CN106512104A - Mineralized collagen-based alveolar bone restoration material and preparation method thereof - Google Patents
Mineralized collagen-based alveolar bone restoration material and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a mineralized collagen-based alveolar bone restoration material and a preparation method thereof. The preparation method comprises the following steps: preparing a collagen solution; adding a calcium ion-containing aqueous solution; adding a phosphate ion-containing aqueous solution; adjusting the pH value to 8; washing a precipitate and performing pumping filtration on the precipitate; performing freeze drying; grinding the material to prepare the mineralized collagen dry powder; preparing a I-type collagen fiber aqueous solution; adding the mineralized collagen dry powder; performing primary freeze drying; crosslinking the material; centrifuging the material, filtering and washing the material; performing secondary freeze drying; and disinfecting the material. The method has the advantages of low cost, high material utilization rate, and easy operation. The mineralized collagen-based alveolar bone restoration material has the advantages of high homogeneity, high mechanical strength, long degradation time, little cross-linking agent residue, high amount of porosity, and excellent guiding performance and inductivity.
Description
Technical field
Patent of the present invention is related to therapeutic treatment field, and especially biomaterial for medical purpose prepares the system for being particularly dental implant material
Standby field, and in particular to a kind of to adopt a kind of New Reclaiming Material based on mineralized collagen and preparation method thereof and in alveolus
Application in Bone Defect Repari.
Background technology
Gear division clinic can run into the bone amount that the case of tooth turnover zone Bone mineral change and Cranial defect, teeth socked infection etc. are caused
It is not enough；Accessory tooth, embedded tooth and malposed tooth are pulled out.This situation can cause the bone amount of tooth turnover zone
Deficiency, can affect the therapeutic scheme of doctor.Bone grafting is guided or is induced using the transplanting of autologous bone, allograph bone or artificial bone
New osteanagenesis, maintain biological characteristicses and skeletonization space, so as to reach the purpose of alveolar bone defect repair.
Most currently used bioactive materials have hydroxyapatite (HA) and xenogenesis inorganic bone two types.Hydroxyl phosphorus
Lime stone (HA) is the important inorganic ingredient of human body bone and tooth, and the hydroxyapatite of the different crystalline phases of sintering or non-sintered is tool
There is the timbering material of good bone guided effect, but degradation rate is fast or slow, it is impossible to match with skeletonization speed.Bio-oss
Bio-oss is that except Deproteinization and other organic principles, antigenicity is low through special handling, high-purity keep porous nature bone
Inorganic structure, the bone- xenograft almost identical with the structure of human body bone.But bone- xenograft still can not overcome the immunity after transplanting completely
Rejection, has potential source of disease to propagate dangerous, while the obstacle in terms of there are medical ethics.
Using bionical thinking, the hydroxyapatite similar with nature bone can be prepared and be combined into uniqueness with collagen
The complex of the hierarchy of rule.The Cui Fuzhai professors of material system of Tsing-Hua University are mentioned in Chinese patent ZL01129699.2
It is using a kind of nm-phase calcium-phosphorus salt/collagen/high-molecular bone for bone renovating material of biomineralization technological invention compound porous
Material and preparation method.Aojing Medicine, Sci. ＆ Tech. Co., Ltd., Beijing is also refer in Chinese patent application 201410275022.9
A kind of mineralized collagen artificial periosteum and preparation method thereof.
The content of the invention
In order to obtain that biocompatibility is more preferable, degradation rate is more controllable, more easy-formation with height porosity and
The frame material of suitable aperture size, the present invention are prepared by way of with nanocrystalline calcium phosphate collagen composite collagen scaffold
The better biological activity repair materials of performance especially biotype alveolar bone repairing material, so as to both maintain biomineralization
The bone guided of collagen, be modified collagen as tissue engineering scaffold again.For the particular/special requirement of the clinical practice of gear division alveolar bone, i.e. material
The shape of different alveolus defects can be moulded with certain flexibility, the present invention makes mineralized collagen as binding agent using bovine collagen
Dry powder dispersion carries out appropriate crosslinking in collagen as tissue engineering scaffold, after molding, increased the mechanical strength and biology of collagen scaffold
Degradation time.In addition, the repair materials of the present invention are not only consistent with nature bone on composition, and because containing mineralized collagen,
So it is also similar to nature bone in structure, it is good artificial bone.In addition, for manufacturing mineralized collagen in prior art
During dry powder, localized ion concentration is excessive or too small, wash time is long, heavy caused by phosphate anion and/or calcium ion residual
Starch wash time-consuming, the low problem of calcium microcosmic salt utilization rate and mineralized collagen base alveolar bone repairing material mechanical property it is low, degraded
When time is short and prepares, mineralized collagen dry powder dispersion is poor, cross-linking agent residual quantity is excessive and is difficult to remove, porosity is low asks
Topic, is improved to prior art and has been optimized, so as to partly even fully solve the problems referred to above.
The present invention provides a kind of method for preparing mineralized collagen dry powder in first aspect, and methods described includes following step
Suddenly：
(1) type i collagen fiber is configured to into the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) in the collagen solution, add calcium ions water-soluble with the amount of 0.08 to 0.12 mole of calcium ion/gram collagen
Liquid, so as to calcium/collagen composite solution is obtained；
(3) phosphorus-containing acid ion aqueous solution is added in the calcium/collagen composite solution with the amount of Ca/P=1.2～1.8,
So as to calcium/phosphorus/collagen composite solution is obtained；
(4) it is 8 calcium/phosphorus/collagen composite solution to be adjusted to pH value, and stirring, precipitation separation, water washing and precipitating are until supernatant
PH value 7.0～7.5, so as to obtain precipitate；
(5) using purification water washing precipitate sucking filtration, obtain filter cake；
(6) filter cake is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then is risen in -10～0 DEG C under vacuum
China, so as to carry out lyophilization to filter cake；
(7) the mineralized collagen dry powder that particle diameter is not more than 80 mesh will be ground to form through cryodesiccated filter cake.
The present invention provides mineralized collagen dry powder obtained in first aspect present invention methods described in second aspect.
The present invention provides the mineralized collagen dry powder described in second aspect present invention in the third aspect and is preparing mineralized collagen
Application in base alveolar bone repairing material.
The present invention provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, methods described in fourth aspect
Comprise the steps：
A () prepares type i collagen fiber aqueous solution of the concentration for 0.0286-0.0667g/ml；
B () adds mineralized collagen dry powder in the collagen fiber aqueous solution, mineralized collagen/collagen mixed solution is obtained,
The mineralized collagen dry powder is 1 with the mass ratio of the type i collagen fiber:4～4:1；
C mineralized collagen/collagen mixed solution is carried out pre-freeze by () at ambient pressure in -30～-20 DEG C, then under vacuum
Distilled in -10～0 DEG C, so as to a lyophilized collagen complex is obtained；
D lyophilized collagen complex is soaked 24 to 48 hours in cross-linking agent solution by (), so as to crosslinked with collagen is obtained
Complex, the cross-linking agent solution are ethanol solution of 0.005 mass % to the glutaraldehyde of 0.25 mass %；
E the crosslinked with collagen complex is centrifuged, filters and wash by (), so as to washing collagen-based composite is obtained；
F washing collagen-based composite is carried out pre-freeze by () at ambient pressure in -30～-20 DEG C, then under vacuum in -10～
0 DEG C is distilled, so as to make secondary lyophilized collagen complex；
G the secondary lyophilized collagen complex is sterilized by (), so as to the mineralized collagen base alveolar repair is obtained
Material.
The present invention additionally provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, the side in terms of the 5th
Method comprises the steps：
(1) type i collagen fiber is configured to into the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) in the collagen solution, add calcium ions water-soluble with the amount of 0.08 to 0.12 mole of calcium ion/gram collagen
Liquid, so as to calcium/collagen composite solution is obtained；
(3) phosphorus-containing acid ion aqueous solution is added in the calcium/collagen composite solution with the amount of Ca/P=1.2～1.8,
So as to calcium/phosphorus/collagen composite solution is obtained；
(4) it is 8 calcium/phosphorus/collagen composite solution to be adjusted to pH value, and stirring, precipitation separation, water washing and precipitating are until supernatant
PH value 7.0～7.5, so as to obtain precipitate；
(5) using purification water washing precipitate sucking filtration, obtain filter cake；
(6) filter cake is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then is risen in -10～0 DEG C under vacuum
China, so as to carry out lyophilization to filter cake；
(7) the mineralized collagen dry powder that particle diameter is not more than 80 mesh will be ground to form through cryodesiccated filter cake；
(8) prepare type i collagen fiber aqueous solution of the concentration for 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in the collagen fiber aqueous solution, mineralized collagen/collagen mixed solution is obtained,
The mineralized collagen dry powder is 1 with the mass ratio of the type i collagen fiber:4～4:1；
(10) mineralized collagen/collagen mixed solution is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then in vacuum condition
Under distilled in -10～0 DEG C, so as to be obtained a lyophilized collagen complex；
(11) a lyophilized collagen complex is soaked 24 to 48 hours in cross-linking agent solution, so as to crosslinked with collagen is obtained
Complex, the cross-linking agent solution are ethanol solution of 0.005 mass % to the glutaraldehyde of 0.25 mass %；
(12) the crosslinked with collagen complex is centrifuged, filters and wash, so as to washing collagen-based composite is obtained；
(13) washing collagen-based composite is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then under vacuum in -10
～0 DEG C is distilled, so as to make secondary lyophilized collagen complex；
(14) the secondary lyophilized collagen complex is sterilized, so as to the mineralized collagen base alveolar repair is obtained
Material.
The present invention provides mineralized collagen abutment obtained in the 4th or the 5th aspect methods described of the invention in terms of the 6th
Groove bone renovating material.
The mineralized collagen base alveolar bone repairing material that the present invention is provided described in sixth aspect present invention in terms of the 7th exists
Repair the application in alveolar bone.
Advantages of the present invention and the unexpected technique effect that brought at least include it is following some：
(1), for 0.0001-0.001g/ml, under the collagen concentration, overall solution viscosity is more for the collagen solution concentration for adopting
Plus it is moderate, viscosity is not resulted in for localized ion concentration is excessive or too small impact.
(2) calcium phosphorus molar feed ratio is adopted for Ca：P=1.2-1.8, improves the utilization ratio of calcium salt and microcosmic salt, reduces
Dissociate in material the residual of calcium salt and microcosmic salt.
(3) adopt pH value for 8 precipitation terminal so that mineralized collagen precipitates quicker and abundant, significantly reduces
Stirring and time of repose.
(4) using the mineralized collagen dry powder below 80 mesh so which is when mixing with collagen adhesives, it is easier to disperse.
(5) irradiation dose for employing below 40kgy carries out sterilizing processing, significantly reduces the mechanical property and drop of material
The solution time.
(6) I-type collagen is employed, the fiber of ungelled state is used as collage raw material, it is to avoid because of solid content in gel
The inconsistent problem for causing inventory inconsistent.
(7) carry out supernatant using sucking filtration mode to separate with precipitate, pH value or can be easier to level off to neutrality.
(8) by way of being segmented lyophilizing so that material can solidify at a lower temperature, distil at slightly higher temperature,
Shorten freeze-drying time.
(9) using collagen fiber as binding agent and timbering material so that material composition maintains consistent with nature bone
Chemical analysis；Dry powder is 1 with the mass ratio of collagen:4～4:1, according to the porous support as used in tissue engineering, different tissues
Cell has a different requirements to aperture, and bone and cartilage tissue engineered, 100-250 μm of aperture is needed, for degradable multiporous
200-400 μm of timbering material is preferred, therefore purified water pore-creating aperture is close to theoretical value under the concentration, and porosity is 70-95%.
(10) by way of washing, secondary freeze drying is centrifuged, the time of washing is shortened, efficiency is improve.
Specific embodiment
As described above, the present invention provides a kind of method for preparing mineralized collagen dry powder, methods described bag in first aspect
Include following steps：
(1) using acid solution by type i collagen fiber be configured to concentration for 0.0001 to 0.001g/ml (such as 0.0001,
0.0005 or 0.001g/ml) collagen solution；
(2) it is molten to the collagen with the amount of 0.08 to 0.12 (such as 0.08,0.10 or 0.12) mole calcium ion/gram collagen
Calcium ions aqueous solution is added in liquid, so as to calcium/collagen composite solution is obtained；
(3) with the amount of Ca/P=1.2～1.8 (such as 1.2,1.4,1.6 or 1.8) in the calcium/collagen composite solution
Phosphorus-containing acid ion aqueous solution is added, so as to calcium/phosphorus/collagen composite solution is obtained；
(4) it is 8 calcium/phosphorus/collagen composite solution to be adjusted to pH value, and stirring, precipitation separation, water washing and precipitating are until supernatant
PH value in 7.0～7.5 (such as 7.0,7.1,7.2,7.3,7.4 or 7.5), so as to obtain precipitate；
(5) using purification water washing precipitate sucking filtration, obtain filter cake；
(6) filter cake is carried out into pre-freeze in -30～-20 DEG C (such as -30, -25 or -20 DEG C) at ambient pressure, then in vacuum bar
Distilled in -10～0 DEG C (such as -10, -5 or 0 DEG C) under part, so as to carry out lyophilization to filter cake；
(7) particle diameter will be ground to form through cryodesiccated filter cake and will be not more than 80 mesh (such as no more than 80,90,100 or 120
Mesh) mineralized collagen dry powder.
Some preferred embodiment in, the acid solution in step (1) be selected from aqueous hydrochloric acid solution, aqueous solution of nitric acid
With the group of acetic acid aqueous solution composition, preferably acetic acid aqueous solution.
Some preferred embodiment in, the concentration of the acid solution be 0.45 to 0.55mM (such as 0.45,0.50 or
0.55mM)。
Some preferred embodiment in, the pH in step (4) is adjusted using sodium hydrate aqueous solution, preferably
It is that the concentration of the sodium hydrate aqueous solution is 0.9M to 1.1M (such as 0.9,1.0 or 1.1M).
Some preferred embodiment in, the calcium ions aqueous solution and/or phosphorus-containing acid ion aqueous solution plus
Entering is carried out by the way of stirring when being slowly added dropwise.
Some preferred embodiment in, in step (4) by pH regulator to 8 after, be stirred for 8 to 24 hours, shape
Into white suspension, stand 12 to 24 hours (such as 12,18 or 24 hours), remove supernatant, then plus purified water to original volume
Stirring 5 to 10 times (such as 5,6,7,8,9 or 10 times), removes supernatant, continuous wash 3 to 7 times, Zhi Daoshang after standing 1 to 3 hour
The pH value of clear liquid is between 7.0～7.5 (7.0,7.1,7.2,7.3,7.4 or 7.5).
Some preferred embodiment in, the concentration of the calcium ions aqueous solution be 0.45M to 0.55M (for example
0.45th, 0.50 or 0.55M).
Some preferred embodiment in, the concentration of the phosphorus-containing acid ion aqueous solution be 0.4M to 0.6M (for example
0.40th, 0.50 or 0.60M).
Some preferred embodiment in, the type i collagen fiber for ungelled state I type bovine collagen fibers.
Some preferred embodiment in, for preparing the calcium salt and phosphorus-containing acid ion of the calcium ions aqueous solution
The calcium salt of aqueous solution and the microcosmic salt for preparing phosphorus-containing acid ion aqueous solution are the other calcium salt of medicinal or pharmaceutic adjuvant grade and phosphorus
Salt.
The present invention provides obtained mineralized collagen dry powder obtained in first aspect present invention methods described in second aspect.
The present invention is provided obtained in first aspect present invention methods described or second aspect present invention in the third aspect
Application of the described mineralized collagen dry powder in mineralized collagen base alveolar bone repairing material is prepared.
The present invention provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, methods described in fourth aspect
Comprise the steps：
A () prepares I type glue of the concentration for 0.0286-0.0667g/ml (such as 0.0286,0.0476 or 0.0667g/ml)
Fibril aqueous solution；
B () adds mineralized collagen dry powder in the collagen fiber aqueous solution, mineralized collagen/collagen mixed solution is obtained,
The mineralized collagen dry powder is 1 with the mass ratio of the type i collagen fiber:4～4:1 (such as 1:4、1:3、1:2、1:1、2:1、
3:1 or 4:1)；
C mineralized collagen/collagen mixed solution is entered by () at ambient pressure in -30～-20 DEG C (such as -30, -25 or -20 DEG C)
Row pre-freeze, then distilled in -10～0 DEG C (such as -10, -5 or 0 DEG C) under vacuum, so as to a lyophilized collagen is obtained
Complex；
(d) by lyophilized collagen complex soak in cross-linking agent solution 24 to 48 hours (such as 24,30,36,42 or
48), so as to be obtained crosslinked with collagen complex, the cross-linking agent solution be 0.005 mass % to 0.25 mass % (such as 0.005,
0.010th, 0.015,0.020 or 0.025 mass %) glutaraldehyde ethanol solution；
E the crosslinked with collagen complex is centrifuged, filters and wash by (), so as to washing collagen-based composite is obtained；
F washing collagen-based composite is carried out pre-freeze in -30～-20 DEG C (such as -30, -25 or -20 DEG C) by () at ambient pressure,
Distilled in -10～0 DEG C (such as -10, -5 or 0 DEG C) under vacuum again, so as to make secondary lyophilized collagen complex；
G the secondary lyophilized collagen complex is sterilized by (), so as to the mineralized collagen base alveolar repair is obtained
Material.
Some preferred embodiment in, also whether be in conjunction including cross-linking agent residual quantity between step (f) and (g)
Detecting step in the range of lattice residue.
Some preferred embodiment in, the qualified residue scope is less than or equal to 25ppm.
Some preferred embodiment in, the detecting step is carried out in the following way：Weigh the secondary jelly of 0.1g
Dry collagen-based composite, detection cross-linking agent residual quantity whether less than or equal to 25ppm (be for example not more than 25,20,15,10 or
5ppm), if greater than 25ppm, then washing in repeat step (e) and the secondary lyophilizing of step (f), until cross-linking agent residual quantity
Less than or equal to 25ppm.
Some preferred embodiment in, it is described sterilizing by using 15kgy to 40kgy (such as 15,20,25,30,
35 or 40kgy) -60 irradiation dose of cobalt is carried out.
Some preferred embodiment in, in step (d), the volume of the cross-linking agent solution is relative to a lyophilizing
The quality of collagen-based composite is 50 to 200ml/g (such as 50,100,150 or 200ml/g).
Some preferred embodiment in, step (b) is uniformly carrying out by dispersion mixing at room temperature.
Some preferred embodiment in, step (e) is carried out in the following way：By crosslinked with collagen complex from crosslinking
Centrifuge loading filter bag is removed and placed in agent solution, at 2000-3000r/min (such as 2000,2500 or 3000r/min)
Centrifugation 2 to 4 times (such as 3 times), 3 to 10 seconds every time (such as 3,5,7 or 10 seconds)；Crosslinked with collagen complex is placed in into chromatographic column again
In, with 48 to 72 (48,54,60, the 66 or 72) hour of purification water wash that flows removing the cross-linking agent of residual.
Some preferred embodiment in, the mineralized collagen dry powder be first aspect present invention described in method system
.
The present invention additionally provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, the side in terms of the 5th
Method comprises the steps：
(1) type i collagen fiber is configured to into the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) in the collagen solution, add calcium ions water-soluble with the amount of 0.08 to 0.12 mole of calcium ion/gram collagen
Liquid, so as to calcium/collagen composite solution is obtained；
(3) phosphorus-containing acid ion aqueous solution is added in the calcium/collagen composite solution with the amount of Ca/P=1.2～1.8,
So as to calcium/phosphorus/collagen composite solution is obtained；
(4) it is 8 calcium/phosphorus/collagen composite solution to be adjusted to pH value, and stirring, precipitation separation, water washing and precipitating are until supernatant
PH value 7.0～7.5, so as to obtain precipitate；
(5) using purification water washing precipitate sucking filtration, obtain filter cake；
(6) filter cake is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then is risen in -10～0 DEG C under vacuum
China, so as to carry out lyophilization to filter cake；
(7) the mineralized collagen dry powder that particle diameter is not more than 80 mesh will be ground to form through cryodesiccated filter cake；
(8) prepare type i collagen fiber aqueous solution of the concentration for 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in the collagen fiber aqueous solution, mineralized collagen/collagen mixed solution is obtained,
The mineralized collagen dry powder is 1 with the mass ratio of the type i collagen fiber:4～4:1；
(10) mineralized collagen/collagen mixed solution is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then in vacuum condition
Under distilled in -10～0 DEG C, so as to be obtained a lyophilized collagen complex；
(11) a lyophilized collagen complex is soaked 24 to 48 hours in cross-linking agent solution, so as to crosslinked with collagen is obtained
Complex, the cross-linking agent solution are ethanol solution of 0.005 mass % to the glutaraldehyde of 0.25 mass %；
(12) the crosslinked with collagen complex is centrifuged, filters and wash, so as to washing collagen-based composite is obtained；
(13) washing collagen-based composite is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then under vacuum in -10
～0 DEG C is distilled, so as to make secondary lyophilized collagen complex；
(14) the secondary lyophilized collagen complex is sterilized, so as to the mineralized collagen base alveolar repair is obtained
Material.
Some preferred embodiment in, the acid solution in step (1) be selected from aqueous hydrochloric acid solution, aqueous solution of nitric acid
With the group of acetic acid aqueous solution composition, preferably acetic acid aqueous solution.
Some preferred embodiment in, the concentration of the acid solution is 0.45 to 0.55mM.
Some preferred embodiment in, the pH in step (4) is adjusted using sodium hydrate aqueous solution, preferably
It is that the concentration of the sodium hydrate aqueous solution is 0.9M to 1.1M.
Some preferred embodiment in, the calcium ions aqueous solution and/or phosphorus-containing acid ion aqueous solution plus
Entering is carried out by the way of stirring when being slowly added dropwise.
Some preferred embodiment in, in step (4) by pH regulator to 8 after, be stirred for 8 to 24 hours, shape
Into white suspension, stand 12 to 24 hours, remove supernatant, then plus purified water stir 5 to 10 times to original volume, standing 1 to 3
Supernatant, continuous wash 3 to 7 times, until the pH value of supernatant is between 7.0～7.5 are removed after hour.
Some preferred embodiment in, the concentration of the calcium ions aqueous solution is 0.45M to 0.55M.
Some preferred embodiment in, the concentration of the phosphorus-containing acid ion aqueous solution is 0.4M to 0.6M.
Some preferred embodiment in, the type i collagen fiber for ungelled state I type bovine collagen fibers.
Some preferred embodiment in, for preparing the calcium salt and phosphorus-containing acid ion of the calcium ions aqueous solution
The calcium salt of aqueous solution and the microcosmic salt for preparing phosphorus-containing acid ion aqueous solution are the other calcium salt of medicinal or pharmaceutic adjuvant grade and phosphorus
Salt.
Some preferred embodiment in, also whether be in including cross-linking agent residual quantity between step (13) and (14)
Detecting step in the range of qualified residue.
Some preferred embodiment in, the qualified residue scope is less than or equal to 25ppm.
Some preferred embodiment in, the detecting step is carried out in the following way：Weigh the secondary jelly of 0.1g
Whether dry collagen-based composite, detection cross-linking agent residual quantity are less than or equal to 25ppm, if greater than 25ppm, then repeat step
(12) the secondary lyophilizing of washing and step (13) in, until cross-linking agent residual quantity is less than or equal to 25ppm.
Some preferred embodiment in, the sterilizing is entered by -60 irradiation dose of cobalt using 15kgy to 40kgy
OK.
Some preferred embodiment in, in step (11), the volume of the cross-linking agent solution is relative to once freezing
The quality of dry collagen-based composite is 50 to 200ml/g.
Some preferred embodiment in, step (9) is uniformly carrying out by dispersion mixing at room temperature.
Some preferred embodiment in, step (12) is carried out in the following way：By crosslinked with collagen complex from friendship
Centrifuge loading filter bag is removed and placed in connection agent solution, is centrifuged 2 to 4 times, every time 3 to 10 seconds in 2000-3000r/min；
Again crosslinked with collagen complex is placed in chromatographic column, with the purification water wash crosslinking to remove residual in 48 to 72 hours flowed
Agent.
In some more specifically embodiment, the side for preparing mineralized collagen base alveolar bone repairing material of the present invention
Method comprises the steps：
(1) acid solution of type i collagen fiber is configured to, in group of the acid therein selected from hydrochloric acid, nitric acid or acetic acid composition
Any one, the concentration of collagen solution is 0.0001-0.001g/ml；
(2) calcium ions aqueous solution is slowly added dropwise while stirring in the acid solution, addition is every gram of collagen Deca
Calcium ion 0.08-0.12mol, so as to form calcium/collagen composite solution；
(3) in calcium/collagen composite solution, Deca contains the aqueous solution of phosphate anion, the phosphate radical of addition while stirring
The amount of ion is Ca with the mol ratio of the amount of the calcium ion for adding：P=1.2-1.8；
(4) in Deca, in the solution of phosphate anion, Deca NaOH solution is 8 to pH value while stirring, is stirred for 8-24
Hour, form white suspension, stand 12-24 hours, remove supernatant, then plus purified water stir to original volume, 1-3 is little for standing
When after remove supernatant, continuous wash 5 to 10 times, until the pH value of supernatant is between 7.0～7.5, is precipitated thing；
(5) pumping and filtering device is opened, pours precipitate into, carry out sucking filtration, with purified water repeated washing filter cake 2-4 time, filtered
Cake；
(6) filter cake is put in freezer dryer carries out pre-freeze at ambient pressure at -30～-20 DEG C, and under vacuum -10
～0 DEG C is distilled so as to realize lyophilization；
(7) will grind through cryodesiccated filter cake, mineralized collagen dry powder will be obtained, the screening of 80 mesh stainless steel meshs will be standby；
(8) type i collagen fiber is placed in reactor, adds purified water, preparing mass-volume concentration at room temperature is
The type i collagen fiber aqueous solution of 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in collagen solution, dispersion mixing is uniform at room temperature, wherein, dry powder and collagen
Mass ratio be 1:4～4:1, mineralized collagen/collagen mixed solution is obtained；
(10) mineralized collagen/collagen mixed solution is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then in vacuum condition
Under distilled in -10～0 DEG C, so as to be obtained a lyophilized collagen complex；
(11) ethanol solution of glutaraldehyde of 0.005-0.25% mass fractions is prepared as cross-linking agent solution, once will freeze
Dry collagen-based composite is complete for the amount of 50-200ml/g with the quality of a lyophilized collagen complex according to the volume of cross-linking agent solution
Complete immersion 24 to 48 hours, so as to crosslinked with collagen complex is obtained；
(12) crosslinked with collagen complex is taken out from cross-linking agent solution, centrifuge loading filter bag, 2000-3000r/
Min is centrifuged 2-4 time, each 3-10 seconds；Again crosslinked with collagen complex is placed in chromatographic column, with flow purification water wash 48 to
72 hours cross-linking agent to remove residual, so as to washing collagen-based composite is obtained；
(13) washing collagen-based composite is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then under vacuum in -10
～0 DEG C is distilled, so as to make secondary lyophilized collagen complex；
(14), after lyophilizing terminates, take out about 0.1g and do cross-linking agent residue analysis, if cross-linking agent residual quantity is more than 25ppm,
Then repeat water wash and secondary lyophilizing operation, until cross-linking agent residual quantity is less than or equal to 25ppm；
(15) the secondary lyophilized collagen complex by cross-linking agent residual quantity less than or equal to 25ppm adopts 15kgy extremely
- 60 irradiation dose of cobalt of 40kgy carries out sterilizing processing, so as to obtain mineralized collagen base alveolar bone repairing material.
The present invention provides fourth aspect present invention or mineralized collagen obtained in the 5th aspect methods described in terms of the 6th
Base alveolar bone repairing material.
Seventh aspect present invention provides the mineralized collagen base alveolar bone repairing material repairing described in sixth aspect present invention
Application in multiple alveolar bone.
In the present invention, the present inventor at least has carried out improving and achieving corresponding technique effect at following aspect：
(1) using every gram of collagen Deca calcium ion 0.08-0.12mol；Sometimes can the very few calcium of Deca in prior art
Ion, sometimes even as low as 0.01mol；The sometimes again excessive calcium ion of Deca, sometimes up to 0.16mol/g；Excessive calcium
Ion remains unnecessary free calcium ions in causing the waste of calcium salt or material so that follow-up cleaning becomes complexity even
Difficulty, if calcium ion is very few, may cause mineralized collagen not enough, and intensity step-down, material are big with osseous tissue difference, affect material
The bone guided and inductivity of material.
(2) collagen solution concentration is adopted for 0.0001-0.001g/ml, under the collagen concentration, overall solution viscosity is more
It is moderate, it is to avoid because viscosity causes localized ion concentration excessive or too small brought impact.
(3) calcium phosphorus molar feed ratio is adopted for Ca：P=1.2-1.8 so that the inorganic salt composition of material more conforms to hydroxyl
The theoretical calcium-phosphorus ratio 1.667 of apatite, improves the utilization rate of calcium salt and microcosmic salt, reduces calcium ion and/or phosphate anion exists
Residual in material, reduces the operation difficulties such as subsequent wash, improves time efficiency, it is to avoid free calcium ions or phosphate anion are residual
The problem for staying the strength of materials for causing not enough.
(4) adopt pH value for 8 precipitation terminal so that mineralized collagen precipitate more it is quick more fully, substantially reduce
Stirring and time of repose；Up to 9 or as little as 7 pH is adopted in prior art even so that only time of repose is even up to 5 days
Time.Further, since the normal Deca NaOH solutions of past Jing are to mixed system pH=6～8, and observe when pH=5～6, mix
Fit system starts precipitation occur, and as pH=7, white suspension occurs in mixed system, therefore the normal Deca NaOH to pH of Jing are left 7
It is right.The present inventor has found that through test Deca NaOH solution is washed to mixed system pH=8, after precipitation again to 7.0-7.5, instead
And sedimentation effect can be improved and required time is reduced.
(5) mixed with high molecular polymer using the mineralized collagen dry powder below 80 mesh, the dry powder under the mesh number is easier
Disperseed in polymer solution system.
(6) irradiation dose for employing below 40kgy carries out sterilizing processing, because material is absorbable polymer and collagen
Deng for the material of radiation sensitive, in order to significantly reduce the mechanical property and degradation time of material, using low irradiation dose
Carry out sterilizing to be very important.
In addition, the present inventor also during quality system foundation has carried out following improvement and has obtained to part raw material
Corresponding technique effect：
(1) I-type collagen is employed, the fiber of ungelled state is used as collage raw material, it is to avoid because containing in gel admittedly
That what is measured is inconsistent, causes inventory inconsistent；NTx is a kind of structural protein found in animal body simultaneously, is autologous
The predominantly organic ingredient of bone.I-type collagen is the major structural protein of spinal animals, is osteoblast in osteogenetic process
The extracellular matrix of secretion, is accelerator, the template of mineralising of the support and bone matrix mineralising of calcium deposition；Cell can be promoted to move
Move, adsorb, break up, and cell growth can be adjusted, approved by U.S. FDA as biomaterial, and there are a series of collagens implantation
Product, including bone implantation product.
(2) the other calcium salt of medicinal or pharmaceutic adjuvant grade, microcosmic salt are employed, it is to avoid because the rank of raw material causes impurity, weight
Metal, ash it is exceeded, also affect material biocompatibility.
In addition, the present inventor also carries out supernatant using sucking filtration mode separating with precipitate, and clean to neutrality.This
Person of good sense's discovery, compared with centrifugation of the prior art, can so cause porous material to obtain contacting with purified water again
Chance, after repeatedly washing, the pH value of lixiviating solution can level off to neutrality for its inside and outside.
Additionally, the present inventor is by way of being segmented lyophilizing, solvent pore-creating are removed, i.e., at ambient pressure (not evacuation) exists
Pre-freeze is first carried out at -30～-20 DEG C in freeze dryer, is then just distilled at -10～0 DEG C in vacuum condition so that material energy
It is enough to solidify at a lower temperature, distil at slightly higher temperature, significantly reduce freeze-drying time.
Further, the present inventor adopts collagen fiber as binding agent and timbering material so that material composition is maintained and day
The consistent chemical analysis of right bone；Dry powder is 1 with the mass ratio of collagen:4～4:1, according to the porous support as used in tissue engineering,
Different histiocytes has a different requirements to aperture, and bone and cartilage tissue engineered, 100-250 μm of aperture is needed, for
200-400 μm of degradable stephanoporate stent material is preferred, therefore purified water pore-creating aperture is close to theoretical value under the concentration, and porosity is
70-95%.
Additionally, cross-linking agent residual is always technical barrier in obtained repair materials.The present inventor is by being centrifuged washing
To remove the cross-linking agent of residual so that cross-linking agent residual quantity is reduced to 25ppm and is possibly realized, and can conveniently realize.
In the technical scheme of the cross-linking agent of the removal residual of the present invention, first pass through centrifugation to remove most of in material loose structure friendship
Connection agent is molten, because chromatographic column drip washing is the process for being crosslinked dilution agent, centrifugally operated can shorten the time of washing, and efficiency even can be with
Improve 1 times！Further, since have passed through water-washing process, therefore secondary freeze drying is carried out.
It is important to note that the numerical range of this specification represent the higher limit of the numerical range, lower limit and
Any numerical value or the subrange being within the numerical range.Therefore, if not otherwise specified, it is related in this manual
The concrete numerical value that be included in the numerical range in is no longer itemized during numerical range just.
Embodiment
Hereafter the present invention will be illustrated by the form of embodiment, but protection scope of the present invention should not be by
It is considered limited to these embodiments.
Embodiment 1
(1) using 0.5M acetic acid solutions 0.0005g/ml type i collagen fiber acid solution；
(2) it is slowly added dropwise the calcium ions aqueous solution of 0.5M in the acid solution while stirring, addition is every gram of glue
Former Deca calcium ion 0.10mol, so as to form calcium/collagen composite solution；
(3) the phosphorus-containing acid ion aqueous solution of Deca 0.5M while stirring in calcium/collagen composite solution, the phosphoric acid of addition
The amount of radical ion is Ca/P=1.5 with the mol ratio of the amount of the calcium ion for adding；
(4) in Deca, in the solution of phosphate anion, the NaOH aqueous solutions of Deca 1M to pH value is 8 while stirring, then is stirred
Mix 16 hours, form white suspension, stand 18 hours, remove supernatant, then plus purified water stir to original volume, standing 2 is little
When after remove supernatant, continuous wash 5 times, after testing, the pH value of supernatant now is 7.2 (being between 7.0～7.5), is obtained
To precipitate；
(5) pumping and filtering device is opened, pours precipitate into, carry out sucking filtration, with purified water repeated washing filter cake 3 times, obtain filter cake；
(6) filter cake is put in freezer dryer carries out pre-freeze at ambient pressure at -25 DEG C, and -5 DEG C are carried out under vacuum
Distil so as to realize lyophilization；
(7) at the filter cake periphery quartering and filter cake middle position carries out 5 points of samplings, observation analysis its homogeneities；So
After will grind through cryodesiccated filter cake, mineralized collagen dry powder is obtained, the screening of 80 mesh stainless steel meshs is standby.
Embodiment 2 to 4
In addition to the content of table 1 below, carried out in the way of substantially the same manner as Example 1.
Embodiment 5
In addition to the content of table 1 below, carried out in the way of substantially the same manner as Example 1, wherein 36 hours sedimentation times
After measure pH.
Embodiment 6
In addition to the content of table 1 below, carried out in the way of substantially the same manner as Example 1, wherein when detection pH reaches 7.2
Required staticly settles the time, if pH is not reaching to 7.2, repeatedly addition water is staticly settled again, and record stands required
Time.
Embodiment 7
(7') mineralized collagen dry powder is prepared in the same manner as example 1；
(8) type i collagen fiber is placed in reactor, adds purified water, preparing mass-volume concentration at room temperature is
The type i collagen fiber aqueous solution of 0.0286g/ml；
(9) mineralized collagen dry powder is added in collagen solution, dispersion mixing is uniform at room temperature, wherein, dry powder and collagen
Mass ratio be 1:4, mineralized collagen/collagen mixed solution is obtained；
(10) mineralized collagen/collagen mixed solution is carried out into pre-freeze in -30 DEG C at ambient pressure, then under vacuum in -
10 DEG C are distilled, so as to a lyophilized collagen complex is obtained；
(11) ethanol solution of glutaraldehyde of 0.005% mass fraction is prepared as cross-linking agent solution, by a lyophilizing glue
Amount of the former complex according to the volume of cross-linking agent solution with the quality of a lyophilized collagen complex for 50ml/g is completely soaked 24
Hour, so as to crosslinked with collagen complex is obtained；
(12) crosslinked with collagen complex is taken out from cross-linking agent solution, centrifuge loading filter bag, 2000r/min centrifugations
2 times, every time 3 seconds；Again crosslinked with collagen complex is placed in chromatographic column, with the purification water wash 48 hours that flows to remove residual
Cross-linking agent, so as to be obtained washing collagen-based composite；
(13) washing collagen-based composite is carried out into pre-freeze in -30 DEG C at ambient pressure, then is carried out in -10 DEG C under vacuum
Distillation, so as to make secondary lyophilized collagen complex；
(14) after lyophilizing terminates, take out about 0.1g and do cross-linking agent residue analysis, as a result find that cross-linking agent residual quantity is less than
25ppm；
(15) secondary lyophilized collagen complex is carried out into sterilizing processing using -60 irradiation dose of cobalt of 40kgy, so as to obtain
Mineralized collagen base alveolar bone repairing material.
Then aperture and the porosity of obtained mineralized collagen base alveolar bone repairing material are measured.
Embodiment 8-9
In addition to content shown in table 2 below, carried out in the way of same as Example 7.
Embodiment 10
In addition to content shown in table 2 below, carried out in the way of substantially the same manner as Example 7, but in step (12),
It is not centrifuged, but directly crosslinked with collagen complex is placed in chromatographic column, with the purification water wash 48 hours that flows to remove
The cross-linking agent for remaining is removed, so as to washing collagen-based composite is obtained.
Embodiment 11
Carried out in the way of substantially the same manner as Example 7, difference is to secondary using -60 irradiation dose of cobalt of 60kgy
Lyophilized collagen complex carries out sterilizing processing, then measures embodiment 7 and the mineralized collagen base alveolar bone obtained by embodiment 11 is repaiied
The mechanical property and degradation time of multiple material.As a result find, the former comprcssive strength is 2 times of the latter, and the latter is often below
0.2MPa, degradation time the former be 1.5 times of the latter, the latter often below 2 months.
Claims (10)
1. a kind of method for preparing mineralized collagen base alveolar bone repairing material, it is characterised in that methods described comprises the steps：
(1) type i collagen fiber is configured to into the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) calcium ions aqueous solution is added in the collagen solution with the amount of 0.08 to 0.12 mole of calcium ion/gram collagen, from
And calcium/collagen composite solution is obtained；
(3) phosphorus-containing acid ion aqueous solution is added in the calcium/collagen composite solution with the amount of Ca/P=1.2～1.8, so as to
Prepared calcium/phosphorus/collagen composite solution；
(4) it is 8 calcium/phosphorus/collagen composite solution to be adjusted to pH value, and stirring, precipitation separation, water washing and precipitating are until the pH of supernatant
It is worth 7.0～7.5, so as to obtain precipitate；
(5) using purification water washing precipitate sucking filtration, obtain filter cake；
(6) filter cake is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then is distilled in -10～0 DEG C under vacuum, from
And lyophilization is carried out to filter cake；
(7) the mineralized collagen dry powder that particle diameter is not more than 80 mesh will be ground to form through cryodesiccated filter cake；
(8) prepare type i collagen fiber aqueous solution of the concentration for 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in the collagen fiber aqueous solution, mineralized collagen/collagen mixed solution is obtained, it is described
Mineralized collagen dry powder is 1 with the mass ratio of the type i collagen fiber:4～4:1；
(10) mineralized collagen/collagen mixed solution is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then under vacuum in -
10～0 DEG C are distilled, so as to a lyophilized collagen complex is obtained；
(11) a lyophilized collagen complex is soaked 24 to 48 hours in cross-linking agent solution, is combined so as to crosslinked with collagen is obtained
Thing, the cross-linking agent solution are ethanol solution of 0.005 mass % to the glutaraldehyde of 0.25 mass %；
(12) the crosslinked with collagen complex is centrifuged, filters and wash, so as to washing collagen-based composite is obtained；
(13) washing collagen-based composite is carried out into pre-freeze in -30～-20 DEG C at ambient pressure, then under vacuum in -10～0 DEG C
Distilled, so as to make secondary lyophilized collagen complex；
(14) the secondary lyophilized collagen complex is sterilized, so as to the mineralized collagen base alveolar repair material is obtained
Material.
2. method according to claim 1, it is characterised in that the acid solution in step (1) selected from aqueous hydrochloric acid solution,
The group of aqueous solution of nitric acid and acetic acid aqueous solution composition, preferably acetic acid aqueous solution.
3. method according to claim 1, it is characterised in that in step (4) by pH regulator to 8 after, be stirred for 8 to
24 hours, form white suspension, stand 12 to 24 hours, remove supernatant, then plus purified water 5 to 10 are stirred to original volume
It is secondary, supernatant, continuous wash 3 to 7 times, until the pH value of supernatant is between 7.0～7.5 are removed after standing 1 to 3 hour.
4. according to the method in any one of claims 1 to 3, it is characterised in that the type i collagen fiber is ungelled
The I type bovine collagen fibers of state.
5. according to the method in any one of claims 1 to 3, it is characterised in that also wrap between step (13) and (14)
Include the detecting step whether cross-linking agent residual quantity is in the range of qualified residue.
6. method according to claim 5, it is characterised in that the qualified residue scope be less than or equal to
25ppm。
7. according to the method in any one of claims 1 to 3, it is characterised in that the sterilizing is by adopting 15kgy extremely
- 60 irradiation dose of cobalt of 40kgy is carried out.
8. according to the method in any one of claims 1 to 3, it is characterised in that in step (11), the cross-linking agent is molten
The volume of liquid is 50 to 200ml/g relative to the quality of a lyophilized collagen complex.
9. according to the method in any one of claims 1 to 3, it is characterised in that step (12) is carried out in the following way：
Crosslinked with collagen complex is removed and placed in into centrifuge loading filter bag from cross-linking agent solution, is centrifuged in 2000-3000r/min
2 to 4 times, every time 3 to 10 seconds；Again crosslinked with collagen complex is placed in chromatographic column, it is little with the purification water wash 48 to 72 for flowing
When removing the cross-linking agent of residual.
10. the mineralized collagen base alveolar bone repairing material by obtained in the method any one of claim 1 to 9.
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