CN109876190A - The preparation method and applications of three dimensional biological marking ink - Google Patents
The preparation method and applications of three dimensional biological marking ink Download PDFInfo
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- CN109876190A CN109876190A CN201910291189.7A CN201910291189A CN109876190A CN 109876190 A CN109876190 A CN 109876190A CN 201910291189 A CN201910291189 A CN 201910291189A CN 109876190 A CN109876190 A CN 109876190A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
The present invention provides the preparation method and applications of a kind of three dimensional biological marking ink, three dimensional biological marking ink.Collagen hydrogel is prepared with gelatin, sodium alginate and I-type collagen in the present invention, loads calf calcined bone powder, the three dimensional biological marking ink being prepared has good biocompatibility, certain mechanical strength and three-dimensional porous stereochemical structure.Through the invention, solve the problems, such as that biocompatibility or the plasticity of bone grafting material product in the related technology are bad, bone-grafting material made of three dimensional biological marking ink using the embodiment of the present invention, biocompatibility is improved for porous apatite ceramics are as bone-grafting material made from marking ink, the plasticity and clinical manipulation of bone grafting material product are improved for the bone-grafting material made from the small bovine calcined bone.
Description
Technical field
The present invention relates to the compound bone-grafting material field of bioactivity, in particular to a kind of three dimensional biological marking ink,
The preparation method and applications of three dimensional biological marking ink.
Background technique
In hydroxyapatite family member, the crystal of nanometer hydroxyapatite (Hydroxyapatite, referred to as HA)
Structure and the mineral particles structure in bone tissue are essentially identical, calcium phosphate (Tricalcium Phosphate, referred to as TCP)
Also occupy certain proportion in bone tissue, degradation rate is much larger than HA, is easy to degrade in vivo.Degradation rate removes and chemical group
Dividing has outside the Pass, also related to its contact area between body fluid.The optimal design principle of porous apatite ceramics is to take into account drop
Solution rate and be conducive to growing into for freshman bone tissue, i.e., the macropore of 300um-500um, have both micron-sized small structure.Although
The molding making technology of Ca-P ceramic has big development, the three-dimensional ceramic printing technique controlled such as computer.However it is reading
It when the structural information of natural bone tissue, is still limited, is shortened and natural bone tissue by measurement & characterization technology and processing technology
Gap is still a long-term effort target.
Small bovine calcined bone (Deprotein Bovine Bone, referred to as DBB) is one of existing bone renovating material, is both protected
Inorganic calcium phosphorus ore substance has been stayed, and has remained the porous structure of bone tissue substantially, has obtained preferable evaluation in clinical application.
But small bovine calcined bone intensity is poor, and due to losing collagen completely through high temperature firing, nanocrystal is changed, no
It is degradable;It is directly applied to also be not easy plastotype in 3 D-printing.
With the development of bone tissue three dimensional biological printing technique, cell and the common stamping ink of bioactive material material are used
The bio-ink that gel and gelatin are constituted, can construct preset micro-structure, and cell survival rate with higher (> 90%).
To guarantee that hydrogel can be squeezed out smoothly, host material need to have certain mobility, and filament is fallen into print platform because of itself weight
Accumulation can deformation occurs for power and superstructure, it is difficult to construct high-precision complex model.Although chemical friendship can be used in research at present
The methods of connection promotes hydrogel rapid shaping, but excessive crosslinking has proven to will affect migration, proliferation and the differentiation of cell.This
Outside, it is limited to the viscosity of printed material, calcium phosphorus (Ca-P) material is usually nanoscale, therefore lacks micron order aperture.
There is good biocompatibility, certain mechanical strength and three-dimensional porous stereochemical structure for building, also to have
Suitable degradation rate maintains the growth of cambium, research can rapid shaping, can the small bovine calcined bone biology of cell mixing beat
Printing ink is exactly vital content in current biological 3 D-printing.
Summary of the invention
The present invention provides the preparation method and applications of a kind of three dimensional biological marking ink, three dimensional biological marking ink,
Biocompatibility or plasticity at least to solve the problems, such as bone grafting material product in the related technology is bad.
In a first aspect, the embodiment of the invention provides a kind of preparation methods of three dimensional biological marking ink, comprising:
Step 1, sodium alginate aqueous solution is prepared;
Step 2, gelatin is added in Xiang Suoshu sodium alginate aqueous solution, the uniform hydrogel of sodium alginate-gelatin is made;
Step 3, it selects Cowhells tendon as raw material, after extracting collagen, removes end peptide, obtain I-type collagen;
Step 4, it selects the epiphysis end of calf Bones and joints head or shin bone as collagen material, collagen material is carried out by modifying agent
It is modified, after calcining, obtain calcined bone powder;
Step 5, the I-type collagen and the calcined bone powder is added in Xiang Suoshu hydrogel, stirs evenly, is prepared into
To three dimensional biological marking ink.
Optionally, the step 3 includes:
Cowhells tendon is chosen, cleans up, using pulverizer, the Cowhells tendon of bulk is cut into small sized Cowhells tendon;
Using homogenate extraction technical treatment Cowhells tendon, acetic acid is added into extract, obtains natural collagen coarse fodder, using
Centrifugation, the purifies and separates for step of dialysing obtain I-type collagen coarse fodder;
By the I-type collagen coarse fodder by pepsin, the type i collagen for removing the non-immunogenicity of end peptide is obtained
Albumen, the as described I-type collagen.
Optionally, the step 4 includes:
It selects the epiphysis end of calf Bones and joints head or shin bone as collagen material, is cleaned through deionized water, with physics or biology
The method of chemistry removes collagen substantially;
Using diammonium hydrogen phosphate, ammonium dihydrogen phosphate or phosphoric acid as modifying agent, to the bone of the collagen material of basic removal collagen
Salt is modified, and the calcium phosphorus atoms ratio Ca/P of its bone salts is made to be down to 1.5~1.67;
Through 800 DEG C of -1100 DEG C of high-temperature calcinations, immunogenicity is removed to get the calcined bone powder.
Optionally, the step 5 includes:
By the ratio of hydrogel/I-type collagen mass ratio 0.2-0.5, the I type glue is added in Xiang Suoshu hydrogel
Former albumen is uniformly mixed, and obtains collagen hydrogel;
Select porosity up to 70%-80%, the calcined bone powder that granularity is 60-100 mesh, according to calcined bone powder/collagen water-setting
The ratio of glue relative mass ratio 0.10-0.25, is added collagen hydrogel for calcined bone powder, is uniformly mixed, and adjust range of viscosities
For 30-107mPa/s, pH to 6.5-7.5 is to get the three dimensional biological marking ink.
Second aspect, the embodiment of the invention provides a kind of three dimensional biological marking ink, the three dimensional biological marking ink
It is to be prepared by method described in first aspect.
The third aspect, the embodiment of the invention provides three dimensional biological marking inks described in a kind of second aspect to beat in three-dimensional
Print the application in bone-grafting material, comprising:
In gnotobasis, three dimensional biological is carried out using the three dimensional biological marking ink and prints plastotype, freeze-drying obtains
To bone grafting material product.
Optionally, carrying out three dimensional biological printing plastotype using the three dimensional biological marking ink includes:
After the sterilized processing of three dimensional biological marking ink, cell and biotic factor are loaded;
Three dimensional biological printing plastotype is carried out using the three dimensional biological marking ink for being loaded with cell and biotic factor;
Wherein, environment temperature when three dimensional biological printing plastotype is 37 DEG C or so.
Optionally, the crosslinking agent used when the three dimensional biological prints plastotype is the CaCl of 100mmol/L2Solution.
The preparation method of the three dimensional biological marking ink, three dimensional biological marking ink that provide through the embodiment of the present invention and its
Using preparing collagen hydrogel with gelatin, sodium alginate and I-type collagen, load calf calcined bone powder, three be prepared
Vitamins marking ink has good biocompatibility, certain mechanical strength and three-dimensional porous stereochemical structure.Three-dimensional is raw
In object marking ink, the compound forging bone of collagen be conducive to osteoblast adherency proliferation, collagen its in several weeks
Interior ingredient will be slowly absorbed, no barrier action.Using CaCl2Solution can will be black in 3-5 minutes as crosslinking agent
Water is changed into homogeneous solid phase, and curing time is suitable for, and product has certain intensity and elasticity.Through the invention, phase is solved
The bad problem of the biocompatibility or plasticity of bone grafting material product in the technology of pass, it is raw using the three-dimensional of the embodiment of the present invention
Bone-grafting material made of object marking ink mentions for porous apatite ceramics are as bone-grafting material made from marking ink
High biocompatibility, improve for the bone-grafting material made from the small bovine calcined bone bone grafting material product plasticity and
Clinical manipulation.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes part of this application, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is collagen infrared signature spectrogram according to an embodiment of the present invention;
Fig. 2 is that the structure of the small bovine calcined bone three dimensional biological printing bone-grafting material of collagen gel according to an embodiment of the present invention is shown
It is intended to;
Fig. 3 is the X ray diffracting spectrum that confirmation bracket hydroxyapatite according to an embodiment of the present invention includes HA and β TCP,
Wherein DBB/Col is small bovine calcined bone and collagen group, and DBB is small bovine calcined bone group;
Fig. 4 is the schematic diagram of material porosity characteristic according to an embodiment of the present invention;
Fig. 5 is that cell tight depends on apatite particle life in three dimensional biological type-script structure according to an embodiment of the present invention
Long schematic diagram;
Fig. 6 is the signal that cell is uniformly distributed growth in three dimensional biological print carriage network according to an embodiment of the present invention
Figure;
Fig. 7 be in three dimensional biological print carriage network according to an embodiment of the present invention cell skeletonization to the schematic diagram of differentiation.
Specific embodiment
The feature and exemplary embodiment of various aspects of the invention is described more fully below, in order to make mesh of the invention
, technical solution and advantage be more clearly understood, with reference to the accompanying drawings and embodiments, the present invention is further retouched in detail
It states.It should be understood that described herein, the specific embodiments are only for explaining the present invention, is not intended to limit the present invention.For ability
For field technique personnel, the present invention can be implemented in the case where not needing some details in these details.It is right below
The description of embodiment is used for the purpose of better understanding the present invention to provide by showing example of the invention.
It should be noted that, in this document, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or equipment for including a series of elements not only includes those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or equipment institute it is intrinsic
Element.In the absence of more restrictions, the element limited by sentence " including ... ", it is not excluded that including described want
There is also other identical elements in the process, method, article or equipment of element.
Embodiment 1
A kind of preparation side of the three dimensional biological marking ink of three dimensional biological printing bone-grafting material is provided in the present embodiment
Method, method includes the following steps:
Step 1, sodium alginate aqueous solution is prepared;
Molecular weight is selected to be dissolved in distilled water, while mechanical stirring in the sodium alginate of 5000-50000 or so, ultrasound is mixed
It is even, obtained aqueous solution concentration 10-30mg/ml.
Step 2, the uniform hydrogel of sodium alginate-gelatin is made using the aqueous solution of step 1;
The viscosity for the aqueous solution that a certain amount of gelatin regulating step 1 obtains, then mixed liquor is stirred under heating and forms water
Gel is cooled to room temperature.Change the ratio of gelatin/sodium alginate, the deflection during colloid storage is adjusted.
Step 3, it selects fresh Cowhells tendon as raw material, after extracting collagen, removes end peptide, obtain type i collagen egg
It is white;
Fresh Cowhells tendon is chosen, is cleaned up, using pulverizer, the Cowhells tendon of bulk is cut into small size raw material;Make
Cowhells tendon is handled with use of flash extraction technology, then uses a certain amount of acetic acid, tentatively obtains natural collagen coarse fodder, using centrifugation, dialysis step
Purifies and separates, obtain type i collagen coarse fodder;The type i collagen coarse fodder of preparation is passed through into pepsin again, obtains and removes end peptide
The I-type collagen of non-immunogenicity.I-type collagen is made of three peptide chains, wherein having α (I), α (II) chain, a β
Chain.The final collagen that obtains is usually white powder object, and relative molecular mass is from about 2kD to 300kD etc., insoluble in cold
Water, diluted acid, dilute alkaline soln have good water-retaining property and emulsibility, and infrared signature spectrogram is detailed in Fig. 1, and wherein curve a is represented
Collagen group, curve b represent Atelocollagen group.
Step 4, select the epiphysis end of newly calve Bones and joints head or shin bone as collagen material, by modifying agent to collagen material
Row is modified, and calcines the modified immunogenicity for completely removing bone material, obtains calcined bone powder;
Selecting the epiphysis end of calf Bones and joints head or shin bone is collagen material, is cleaned through deionized water, with physics or bioid
Method removes collagen substantially.Using diammonium hydrogen phosphate, ammonium dihydrogen phosphate or phosphoric acid as modifying agent, to the matrix (bone of bone
Salt) it is modified, so that the Ca/P atomic ratio of bone matrix is down to 1.5~1.67.Through 800 DEG C of -1100 DEG C of high-temperature calcinations, completely remove
Immunogenicity;Meanwhile HA/ β-is changed by HA by X-ray diffraction (XRD) map visible bone matrix crystal phase of bone block calcining front and back
The double structure of TCP.Electron scanning Electronic Speculum (SEM) observation show calcined bone powder not only containing 300 μm~500 μm macropore but also contain
There is micron-sized microcellular structure.
Step 5, I-type collagen and calcined bone powder are added in the hydrogel prepared to step 2, it is three-dimensional to stir evenly preparation
Biometric print ink.
Hydrogel is mixed into I-type collagen according to mass ratio 0.2-0.5 ratio, obtains collagen hydrogel.Select hole
Rate (volume ratio) reaches 70%-80%, and granularity is the calf calcined bone powder of 60-100 mesh, according to calcined bone powder/collagen hydrogel phase
To the ratio of mass ratio 0.10-0.25, collagen hydrogel is added in calcined bone powder, is uniformly mixed and is prepared into three dimensional biological stamping ink
Water, by adjusting the mode of mixed proportion and environment temperature, adjustment range of viscosities is 30-107mPa/s;Adjust pH to 6.5-
7.5.In addition, changing the additional proportion of calcined bone powder, the deflection during colloid is stored and intensity is adjusted.
Embodiment 2
A kind of three dimensional biological of three dimensional biological printing bone-grafting material provided by embodiment 1 is be provided in the present embodiment
The three dimensional biological marking ink that the preparation method of marking ink is prepared.
Wherein, state in use three dimensional biological marking ink carry out when three dimensional biological printing plastotype the crosslinking agent that uses for
The CaCl of 100mmol/L2Solution.
The collagen gel system that the three dimensional biological marking ink of the present embodiment has is with gelatin/acid sodium and I-type collagen
The network structure for forming interpenetrating is bracket, and porous C a-P bone meal is filled in this network structure, in system cell and biology because
It is sub then using protein gel body as carrier, substantially improve bioactivity, the mechanical performance of bone-grafting material, for bone defect healing treatment mention
For a kind of New Reclaiming Material.
Embodiment 3
A kind of three dimensional biological marking ink answering in three dimensional biological printing bone-grafting material is additionally provided in the present embodiment
With, comprising:
In gnotobasis, three dimensional biological printing plastotype is carried out using three dimensional biological marking ink prepared by embodiment 1 ,-
It is freeze-dried under the conditions of 20 DEG C, obtains bone-grafting material made of series initialization three-dimensional gelatine raw material.
Wherein, the crosslinking agent used when the three dimensional biological prints plastotype is the CaCl of 100mmol/L2Solution.
Embodiment 4
Another three dimensional biological marking ink is additionally provided in the present embodiment in three dimensional biological printing bone-grafting material
Using, comprising:
After the sterilized processing of three dimensional biological marking ink prepared by embodiment 1, with biotic factor or mixing with cells, that is, make
There must be the three dimensional biological marking ink of bioactie agent or cell;Various biological activity plantings can be printed as using the ink
Bone material.
Wherein, the crosslinking agent used when the three dimensional biological prints plastotype is the CaCl of 100mmol/L2Solution.Physiology
Homogeneous solid phase can be changed within 3-5 minutes through 100mmol/L CaCl2 solution crosslinking at 37 DEG C of body temperature, there is some strength and bullet
Property.
The curing time of material is affected for three dimensional biological printing, should have suitable curing time, time mistake
It is short to will lead to premature solidification, block printing head;Curing time is too long, then causes plastotype undesirable.Ideal curing time answers
When in 7-10 minutes this section.This is because bone meal has a large amount of holes, adsorption moisture, experiment shows that 10%- is added
When 25% calf calcined bone powder, compression strength 0.1-0.3Mpa or so has hydroxyapatite/two kinds of β tricalcium phosphate object phase
(Fig. 3), porosity 60%-80%, pore size is about in 150 μm of -500um (Fig. 4).Biocompatibility in vitro experiments have shown that, three
In vitamins type-script support network, osteoblast is uniformly distributed in supporting structure (Fig. 5), and cell tight depends on phosphorus ash
Stone particle grows (Fig. 6), and to skeletonization to differentiation (Fig. 7).
Bioactivity bone-grafting material prepared by the present embodiment has certain form and mechanical strength, while can be sustained promotion
The bioactie agent of bon e formation, and it is able to satisfy the design requirement of clinical bone defect healing personalization, it is the defects such as Maxillary region bone
Repairing and treating a kind of New Reclaiming Material is provided.
In embodiment 3 and embodiment 4, gelatin, sodium alginate is selected to make aqueous gel body, with good biocompatibility
CaCl2Mosanom is formed through network structure (gelatin and sodium alginate being formed except CaCl2's through network structure for crosslinking agent
Outside crosslinked action, there are also the complexings of Mg2+ and colloid, and hydrogel is collectively formed), and loaded using this gel network structure
I-type collagen and porous calf calcined bone powder.Three dimensional biological printing behaviour can be adjusted by adjusting colloid composition and concentration
Make temperature and crosslinking time, cell and bioactie agent is added, and adjust the addition of I-type collagen and calf calcined bone powder
Ratio can form serial three dimensional biological printing bone-grafting material, and have excellent bioactivity, biocompatibility, mechanical resistance
Property and sustained release performance.It can be widely used for periodontal, the reparation that the Bone Defect Repari reparation of plantation and alveolar ridge atrophy absorb and build again.
In conclusion the above embodiment of the present invention is poor for existing three dimensional biological printing bone-grafting material intensity, it is difficult to
The deficiencies of degradation, bioactivity is poor, lacks natural micropore structure, selecting modified calcining osseous granules is matrix, bright with mosanom-
Glue mixes I-type collagen and obtains collagen hydrogel, and collagen hydrogel is used to the CaCl of good biocompatibility2Crosslinking, cell
It can then be loaded in collagen hydrogel with biotic factor.Thus the bone-grafting material intensity printed is higher, is easy to degrade same
When have self-bone grafting characteristic.
It should be clear that the invention is not limited to specific configuration as described above and processing.For brevity,
It is omitted here the detailed description to known method.In the above-described embodiments, several specific step conducts have been described and illustrated
Example.But method process of the invention is not limited to described and illustrated specific steps, those skilled in the art can be with
It after understanding spirit of the invention, is variously modified, modification and addition, or the sequence between changing the step.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of preparation method of three dimensional biological marking ink characterized by comprising
Step 1, sodium alginate aqueous solution is prepared;
Step 2, gelatin is added in Xiang Suoshu sodium alginate aqueous solution, the uniform hydrogel of sodium alginate-gelatin is made;
Step 3, it selects Cowhells tendon as raw material, after extracting collagen, removes end peptide, obtain I-type collagen;
Step 4, the epiphysis end of calf Bones and joints head or shin bone is selected to change by modifying agent to collagen material as collagen material
Property, after calcining, obtain calcined bone powder;
Step 5, the I-type collagen and the calcined bone powder is added in Xiang Suoshu hydrogel, stirs evenly, is prepared three
Vitamins marking ink.
2. the method according to claim 1, wherein the step 3 includes:
Cowhells tendon is chosen, cleans up, using pulverizer, the Cowhells tendon of bulk is cut into small sized Cowhells tendon;
Using homogenate extraction technical treatment Cowhells tendon, acetic acid is added into extract, obtains natural collagen coarse fodder, using from
The heart, the purifies and separates for step of dialysing obtain I-type collagen coarse fodder;
By the I-type collagen coarse fodder by pepsin, the type i collagen egg for removing the non-immunogenicity of end peptide is obtained
It is white, the as described I-type collagen.
3. the method according to claim 1, wherein the step 4 includes:
It selects the epiphysis end of calf Bones and joints head or shin bone as collagen material, is cleaned through deionized water, with physics or biochemistry
Method remove collagen substantially;
Using diammonium hydrogen phosphate, ammonium dihydrogen phosphate or phosphoric acid as modifying agent, to the bone salts of the collagen material of basic removal collagen into
Row is modified, and the calcium phosphorus atoms ratio Ca/P of its bone salts is made to be down to 1.5~1.67;
Through 800 DEG C of -1100 DEG C of high-temperature calcinations, immunogenicity is removed to get the calcined bone powder.
4. the method according to claim 1, wherein the step 5 includes:
By the ratio of hydrogel/I-type collagen mass ratio 0.2-0.5, the type i collagen egg is added in Xiang Suoshu hydrogel
It is white, it is uniformly mixed, obtains collagen hydrogel;
Select porosity up to 70%-80%, the calcined bone powder that granularity is 60-100 mesh, according to calcined bone powder/collagen hydrogel phase
To the ratio of mass ratio 0.10-0.25, collagen hydrogel is added in calcined bone powder, is uniformly mixed, and adjusting range of viscosities is 30-
107mPa/s, pH are to 6.5-7.5 to get the three dimensional biological marking ink.
5. a kind of three dimensional biological marking ink, which is characterized in that the three dimensional biological marking ink is to pass through Claims 1-4
Any one of described in method preparation.
6. application of the three dimensional biological marking ink in 3 D-printing bone-grafting material, feature described in a kind of claim 5 exist
In including:
In gnotobasis, three dimensional biological is carried out using the three dimensional biological marking ink and prints plastotype, freeze-drying is planted
Bone material product.
7. application according to claim 6, which is characterized in that carry out three dimensional biological using the three dimensional biological marking ink
Printing plastotype includes:
After the sterilized processing of three dimensional biological marking ink, cell and biotic factor are loaded;
Three dimensional biological printing plastotype is carried out using the three dimensional biological marking ink for being loaded with cell and biotic factor;
Wherein, environment temperature when three dimensional biological printing plastotype is 37 DEG C or so.
8. application according to claim 6 or 7, which is characterized in that the friendship used when the three dimensional biological prints plastotype
Join the CaCl that agent is 100mmol/L2Solution.
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