CN110368531A - Mineralized collagen base alveolar bone repairing material and preparation method thereof - Google Patents
Mineralized collagen base alveolar bone repairing material and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to mineralized collagen base alveolar bone repairing materials and preparation method thereof；The preparation method includes the following steps: to prepare collagen solution；Add calcium ions aqueous solution；Add phosphorus-containing acid ion aqueous solution；Being adjusted to pH value is 8；Washing precipitate simultaneously filters；Freeze-drying；It grinds and mineralized collagen dry powder is made；Prepare type i collagen fiber aqueous solution；Add mineralized collagen dry powder；Primary freeze drying；Crosslinking；It is centrifuged, filters and wash；Secondary freeze drying；Sterilizing.The method has many advantages, such as that at low cost, stock utilization is high, easy to operate, and mineralized collagen base alveolar bone repairing material obtained by this method is high with homogenieity, mechanical strength is high, degradation time is long, residual crosslinker is few, porosity is high, guided bone and inductivity are excellent.
Description
The application is
" on October 31st, 2016 ", application No. is the divisional applications of the application for a patent for invention of " 201610928747.2 ".
Technical field
The invention patent relates to therapeutic treatment fields, the especially system of biomaterial for medical purpose preparation especially dental implant material
Standby field, and in particular to it is a kind of use it is a kind of using mineralized collagen as New Reclaiming Material of matrix and preparation method thereof and in alveolus
Application in Bone Defect Repari.
Background technique
Gear division clinic can encounter the case of tooth turnover zone Bone mineral change and bone defect, bone amount caused by teeth socked infection etc.
It is insufficient；Accessory tooth, impacted tooth and malposed tooth, which are pulled out, also often results in bone defect.This situation will cause the bone amount of tooth turnover zone
Deficiency will 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 maintains biological characteristics and skeletonization space, to achieve the purpose that alveolar bone defect repair.
Currently most used bioactive materials have hydroxyapatite (HA) and xenogenesis inorganic bone two types.Hydroxyl phosphorus
Lime stone (HA) is the important inorganic component part of human body bone and tooth, and the hydroxyapatite of sintering or non-sintered different crystal phases is tool
There is the timbering material of good bone guided effect, but degradation rate is fast or slow, cannot match with skeletonization rate.Bio-oss
Bio-oss is by specially treated, and except deproteinized and other organic principles, antigenic low, high-purity simultaneously keeps porous nature bone
Inorganic structure, the bone- xenograft almost the same with the structure of human body bone.But bone- xenograft cannot still overcome being immunized after transplanting completely
Rejection has potential pathogeny to propagate dangerous, exists simultaneously the obstacle in terms of Medicine Ethics.
Using bionical thinking, can prepare the hydroxyapatite and collagen similar with nature bone and be combined into has uniqueness
The compound of the hierarchical structure of rule.The Cui Fuzhai professor of material system, Tsinghua University mentions in Chinese patent ZL01129699.2
It is compound porous using a kind of biomineralization technological invention nm-phase calcium-phosphorus salt/collagen/high-molecular bone for bone renovating material
Material and preparation method.Aojing Medicine, Sci. & Tech. Co., Ltd., Beijing also refers in Chinese patent application 201410275022.9
A kind of mineralized collagen artificial periosteum and preparation method thereof.
Summary of the invention
Biocompatibility is more preferable in order to obtain, degradation rate is more controllable, it is more easily molded have 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 more good bioactivity repair materials of performance especially bion alveolar bone repairing material, to both maintain biomineralization
The bone guided of collagen, and it has been modified collagen as tissue engineering scaffold.For the particular/special requirement of the clinical application 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 adhesive using bovine collagen
Dry powder dispersion carries out crosslinking appropriate in collagen as tissue engineering scaffold after molding, increase the mechanical strength and biology of collagen scaffold
Degradation time.In addition, repair materials of the invention are not only consistent with nature bone on composition, but also because containing mineralized collagen,
So it is also similar to nature bone in structure, it is good artificial bone.In addition, for mineralized collagen is manufactured in the prior art
Wash time caused by localized ion concentration is too large or too small when dry powder, phosphate anion and/or calcium ion remain is too long, heavy
The problems such as starch washing is time-consuming, calcium microcosmic salt utilization rate is low and mineralized collagen base alveolar bone repairing material mechanical property are low, degrade
Mineralized collagen dry powder dispersion is poor when time is short and preparation, residual crosslinker amount it is excessive be difficult to remove, porosity is low etc. asks
Topic, improves the prior art and has been optimized, to partly even fully solve the above problem.
The present invention provides a kind of method for preparing mineralized collagen dry powder in first aspect, and the method includes walking as follows
It is rapid:
(1) type i collagen fiber is configured to the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) that calcium ions are added into the collagen solution with 0.08 to 0.12 mole of calcium ion/gram collagen amount is water-soluble
Liquid, so that calcium/collagen composite solution be made；
(3) phosphorus-containing acid ion aqueous solution is added into the calcium/collagen composite solution with the amount of Ca/P=1.2~1.8,
To which calcium/phosphorus/collagen composite solution be made；
(4) calcium/phosphorus/collagen composite solution is adjusted to pH value is 8, and stirring, precipitation and separation, water washing and precipitating are until supernatant
PH value 7.0~7.5, to obtain sediment；
(5) using purifying water washing precipitate and filtering, filter cake is obtained；
(6) it under normal pressure by filter cake in -30~-20 DEG C of progress pre-freezes, then is risen under vacuum conditions in -10~0 DEG C
China, to be freeze-dried to filter cake；
(7) the mineralized collagen dry powder that partial size is not more than 80 mesh will be ground by the filter cake of freeze-drying.
The present invention provides mineralized collagen dry powder made from first aspect present invention the method in second aspect.
The present invention provides mineralized collagen dry powder described in second aspect of the present invention in preparation mineralized collagen in the third aspect
Application in base alveolar bone repairing material.
The present invention provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, the method in fourth aspect
Include the following steps:
(a) the type i collagen fiber aqueous solution that preparation concentration is 0.0286-0.0667g/ml；
(b) mineralized collagen dry powder is added in Xiang Suoshu collagenous fibres aqueous solution, mineralized collagen/collagen mixed solution is made,
The mass ratio of the mineralized collagen dry powder and the type i collagen fiber is 1:4~4:1；
(c) under normal pressure by mineralized collagen/collagen mixed solution in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions
It distils in -10~0 DEG C, so that a lyophilized collagen compound be made；
(d) a lyophilized collagen compound is impregnated 24 to 48 hours in cross-linking agent solution, so that crosslinked with collagen be made
Compound, the cross-linking agent solution are the ethanol solution of the glutaraldehyde of 0.005 mass % to 0.25 mass %；
(e) the crosslinked with collagen compound is centrifuged, filtered and washed, so that washing collagen-based composite be made；
(f) under normal pressure will washing collagen-based composite in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions in -10~
0 DEG C distils, to make secondary lyophilized collagen compound；
(g) the secondary lyophilized collagen compound is sterilized, so that the mineralized collagen base alveolar repair be made
Material.
The present invention additionally provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, the side at the 5th aspect
Method includes the following steps:
(1) type i collagen fiber is configured to the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) that calcium ions are added into the collagen solution with 0.08 to 0.12 mole of calcium ion/gram collagen amount is water-soluble
Liquid, so that calcium/collagen composite solution be made；
(3) phosphorus-containing acid ion aqueous solution is added into the calcium/collagen composite solution with the amount of Ca/P=1.2~1.8,
To which calcium/phosphorus/collagen composite solution be made；
(4) calcium/phosphorus/collagen composite solution is adjusted to pH value is 8, and stirring, precipitation and separation, water washing and precipitating are until supernatant
PH value 7.0~7.5, to obtain sediment；
(5) using purifying water washing precipitate and filtering, filter cake is obtained；
(6) it under normal pressure by filter cake in -30~-20 DEG C of progress pre-freezes, then is risen under vacuum conditions in -10~0 DEG C
China, to be freeze-dried to filter cake；
(7) the mineralized collagen dry powder that partial size is not more than 80 mesh will be ground by the filter cake of freeze-drying；
(8) the type i collagen fiber aqueous solution that preparation concentration is 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in Xiang Suoshu collagenous fibres aqueous solution, mineralized collagen/collagen mixed solution is made,
The mass ratio of the mineralized collagen dry powder and the type i collagen fiber is 1:4~4:1；
(10) under normal pressure by mineralized collagen/collagen mixed solution in -30~-20 DEG C of progress pre-freezes, then in vacuum condition
Under distil in -10~0 DEG C, thus be made a lyophilized collagen compound；
(11) a lyophilized collagen compound is impregnated 24 to 48 hours in cross-linking agent solution, so that crosslinked with collagen be made
Compound, the cross-linking agent solution are the ethanol solution of the glutaraldehyde of 0.005 mass % to 0.25 mass %；
(12) the crosslinked with collagen compound is centrifuged, filtered and washed, so that washing collagen-based composite be made；
(13) collagen-based composite will be washed under normal pressure in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions in -10
~0 DEG C distils, to make secondary lyophilized collagen compound；
(14) the secondary lyophilized collagen compound is sterilized, so that the mineralized collagen base alveolar repair be made
Material.
The present invention provides mineralized collagen abutment made from the 4th or the 5th aspect the method for the invention at the 6th aspect
Slot bone renovating material.
The present invention provides mineralized collagen base alveolar bone repairing material described in sixth aspect present invention at the 7th aspect and exists
Prepare the application in the material for repairing alveolar bone.
Advantages of the present invention and brought unexpected technical effect include at least following several points:
(1) the collagen solution concentration used is 0.0001-0.001g/ml, and under the collagen concentration, whole solution viscosity is more
Add moderate, not will cause viscosity influence excessive for localized ion concentration or too small.
(2) it uses calcium phosphorus molar feed ratio for Ca:P=1.2-1.8, improves the utilization efficiency of calcium salt and microcosmic salt, reduce
Dissociate in material the residual of calcium salt and microcosmic salt.
(3) pH value is used to significantly reduce for 8 precipitating terminal so that mineralized collagen precipitates quicker and abundant
Stirring and time of repose.
(4) 80 mesh mineralized collagen dry powder below is used, so that it with collagen adhesives when mixing, it is easier to disperse.
(5) it uses 40kgy irradiation dose below and carries out sterilizing processing, significantly reduce the mechanical property and drop of material
Solve the time.
(6) I-type collagen is used, the fiber of ungelled state is avoided as collage raw material because of solid content in gel
The inconsistent problem for causing inventory inconsistent.
(7) separation of supernatant and sediment is carried out using suction filtration mode, pH value can or be easier to level off to neutrality.
(8) it by way of segmentation freeze-drying, allows the material to solidify at a lower temperature, distil at slightly higher temperature,
Shorten freeze-drying time.
(9) using collagenous fibres as adhesive and timbering material so that material composition maintain it is consistent with nature bone
Chemical analysis；The mass ratio of dry powder and collagen is 1:4~4:1, according to the porous support as used in tissue engineering, different tissues
Cell has 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 centrifugation washing, secondary freeze drying, the time of washing is shortened, efficiency is improved.
Specific embodiment
As described above, the present invention provides a kind of method for preparing mineralized collagen dry powder, the method packet in first aspect
Include following steps:
(1) using acid solution by type i collagen fiber be configured to concentration be 0.0001 to 0.001g/ml (such as 0.0001,
0.0005 or 0.001g/ml) collagen solution；
(2) 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 that calcium/collagen composite solution be made；
(3) with the amount of Ca/P=1.2~1.8 (such as 1.2,1.4,1.6 or 1.8) into the calcium/collagen composite solution
Phosphorus-containing acid ion aqueous solution is added, so that calcium/phosphorus/collagen composite solution be made；
(4) calcium/phosphorus/collagen composite solution is adjusted to pH value is 8, and stirring, precipitation and 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), to obtain sediment；
(5) using purifying water washing precipitate and filtering, filter cake is obtained；
(6) under normal pressure by filter cake in -30~-20 DEG C (such as -30, -25 or -20 DEG C) progress pre-freezes, then in vacuum item
It distils under part in -10~0 DEG C (such as -10, -5 or 0 DEG C), to be freeze-dried to filter cake；
(7) partial size will be ground by the filter cake of freeze-drying no more than 80 mesh (such as no more than 80,90,100 or 120
Mesh) mineralized collagen dry powder.
In some preferred embodiments, the acid solution in step (1) is selected from by aqueous hydrochloric acid solution, aqueous solution of nitric acid
With the group of acetic acid aqueous solution composition, preferably acetic acid aqueous solution.
In some preferred embodiments, the concentration of the acid solution be 0.45 to 0.55mM (such as 0.45,0.50 or
0.55mM)。
In some preferred embodiments, 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).
In some preferred embodiments, the calcium ions aqueous solution and/or phosphorus-containing acid ion aqueous solution plus
Enter and is carried out by the way of being stirred when being slowly added dropwise.
In some preferred embodiments, pH is adjusted to 8 and then stirring 8 to 24 hours, shape in step (4)
At white suspension, stand 12 to 24 hours (such as 12,18 or 24 hours), remove supernatant, then plus purified water to original volume after
It stirs 5 to 10 times (such as 5,6,7,8,9 or 10 times), removes supernatant after standing 1 to 3 hour, continuous wash 3 to 7 times, Zhi Daoshang
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).
In some preferred embodiments, the concentration of the calcium ions aqueous solution be 0.45M to 0.55M (such as
0.45,0.50 or 0.55M).
In some preferred embodiments, the concentration of the phosphorus-containing acid ion aqueous solution be 0.4M to 0.6M (such as
0.40,0.50 or 0.60M).
In some preferred embodiments, the type i collagen fiber is the I type bovine collagen fibers of ungelled state.
In some preferred embodiments, the calcium salt and phosphorus-containing acid ion of the calcium ions aqueous solution are used to prepare
The calcium salt of aqueous solution and the microcosmic salt for being used to prepare phosphorus-containing acid ion aqueous solution are the other calcium salt of medicinal or pharmaceutic adjuvant grade and phosphorus
Salt.
The present invention provides mineralized collagen dry powder obtained made from first aspect present invention the method in second aspect.
The present invention provides made from first aspect present invention the method or second aspect of the present invention in the third aspect
Application of the mineralized collagen dry powder in preparation mineralized collagen base alveolar bone repairing material.
The present invention provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, the method in fourth aspect
Include the following steps:
(a) preparation concentration is the I type glue of 0.0286-0.0667g/ml (such as 0.0286,0.0476 or 0.0667g/ml)
Fibrinogen aqueous solution；
(b) mineralized collagen dry powder is added in Xiang Suoshu collagenous fibres aqueous solution, mineralized collagen/collagen mixed solution is made,
The mass ratio of the mineralized collagen dry powder and the type i collagen fiber be 1:4~4:1 (such as 1:4,1:3,1:2,1:1,2:1,
3:1 or 4:1)；
(c) under normal pressure by mineralized collagen/collagen mixed solution in -30~-20 DEG C (such as -30, -25 or -20 DEG C) into
Row pre-freeze, then distil under vacuum conditions in -10~0 DEG C (such as -10, -5 or 0 DEG C), so that a lyophilized collagen be made
Compound；
(d) by a lyophilized collagen compound impregnated in cross-linking agent solution 24 to 48 hours (such as 24,30,36,42 or
48), thus be made crosslinked with collagen compound, the cross-linking agent solution be 0.005 mass % to 0.25 mass % (such as 0.005,
0.010,0.015,0.020 or 0.025 mass %) glutaraldehyde ethanol solution；
(e) the crosslinked with collagen compound is centrifuged, filtered and washed, so that washing collagen-based composite be made；
(f) under normal pressure will washing collagen-based composite in -30~-20 DEG C (such as -30, -25 or -20 DEG C) progress pre-freezes,
It distils under vacuum conditions in -10~0 DEG C (such as -10, -5 or 0 DEG C) again, to make secondary lyophilized collagen compound；
(g) the secondary lyophilized collagen compound is sterilized, so that the mineralized collagen base alveolar repair be made
Material.
In some preferred embodiments, in step (f) and (g) between further include whether residual crosslinker amount is in conjunction
Detecting step within the scope of lattice residue.
In some preferred embodiments, the qualified residue range is less than or equal to 25ppm.
In some preferred embodiments, the detecting step carries out in the following way: weighing the secondary jelly of 0.1g
Dry collagen-based composite, detection residual crosslinker amount whether be less than or equal to 25ppm (such as no more than 25,20,15,10 or
5ppm), if it is greater than 25ppm, then the repeatedly secondary freeze-drying of the washing in step (e) and step (f), until residual crosslinker amount
Less than or equal to 25ppm.
In some preferred embodiments, it is described sterilizing by using 15kgy to 40kgy (such as 15,20,25,30,
35 or 40kgy) -60 irradiation dose of cobalt carry out.
In some preferred embodiments, in step (d), the volume of the cross-linking agent solution is relative to primary freeze-drying
The quality of collagen-based composite is 50 to 200ml/g (such as 50,100,150 or 200ml/g).
In some preferred embodiments, step (b) is uniformly carried out by dispersion mixing at room temperature.
In some preferred embodiments, step (e) carries out in the following way: by crosslinked with collagen compound from crosslinking
Centrifuge loading filter bag is removed and placed in agent solution, at 2000-3000r/min (such as 2000,2500 or 3000r/min)
It is centrifuged 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 compound is placed in chromatographic column again
In, 48 to 72 (48,54,60,66 or 72) hours were eluted to remove remaining crosslinking agent with the purified water of flowing.
In some preferred embodiments, the mineralized collagen dry powder is method system described in first aspect present invention
?.
The present invention additionally provides a kind of method for preparing mineralized collagen base alveolar bone repairing material, the side at the 5th aspect
Method includes the following steps:
(1) type i collagen fiber is configured to the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) that calcium ions are added into the collagen solution with 0.08 to 0.12 mole of calcium ion/gram collagen amount is water-soluble
Liquid, so that calcium/collagen composite solution be made；
(3) phosphorus-containing acid ion aqueous solution is added into the calcium/collagen composite solution with the amount of Ca/P=1.2~1.8,
To which calcium/phosphorus/collagen composite solution be made；
(4) calcium/phosphorus/collagen composite solution is adjusted to pH value is 8, and stirring, precipitation and separation, water washing and precipitating are until supernatant
PH value 7.0~7.5, to obtain sediment；
(5) using purifying water washing precipitate and filtering, filter cake is obtained；
(6) it under normal pressure by filter cake in -30~-20 DEG C of progress pre-freezes, then is risen under vacuum conditions in -10~0 DEG C
China, to be freeze-dried to filter cake；
(7) the mineralized collagen dry powder that partial size is not more than 80 mesh will be ground by the filter cake of freeze-drying；
(8) the type i collagen fiber aqueous solution that preparation concentration is 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in Xiang Suoshu collagenous fibres aqueous solution, mineralized collagen/collagen mixed solution is made,
The mass ratio of the mineralized collagen dry powder and the type i collagen fiber is 1:4~4:1；
(10) under normal pressure by mineralized collagen/collagen mixed solution in -30~-20 DEG C of progress pre-freezes, then in vacuum condition
Under distil in -10~0 DEG C, thus be made a lyophilized collagen compound；
(11) a lyophilized collagen compound is impregnated 24 to 48 hours in cross-linking agent solution, so that crosslinked with collagen be made
Compound, the cross-linking agent solution are the ethanol solution of the glutaraldehyde of 0.005 mass % to 0.25 mass %；
(12) the crosslinked with collagen compound is centrifuged, filtered and washed, so that washing collagen-based composite be made；
(13) collagen-based composite will be washed under normal pressure in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions in -10
~0 DEG C distils, to make secondary lyophilized collagen compound；
(14) the secondary lyophilized collagen compound is sterilized, so that the mineralized collagen base alveolar repair be made
Material.
In some preferred embodiments, the acid solution in step (1) is selected from by aqueous hydrochloric acid solution, aqueous solution of nitric acid
With the group of acetic acid aqueous solution composition, preferably acetic acid aqueous solution.
In some preferred embodiments, the concentration of the acid solution is 0.45 to 0.55mM.
In some preferred embodiments, 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.
In some preferred embodiments, the calcium ions aqueous solution and/or phosphorus-containing acid ion aqueous solution plus
Enter and is carried out by the way of being stirred when being slowly added dropwise.
In some preferred embodiments, pH is adjusted to 8 and then stirring 8 to 24 hours, shape in step (4)
At white suspension, stand 12 to 24 hours, remove supernatant, then plus purified water to original volume after stir 5 to 10 times, standing 1 to 3
Supernatant is removed after hour, continuous wash 3 to 7 times, until the pH value of supernatant is between 7.0~7.5.
In some preferred embodiments, the concentration of the calcium ions aqueous solution is 0.45M to 0.55M.
In some preferred embodiments, the concentration of the phosphorus-containing acid ion aqueous solution is 0.4M to 0.6M.
In some preferred embodiments, the type i collagen fiber is the I type bovine collagen fibers of ungelled state.
In some preferred embodiments, the calcium salt and phosphorus-containing acid ion of the calcium ions aqueous solution are used to prepare
The calcium salt of aqueous solution and the microcosmic salt for being used to prepare phosphorus-containing acid ion aqueous solution are the other calcium salt of medicinal or pharmaceutic adjuvant grade and phosphorus
Salt.
In some preferred embodiments, further include whether residual crosslinker amount is between step (13) and (14)
Detecting step within the scope of qualified residue.
In some preferred embodiments, the qualified residue range is less than or equal to 25ppm.
In some preferred embodiments, the detecting step carries out in the following way: weighing the secondary jelly of 0.1g
Dry collagen-based composite, whether detection residual crosslinker amount is less than or equal to 25ppm, if it is greater than 25ppm, then repeatedly step
(12) the secondary freeze-drying of washing and step (13) in, until residual crosslinker amount is less than or equal to 25ppm.
In some preferred embodiments, it is described sterilizing by using 15kgy to 40kgy -60 irradiation dose of cobalt into
Row.
In some preferred embodiments, in step (11), the volume of the cross-linking agent solution is frozen relative to primary
The quality of dry collagen-based composite is 50 to 200ml/g.
In some preferred embodiments, step (9) is uniformly carried out by dispersion mixing at room temperature.
In some preferred embodiments, step (12) carries out in the following way: by crosslinked with collagen compound from friendship
It is removed and placed in centrifuge loading filter bag in connection agent solution, is centrifuged 2 to 4 times, every time 3 to 10 seconds in 2000-3000r/min；
Crosslinked with collagen compound is placed in chromatographic column again, elutes 48 to 72 hours with the purified water of flowing to remove remaining crosslinking
Agent.
In some more specific embodiments, the side for being used to prepare mineralized collagen base alveolar bone repairing material of the invention
Method includes the following steps:
(1) it is configured to the acid solution of type i collagen fiber, acid therein is in the group being made of hydrochloric acid, nitric acid or acetic acid
Any one, the concentration of collagen solution is 0.0001-0.001g/ml；
(2) calcium ions aqueous solution is slowly added dropwise in Xiang Suoshu acid solution while stirring, additional amount is that every gram of collagen is added dropwise
Calcium ion 0.08-0.12mol, to form calcium/collagen composite solution；
(3) aqueous solution containing phosphate anion, the phosphate radical of addition is added dropwise while stirring in calcium/collagen composite solution
The molar ratio of the amount of the calcium ion of the amount and addition of ion is Ca:P=1.2-1.8；
(4) it is 8 that NaOH solution to pH value is added dropwise while stirring in the solution that phosphate anion has been added dropwise, and is stirred for 8-24
Hour, form white suspension, stand 12-24 hour, remove supernatant, then plus purified water to original volume after stir, standing 1-3 is small
When after remove supernatant, continuous wash 5 to 10 times, until the pH value of supernatant is between 7.0~7.5, obtain sediment；
(5) pumping and filtering device is opened, sediment is poured into, is filtered, is washed repeatedly filter cake 2-4 times, is filtered with purified water
Cake；
(6) filter cake is put into freeze drier and carries out pre-freeze under normal pressure at -30~-20 DEG C, under vacuum conditions -10
~0 DEG C carries out distillation to realize freeze-drying；
(7) filter cake by freeze-drying is ground, mineralized collagen dry powder is made, the screening of 80 mesh stainless steel mesh is spare；
(8) type i collagen fiber is placed in reaction kettle, purified water is added, 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 into collagen solution, dispersion mixing is uniform at room temperature, wherein dry powder and collagen
Mass ratio be 1:4~4:1, be made mineralized collagen/collagen mixed solution；
(10) under normal pressure by mineralized collagen/collagen mixed solution in -30~-20 DEG C of progress pre-freezes, then in vacuum condition
Under distil in -10~0 DEG C, thus be made a lyophilized collagen compound；
(11) ethanol solution of the glutaraldehyde of 0.005-0.25% mass fraction is prepared as cross-linking agent solution, will once be frozen
Dry collagen-based composite is complete for the amount of 50-200ml/g according to the volume of cross-linking agent solution and the quality of a lyophilized collagen compound
Complete immersion 24 to 48 hours, so that crosslinked with collagen compound be made；
(12) crosslinked with collagen compound is taken out from cross-linking agent solution, centrifuge loading filter bag, 2000-3000r/
Min is centrifuged 2-4 times, 3-10 seconds each；Crosslinked with collagen compound is placed in chromatographic column again, with the purified water of flowing elution 48 to
Collagen-based composite is washed to remove remaining crosslinking agent, to be made within 72 hours；
(13) collagen-based composite will be washed under normal pressure in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions in -10
~0 DEG C distils, to make secondary lyophilized collagen compound；
(14) it after being lyophilized, takes out about 0.1g and does residual crosslinker test, if residual crosslinker amount is greater than 25ppm,
Water elution and secondary freeze-drying process are then repeated, until residual crosslinker amount is less than or equal to 25ppm；
(15) the secondary lyophilized collagen compound by residual crosslinker amount less than or equal to 25ppm uses 15kgy extremely
- 60 irradiation dose of cobalt of 40kgy carries out sterilizing processing, to obtain mineralized collagen base alveolar bone repairing material.
The present invention provides mineralized collagen made from fourth aspect present invention or the 5th aspect the method at the 6th aspect
Base alveolar bone repairing material.
Seventh aspect present invention provides mineralized collagen base alveolar bone repairing material described in sixth aspect present invention and is repairing
Application in multiple alveolar bone.
In the present invention, the present inventor at least improve at following aspect and has achieved corresponding technical effect:
(1) calcium ion 0.08-0.12mol is added dropwise using every gram of collagen；Very few calcium can sometimes be added dropwise in the prior art
Ion, sometimes even as low as 0.01mol；Excessive calcium ion sometimes is added dropwise again, sometimes up to 0.16mol/g；Excessive calcium
Ion, which will lead to, remains extra free calcium ions in the waste or material of calcium salt, so that subsequent cleaning becomes complexity even
Difficulty may cause mineralized collagen deficiency if calcium ion is very few, and intensity is lower, and material and bone tissue difference are big, influence material
The bone guided and inductivity of material.
(2) use collagen solution concentration for 0.0001-0.001g/ml, under the collagen concentration, whole solution viscosity is more
It is moderate, caused by avoiding because of viscosity localized ion concentration it is excessive or too small brought by influence.
(3) use calcium phosphorus molar feed ratio for Ca:P=1.2-1.8, so that the inorganic salts composition of material is more in line with 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, avoids free calcium ions or phosphate anion residual
The problem of the deficiency of the strength of materials caused by staying.
(4) pH value is used to substantially reduce for 8 precipitating terminal so that mineralized collagen precipitates more quickly more abundant
Stirring and time of repose；In the prior art even using up to 9 or down to 7 pH so that only time of repose is even up to 5 days
Time.In addition, since NaOH solution is often added dropwise to mixed system pH=6~8 in the past, and observe as pH=5~6, it mixes
Zoarium system starts to precipitate, and as pH=7, white suspension occurs in mixed system, therefore NaOH to pH is often added dropwise on 7 left sides
It is right.The present inventor has found through overtesting, and NaOH solution is added dropwise to mixed system pH=8, is washed again after precipitating to 7.0-7.5, instead
And the time required to sedimentation effect can be improved and reduce.
(5) it is mixed using 80 mesh mineralized collagen dry powder below with high molecular polymer, the dry powder under the mesh number is easier
Dispersed in polymer solution system.
(6) it uses 40kgy irradiation dose below and carries out sterilizing processing, because material is absorbable polymer and collagen
Deng the material for radiation sensitive, in order to significantly reduce the mechanical property and degradation time of material, using low irradiation dose
Carrying out sterilizing is very important.
In addition, the present inventor also in quality system establishment process, has carried out following improvement to part raw material and has obtained
Corresponding technical effect:
(1) I-type collagen is used, the fiber of ungelled state is avoided as collage raw material because containing admittedly in gel
That measures is inconsistent, causes inventory inconsistent；Type I collagen is a kind of structural proteins found in animal body simultaneously, is self
The main organic component of bone.I-type collagen is the major structural protein of spinal animals, is osteoblast in osteogenetic process
The extracellular matrix of secretion is the bracket of calcium deposition and the template of the promotor of bone matrix mineralising, mineralising；Cell can be promoted to move
It moves, absorption, differentiation, and is adjustable cell growth, approved by U.S. FDA as biomaterial, and there are a series of collagens to be implanted into
Product, including bone implant product.
(2) the other calcium salt of medicinal or pharmaceutic adjuvant grade, microcosmic salt are used, is avoided because the rank of raw material causes impurity, again
Metal, ash content it is exceeded, also influence material biocompatibility.
In addition, the present inventor also uses suction filtration mode to carry out the separation of supernatant and sediment, and clean to neutrality.This hair
Bright people's discovery, compared with centrifugation in the prior art, can so make porous material obtain contacting with purified water again
Chance, after repeatedly washing, the pH value of leaching liquor can level off to neutrality for inside and outside.
In addition, the present inventor is removed solvent and pore-creating, i.e., (is not vacuumized) under normal pressure by way of segmentation freeze-drying
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, enables material
It is enough to solidify at a lower temperature, it distils at slightly higher temperature, shortens freeze-drying time significantly.
In addition, the present inventor uses collagenous fibres as adhesive and timbering material, so that material composition maintains and day
The consistent chemical analysis of right bone；The mass ratio of dry powder and collagen is 1:4~4:1, according to the porous support as used in tissue engineering,
Different histocytes has different requirements to aperture, and bone and cartilage tissue engineered, needs 100-250 μm of aperture, 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%.
In addition, residual crosslinker is always technical problem in obtained repair materials.The present inventor passes through centrifugation washing
Remaining crosslinking agent is removed, for residual crosslinker amount to be reduced to 25ppm, and can conveniently realize.
In the technical solution of the remaining crosslinking agent of removal of the invention, first passes through centrifugation and largely handed over removing in material porous structure
It is molten to join agent, because chromatographic column elution is the diluted process of crosslinking agent, centrifugally operated can shorten the time of washing, and efficiency even can be with
Improve 1 times！In addition, having carried out secondary freeze drying due to have passed through water-washing process.
It is important to note that the numberical range of this specification indicate the upper limit value of the numberical range, lower limit value and
Any numerical value or the subrange being within the numberical range.Therefore, if not otherwise specified, it is related in the present specification
Just no longer itemizing when numberical range includes specific value in the numberical range.
Embodiment
The present invention will be hereafter illustrated by way of embodiment, but protection scope of the present invention should not be by
It is considered limited to these embodiments.
Embodiment 1
(1) using the acid solution of the type i collagen fiber of the acetic acid solutions 0.0005g/ml of 0.5M；
(2) the calcium ions aqueous solution of 0.5M is slowly added dropwise in Xiang Suoshu acid solution while stirring, additional amount is every gram of glue
Calcium ion 0.10mol is added dropwise in original, to form calcium/collagen composite solution；
(3) the phosphorus-containing acid ion aqueous solution of 0.5M, the phosphoric acid of addition is added dropwise while stirring in calcium/collagen composite solution
The molar ratio of the amount of the calcium ion of the amount and addition of radical ion is Ca/P=1.5；
(4) NaOH aqueous solution to the pH value that 1M is added dropwise while stirring in the solution that phosphate anion has been added dropwise is 8, then is stirred
Mix 16 hours, form white suspension, stand 18 hours, remove supernatant, then plus purified water to original volume after stir, it is small to stand 2
When after remove supernatant, continuous wash 5 times, through detecting, the pH value of supernatant at this time is 7.2 (being between 7.0~7.5), is obtained
To sediment；
(5) pumping and filtering device is opened, sediment is poured into, is filtered, is washed repeatedly filter cake 3 times with purified water, obtains filter cake；
(6) filter cake is put into freeze drier and carries out pre-freeze under normal pressure at -25 DEG C, under vacuum conditions -5 DEG C of progress
Distillation is to realize freeze-drying；
(7) at the quartering of filter cake periphery and filter cake central location carry out five point samplings, its homogenieity of observation analysis；So
The filter cake by freeze-drying is ground afterwards, mineralized collagen dry powder is made, the screening of 80 mesh stainless steel mesh is spare.
Embodiment 2 to 4
Other than the content of the following table 1, carried out in mode substantially the same manner as Example 1.
Embodiment 5
It other than the content of the following table 1, is carried out in mode substantially the same manner as Example 1, wherein sedimentation time 36 hours
After measure pH.
Embodiment 6
It other than the content of the following table 1, is carried out in mode substantially the same manner as Example 1, wherein when detection pH reaches 7.2
It is required to staticly settle the time, if pH does not reach 7.2, repeats addition water and staticly settle again, 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 reaction kettle, purified water is added, 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 into collagen solution, dispersion mixing is uniform at room temperature, wherein dry powder and collagen
Mass ratio be 1:4, be made mineralized collagen/collagen mixed solution；
(10) under normal pressure by mineralized collagen/collagen mixed solution in -30 DEG C of progress pre-freezes, then under vacuum conditions in -
10 DEG C distil, so that a lyophilized collagen compound be made；
(11) ethanol solution of the glutaraldehyde of 0.005% mass fraction is prepared as cross-linking agent solution, and glue will once be lyophilized
Former compound is completely soaked 24 according to the amount that the volume of cross-linking agent solution and the quality of a lyophilized collagen compound are 50ml/g
Hour, so that crosslinked with collagen compound be made；
(12) crosslinked with collagen compound is taken out from cross-linking agent solution, centrifuge loading filter bag, 2000r/min centrifugation
2 times, every time 3 seconds；Crosslinked with collagen compound is placed in chromatographic column again, elutes 48 hours with the purified water of flowing to remove residual
Crosslinking agent, thus be made washing collagen-based composite；
(13) washing collagen-based composite is carried out in -30 DEG C of progress pre-freezes, then under vacuum conditions in -10 DEG C under normal pressure
Distillation, to make secondary lyophilized collagen compound；
(14) it after being lyophilized, takes out about 0.1g and does residual crosslinker test, as a result, it has been found that residual crosslinker amount is less than
25ppm；
(15) secondary lyophilized collagen compound is subjected to sterilizing processing using -60 irradiation dose of cobalt of 40kgy, 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
Other than the content shown in the following table 2, carried out in mode same as Example 7.
Embodiment 10
Other than the content shown in the following table 2, carried out in mode substantially the same manner as Example 7, but in step (12),
Without centrifugation, but directly crosslinked with collagen compound is placed in chromatographic column, elutes 48 hours with the purified water of flowing to remove
Remaining crosslinking agent is removed, so that washing collagen-based composite be made.
Embodiment 11
It is carried out in mode substantially the same manner as Example 7, difference is using -60 irradiation dose of cobalt of 60kgy to secondary
Lyophilized collagen compound carries out sterilizing processing, then measures embodiment 7 and the resulting mineralized collagen base alveolar bone of embodiment 11 is repaired
The mechanical property and degradation time of multiple material.As a result, it has been found that the former compression strength is 2 times of the latter, the latter is often below
0.2MPa, the former is 1.5 times of the latter to degradation time, and the latter is often below 2 months.
Claims (11)
1. a kind of method for preparing mineralized collagen base alveolar bone repairing material, which is characterized in that described method includes following steps:
(1) type i collagen fiber is configured to the collagen solution that concentration is 0.0001 to 0.001g/ml using acid solution；
(2) calcium ions aqueous solution is added into the collagen solution with 0.08 to 0.12 mole of calcium ion/gram collagen amount, from
And calcium/collagen composite solution is made；
(3) phosphorus-containing acid ion aqueous solution is added into the calcium/collagen composite solution with the amount of Ca/P=1.2~1.8, thus
Calcium/phosphorus/collagen composite solution is made；
(4) by calcium/phosphorus/collagen composite solution be adjusted to pH value be 8, stirring, precipitation and separation, water washing and precipitating until supernatant pH
Value is 7.0~7.5, to obtain sediment, wherein pH is adjusted to 8 and then stirring 8 to 24 hours, it is suspended to form white
Liquid, stand 12 to 24 hours, remove supernatant, then plus purified water to original volume after stir 5 to 10 times, standing 1 to 3 hour after remove
Supernatant, continuous wash 3 to 7 times, until the pH value of supernatant is between 7.0~7.5.
(5) using purifying water washing precipitate and filtering, filter cake is obtained；
(6) it under normal pressure by filter cake in -30~-20 DEG C of progress pre-freezes, then distils under vacuum conditions in -10~0 DEG C, from
And filter cake is freeze-dried；
(7) the mineralized collagen dry powder that partial size is not more than 80 mesh will be ground by the filter cake of freeze-drying；
(8) the type i collagen fiber aqueous solution that preparation concentration is 0.0286-0.0667g/ml；
(9) mineralized collagen dry powder is added in Xiang Suoshu collagenous fibres aqueous solution, mineralized collagen/collagen mixed solution is made, it is described
The mass ratio of mineralized collagen dry powder and the type i collagen fiber is 1:4~4:1；
(10) under normal pressure by mineralized collagen/collagen mixed solution in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions in -
10~0 DEG C distil, so that a lyophilized collagen compound be made；
(11) a lyophilized collagen compound is impregnated 24 to 48 hours in cross-linking agent solution, so that it is compound that crosslinked with collagen is made
Object, the cross-linking agent solution are the ethanol solution of the glutaraldehyde of 0.005 mass % to 0.25 mass %；
(12) the crosslinked with collagen compound is centrifuged, filtered and washed, so that washing collagen-based composite be made；
(13) collagen-based composite will be washed under normal pressure in -30~-20 DEG C of progress pre-freezes, then under vacuum conditions in -10~0 DEG C
It distils, to make secondary lyophilized collagen compound；
(14) the secondary lyophilized collagen compound is sterilized, so that the mineralized collagen base alveolar repair material be made
Material.
2. the method according to claim 1, wherein the acid solution in step (1) be selected from by aqueous hydrochloric acid solution,
The group of aqueous solution of nitric acid and acetic acid aqueous solution composition, preferably acetic acid aqueous solution.
3. according to the method described in claim 1, it is characterized by:
PH in step (4) is adjusted using the sodium hydrate aqueous solution that concentration is 0.9M to 1.1M；
The concentration of the acid solution is 0.45 to 0.55mM；
The concentration of the calcium ions aqueous solution is 0.45M to 0.55M；And/or
The concentration of the phosphorus-containing acid ion aqueous solution is 0.4M to 0.6M.
4. according to the method in any one of claims 1 to 3, which is characterized 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, which is characterized in that also wrapped between step (13) and (14)
Include the detecting step whether residual crosslinker amount is within the scope of qualified residue.
6. according to the method described in claim 5, it is characterized in that, it is described qualification residue range be less than or equal to
25ppm。
7. according to the method in any one of claims 1 to 3, which is characterized in that described to sterilize by using 15kgy extremely
- 60 irradiation dose of cobalt of 40kgy carries out.
8. according to the method in any one of claims 1 to 3, which is characterized in that in step (11), the crosslinking agent is molten
The volume of liquid is 50 to 200ml/g relative to the quality of a lyophilized collagen compound.
9. according to the method in any one of claims 1 to 3, which is characterized in that step (12) carries out in the following way:
Crosslinked with collagen compound is removed and placed in 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；Crosslinked with collagen compound is placed in chromatographic column again, it is small with the purified water elution 48 to 72 of flowing
When to remove remaining crosslinking agent.
10. mineralized collagen base alveolar bone repairing material made from the method as described in any one of claims 1 to 9.
11. mineralized collagen base alveolar bone repairing material made from the method as described in any one of claims 1 to 9 is used in preparation
Application in the material for repairing alveolar bone.
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| CN113117149A (en) * | 2020-01-15 | 2021-07-16 | 北京化工大学 | Bone-like hydroxyapatite-collagen composite scaffold and preparation method thereof |
| CN113913961A (en) * | 2021-11-16 | 2022-01-11 | 清华大学 | Mineralized collagen nanofiber doped with active elements and preparation method thereof |
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| WO2023185212A1 (en) * | 2022-03-29 | 2023-10-05 | 上海市第十人民医院 | Mineralized collagen material, preparation method therefor, and application thereof |
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| CN108096629B (en) * | 2018-01-29 | 2021-08-06 | 奥精医疗科技股份有限公司 | Polymethyl methacrylate bone cement and preparation method thereof |
| CN115869467A (en) * | 2022-12-12 | 2023-03-31 | 上海典范医疗科技有限公司 | Preparation method of mineralized collagen for bone repair material |
| CN117138113A (en) * | 2023-08-25 | 2023-12-01 | 重庆生物智能制造研究院 | Mineralized collagen material and preparation method and application thereof |
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