusion forming, is then placed in dehydrated alcohol and is dehydrated, obtain style B；
(3) style B is put into glutaraldehyde solution and is crosslinked, then taken out to be placed in distilled water and clean, then in air
It is dried to obtain style C；
(4) style C is placed in human body fluid simulated solution (SBF simulated body fluid), is subsequently placed in 37.5 DEG C of constant temperature training
It supports in case and stands 14-30 days (i.e. mineralising time), change within every 2 ~ 4 days a not good liquor, the sample that mineralising terminates is cleaned with deionized water, is dry
Imitation human bone material is obtained after dry.
Preferably, standing in step (1) of the present invention to the viscosity increased time is 10 ~ 30 min.
Preferably, the cleaning process in step (3) of the present invention are as follows: take out style and be placed in distilled water and impregnate 2-6 times, every time
10-30min。
Preferably, the mass percent concentration of glutaraldehyde solution is 1-10wt% in step (3) of the present invention, and crosslinking time is
6-24 h。
Preferably, temperature dry in step (3) of the present invention is 40-60 DEG C.
The imitation human bone material that the present invention is prepared has regular clear opening, and pore-size distribution is at 300-450 μm.Porosity exists
Within the scope of 50%-95%.The porous support axial compression strength reaches 12-14MPa, and lateral compression strength reaches 7-9 MPa.
Beneficial effects of the present invention:
(1) present invention uses extrinsion pressing to prepare the porous support materials using gelatin as raw material, which is able to satisfy
The hole configurations and pore size that bone tissue engineer requires, are capable of providing the three dimensional scaffold structure of a tissue growth.
(2) present invention constructs condition similar with human physiological environment, and simulate using the method for external biomimetic mineralization
The mineralising of people's bone in vivo attempts the hydroxyl being close in the matrix surface fabricated in situ of gelatin with the biological mineral matter in natural bone
Base phosphorite crystal constructs imitative bone composite organic-inorganic material.
Detailed description of the invention
Fig. 1 is the SEM picture of the isinglass multi-hole bracket prepared in embodiment 2.
Specific embodiment
Invention is further described in detail in the following with reference to the drawings and specific embodiments, but protection scope of the present invention is simultaneously
It is not limited to the content.
Human body fluid simulated solution (SBF simulated body fluid) of the present invention is prepared to obtain by conventional method and conventional raw material.This
SBF simulated body fluid described in inventive embodiments is prepared to obtain by following methods: measure the deionized water of 800ml as buffer,
And be placed in water-bath and be heated to 35 DEG C -37 DEG C, solution is stirred continuously by blender, and constantly measure real-time PH with PH meter
Value, 8g NaCl powder, 0.35g NaHCO are sequentially added when PH reaches 6.5-7.53Powder, 0.2g KCl powder, 0.2g
K2HPO4*3H2O powder, 0.3g MgCl2*6H2O powder, HCl 30ml, the 0.3g CaCl of 1.0mol/ml2Powder, 0.1g
Na2SO4Powder；6g TRIS(CH is added on a small quantity in batches again2OH)3CNH2Powder stablizes PH 7.0 or so, directly with HCl solution
To TRIS(CH2OH)3CNH2Until powder all adds；Last constant volume drops to room temperature to solution temperature into 1000ml volumetric bottle
Deionized water is added dropwise afterwards and obtains human body fluid simulated solution to graduation mark.
Embodiment 1
A kind of this implementation fabricated in situ imitation human bone material and preparation method thereof specific steps include:
(1) by 0.01g Ca(OH)2It is added in 10ml distilled water, is placed on magnetic stirring apparatus and is heated to 40 DEG C or so,
The gelatin particle of 2g is added into above-mentioned solution again, it is 2% glycerine 1ml as lubricant, constant temperature stirring that concentration, which is added,.To bright
After glue particle is completely soluble in water, colloidal sol is placed on to stand at room temperature when (10min) is significantly increased to viscosity and is poured into
Cooling is stood in the needle tubing of 30ml, obtains Sol A.
(2) Sol A is subjected to extrusion forming, the sample for being cut after every bracket for squeezing out 3cm long, and being cut with thin wire
It is directly placed into dehydrated alcohol and is dehydrated, obtain style B.
(3) style B is cut into the small rack of 3x3, small rack is put into the glutaraldehyde solution that concentration is 10% and is crosslinked 6h
Afterwards, it takes out to be placed in distilled water and impregnate 6 times, each 10min is placed on filter paper after having cleaned and is air-dried, and obtains style C.
(4) style C is placed in the plastic centrifuge tube of 50ml, the SBF simulated body fluid of every centrifuge tube addition 30ml, so
It is placed in 37.5 DEG C of constant incubator and stands 30 days, change a not good liquor every three days, the sample deionized water that mineralising is terminated
Careful cleaning is placed in drying in 60 DEG C of drying box, obtains final timbering material.
The present embodiment is measured by the axial compression strength to the gelatin bracket with lateral compression strength, shows axial direction
Compression strength and lateral compression strength are respectively 11.52MPa, 7.56 MPa；Its porosity is measured, shows that porosity is
75%。
Embodiment 2
A kind of this implementation fabricated in situ imitation human bone material and preparation method thereof specific steps include:
(1) by 0.02g Ca(OH)2It is added in 10ml distilled water, is placed on magnetic stirring apparatus and is heated to 40 DEG C or so,
The gelatin particle of 3.5g is added into above-mentioned solution again, it is 4% glycerine 1ml as lubricant, constant temperature stirring that concentration, which is added,.To
After gelatin particle is completely soluble in water, colloidal sol is placed on to stand at room temperature when (20min) is significantly increased to viscosity and is poured into
Cooling is stood in the needle tubing of 30ml, obtains Sol A.
(2) Sol A is subjected to extrusion forming, the sample for being cut after every bracket for squeezing out 3cm long, and being cut with thin wire
It is directly placed into dehydrated alcohol and is dehydrated, obtain style B.
(3) style B is cut into the small rack of 3x3, small rack is put into the glutaraldehyde solution that concentration is 5% and is crosslinked 12h
Afterwards, it takes out to be placed in distilled water and impregnate 3 times, each 20min is placed on filter paper after having cleaned and is air-dried, and obtains style C.
(4) style C is placed in the plastic centrifuge tube of 50ml, the SBF simulated body fluid of every centrifuge tube addition 30ml, so
It is placed in 37.5 DEG C of constant incubator and stands 25 days, change a not good liquor every three days, the sample deionized water that mineralising is terminated
Careful cleaning is placed in drying in 40 DEG C of drying box, obtains final timbering material.
The present embodiment is measured by the axial compression strength to the gelatin bracket with lateral compression strength, shows axial direction
Compression strength and lateral compression strength are respectively 13.50MPa, 8.5 MPa.Its porosity is measured, shows that porosity is
85%.The SEM picture of the gelatin bracket prepared in the present embodiment is as shown in Figure 1, as can be seen from Figure, which has rule
The hole configurations of clear opening, aperture 300-450um.
Embodiment 3
A kind of this implementation fabricated in situ imitation human bone material and preparation method thereof specific steps include:
(1) by 0.03g Ca(OH)2It is added in 10ml distilled water, is placed on magnetic stirring apparatus and is heated to 40 DEG C or so,
The gelatin particle of 4g is added into above-mentioned solution again, 5% glycerine 1ml is added as lubricant, constant temperature stirring.To gelatin particle
After completely soluble in water, colloidal sol is placed on to the needle for standing pour into 30ml when (30min) is significantly increased to viscosity at room temperature
Cooling is stood in pipe, obtains Sol A.
(2) Sol A is subjected to extrusion forming, the sample for being cut after every bracket for squeezing out 3cm long, and being cut with thin wire
It is directly placed into dehydrated alcohol and is dehydrated, obtain style B.
(3) style B is cut into the small rack of 3x3, small rack is put into the glutaraldehyde solution that concentration is 1% and is crosslinked for 24 hours
Afterwards, it takes out to be placed in distilled water and impregnate 5 times, each 30min is placed on filter paper after having cleaned and is air-dried, and obtains style C.
(4) style C is placed in the plastic centrifuge tube of 50ml, the SBF simulated body fluid of every centrifuge tube addition 30ml, so
It is placed in 37.5 DEG C of constant incubator and stands 14 days, change a not good liquor every three days, the sample deionized water that mineralising is terminated
Careful cleaning is placed in drying in 30 DEG C of drying box, obtains final timbering material.
The present embodiment is measured by the axial compression strength to the gelatin bracket with lateral compression strength, shows axial direction
Compression strength and lateral compression strength are respectively 14.50MPa, 9.47 MPa.Its porosity is measured, shows that porosity is
90%。
According to standard GB/T/T14233.3-2005, using selection 293T cell (people's kidney of transfection Adenovirus E1A gene
Epithelial cell), CCK-8 method it is prepared by embodiment 1 porous calcium phosphate/gelatin-compounded timbering material carry out cytotoxicity test,
Experimental result is as shown in table 1.In conjunction with standard GB/T/T14233.3-2005 relevant regulations, cytotoxicity result shows bracket
For concentration in 0.1-0.2g/ml, for relative growth rate between 145-165, cytotoxicity is 0 grade.Thus illustrate 1 institute of embodiment
The porous calcium phosphate of preparation/gelatin-compounded timbering material no cytotoxicity.
The relative growth rate of 1 gelatin bracket of table
Claims (4)
1. a kind of preparation method of imitation human bone material, which is characterized in that specifically includes the following steps:
(1) Ca (OH) that mass percent concentration is 0.1% ~ 0.3% is prepared2Solution, in the ratio of 200g/L ~ 400g/L in Ca
(OH)2Gelatin particle is added in solution, it is 1%-6% that mass percent concentration then, which is added, in the ratio of 100ml/L ~ 150ml/L
Glycerine, after gelatin particle is completely soluble in water, by colloidal sol be placed at room temperature stand 10 ~ 30 min after pour into extruding container
Middle standing cooling, obtains Sol A；
(2) Sol A is subjected to extrusion forming, is then placed in dehydrated alcohol and is dehydrated, obtain style B；
(3) style B is put into glutaraldehyde solution and is crosslinked, then taken out to be placed in distilled water and clean, be dried to obtain in air
Style C；
(4) style C is placed on human body fluid simulated solution, is subsequently placed in 37.5 DEG C of constant incubator and stands 14-30 days, often
A not good liquor is changed within 2 ~ 4 days, the sample that mineralising terminates is cleaned with deionized water, obtains imitation human bone material after drying.
2. the preparation method of imitation human bone material according to claim 1, it is characterised in that: the cleaning process in step (3) are as follows:
It takes out style and is placed in distilled water and impregnate 2-6 times, each 10-30min.
3. the preparation method of imitation human bone material according to claim 1, it is characterised in that: glutaraldehyde solution in step (3)
Mass percent concentration is 1-10wt%, and crosslinking time is 6-24 h.
4. the preparation method of imitation human bone material according to claim 1, it is characterised in that: dry temperature is in step (3)
40-60℃。
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Citations (5)
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|---|---|---|---|---|
| CN1799647A (en) * | 2005-12-13 | 2006-07-12 | 天津大学 | Nanometer hydroxyapatite/chitosan/gelatin porous scaffold material and preparation method thereof |
| CN102380128A (en) * | 2011-10-31 | 2012-03-21 | 昆明理工大学 | Hydroxyapatite, sodium hyaluronate and konjac glucomannan composite material and preparation method thereof |
| CN104107454A (en) * | 2013-04-16 | 2014-10-22 | 中国人民解放军第二军医大学 | Bone tissue porous scaffold material and preparation method thereof |
| CN104740682A (en) * | 2015-04-16 | 2015-07-01 | 济宁学院 | Hydroxylapatite/gelatin-carboxymethyl chitosan composite scaffold and preparation method thereof |
| CN105233335A (en) * | 2015-11-06 | 2016-01-13 | 四川大学 | Porous polyaryletherketone material with bioactivity, and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW574302B (en) * | 2001-08-10 | 2004-02-01 | Ind Tech Res Inst | A method for producing cross-linked hyaluronic acid-protein bio-composites |
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|---|---|---|---|---|
| CN1799647A (en) * | 2005-12-13 | 2006-07-12 | 天津大学 | Nanometer hydroxyapatite/chitosan/gelatin porous scaffold material and preparation method thereof |
| CN102380128A (en) * | 2011-10-31 | 2012-03-21 | 昆明理工大学 | Hydroxyapatite, sodium hyaluronate and konjac glucomannan composite material and preparation method thereof |
| CN104107454A (en) * | 2013-04-16 | 2014-10-22 | 中国人民解放军第二军医大学 | Bone tissue porous scaffold material and preparation method thereof |
| CN104740682A (en) * | 2015-04-16 | 2015-07-01 | 济宁学院 | Hydroxylapatite/gelatin-carboxymethyl chitosan composite scaffold and preparation method thereof |
| CN105233335A (en) * | 2015-11-06 | 2016-01-13 | 四川大学 | Porous polyaryletherketone material with bioactivity, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
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