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 PDF

Info

Publication number
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
Authority
CN
China
Prior art keywords
collagen
dimensional biological
bone
marking ink
hydrogel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910291189.7A
Other languages
Chinese (zh)
Inventor
吕培军
李箐
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University School of Stomatology
Original Assignee
Peking University School of Stomatology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University School of Stomatology filed Critical Peking University School of Stomatology
Priority to CN201910291189.7A priority Critical patent/CN109876190A/en
Priority to PCT/CN2019/086571 priority patent/WO2020206799A1/en
Publication of CN109876190A publication Critical patent/CN109876190A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Manufacturing & Machinery (AREA)
  • Dispersion Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Inorganic Chemistry (AREA)
  • Materials For Medical Uses (AREA)

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

The preparation method and applications of three dimensional biological marking ink
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.
CN201910291189.7A 2019-04-11 2019-04-11 The preparation method and applications of three dimensional biological marking ink Pending CN109876190A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201910291189.7A CN109876190A (en) 2019-04-11 2019-04-11 The preparation method and applications of three dimensional biological marking ink
PCT/CN2019/086571 WO2020206799A1 (en) 2019-04-11 2019-05-13 Method for preparing three-dimensional bioprinting ink and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910291189.7A CN109876190A (en) 2019-04-11 2019-04-11 The preparation method and applications of three dimensional biological marking ink

Publications (1)

Publication Number Publication Date
CN109876190A true CN109876190A (en) 2019-06-14

Family

ID=66937043

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910291189.7A Pending CN109876190A (en) 2019-04-11 2019-04-11 The preparation method and applications of three dimensional biological marking ink

Country Status (2)

Country Link
CN (1) CN109876190A (en)
WO (1) WO2020206799A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110393823A (en) * 2019-08-09 2019-11-01 苏州苏新瑞可医疗科技有限公司 A kind of hydrogel ink of 3D biometric print and preparation method thereof
CN110772669A (en) * 2019-11-04 2020-02-11 中南大学湘雅三医院 Biological ink for 3D printing of artificial skin
CN112870452A (en) * 2020-03-12 2021-06-01 深圳市第二人民医院(深圳市转化医学研究院) Manufacturing method of 3D printing gelatin-hydroxyapatite composite hydrogel scaffold
CN114366856A (en) * 2021-12-17 2022-04-19 同济大学 Method for compounding gelatin and collagen with 3D printing biological scaffold

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112220964A (en) * 2020-10-19 2021-01-15 西安点云生物科技有限公司 Composite biological ceramic powder, composite biological ceramic artificial bone prepared from composite biological ceramic powder and preparation method of composite biological ceramic artificial bone

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103920193A (en) * 2014-04-04 2014-07-16 北京大学口腔医院 Preparation method of bioactive factor loaded bone-like ceramic composite material
WO2018078130A1 (en) * 2016-10-28 2018-05-03 Paul Gatenholm Preparation and applications of 3d bioprinting bioinks for repair of bone defects, based on cellulose nanofibrils hydrogels with natural or synthetic calcium phosphate particles
CN108192941A (en) * 2018-03-07 2018-06-22 广州创尔生物技术股份有限公司 A kind of method of quality control of biologically active collagen
CN108297396A (en) * 2018-02-07 2018-07-20 华中科技大学鄂州工业技术研究院 A method of it squeezing out sedimentation type 3D printing and prepares three-dimensional bone tissue engineering scaffold
CN108888803A (en) * 2018-07-11 2018-11-27 蒋青 A kind of biological support and preparation method thereof, purposes and aquogel system
CN109381749A (en) * 2018-10-23 2019-02-26 杭州捷诺飞生物科技股份有限公司 Bone tissue reparation ink, composition, bracket and preparation method and kit

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102406965B (en) * 2011-12-01 2015-06-24 广西南宁博恩康生物科技有限公司 Injectable gel material for treating bone defect and preparation method thereof
JP6628416B2 (en) * 2014-08-04 2020-01-08 国立大学法人千葉大学 Cell culture method
KR20170097937A (en) * 2016-02-19 2017-08-29 포항공과대학교 산학협력단 Inkjet printer head assembly and method for fabricating hydrogel structure using inkjet printer comprising the same
CN107376017B (en) * 2017-08-24 2020-10-09 浙江大学 3D printed sodium alginate-I type collagen-ceramic composite scaffold, preparation method and application
CN108815574B (en) * 2018-07-17 2021-04-06 深圳市晶莱新材料科技有限公司 Bone repair hydrogel scaffold and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103920193A (en) * 2014-04-04 2014-07-16 北京大学口腔医院 Preparation method of bioactive factor loaded bone-like ceramic composite material
WO2018078130A1 (en) * 2016-10-28 2018-05-03 Paul Gatenholm Preparation and applications of 3d bioprinting bioinks for repair of bone defects, based on cellulose nanofibrils hydrogels with natural or synthetic calcium phosphate particles
CN108297396A (en) * 2018-02-07 2018-07-20 华中科技大学鄂州工业技术研究院 A method of it squeezing out sedimentation type 3D printing and prepares three-dimensional bone tissue engineering scaffold
CN108192941A (en) * 2018-03-07 2018-06-22 广州创尔生物技术股份有限公司 A kind of method of quality control of biologically active collagen
CN108888803A (en) * 2018-07-11 2018-11-27 蒋青 A kind of biological support and preparation method thereof, purposes and aquogel system
CN109381749A (en) * 2018-10-23 2019-02-26 杭州捷诺飞生物科技股份有限公司 Bone tissue reparation ink, composition, bracket and preparation method and kit

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHENGJIE WU等: "Bioprinting three-dimentinal cell-laden tissue constructs with controllable degradation", 《SCIENTIFIC REPORTS》 *
李箐等: "两种骨/胶原三维生物打印支架材料及其生物性的比较研究", 《第十六次全国口腔医药数字化学术会议暨中华口腔医学会第四届口腔医学计算机专业委员会第二次全体会议论文汇编》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110393823A (en) * 2019-08-09 2019-11-01 苏州苏新瑞可医疗科技有限公司 A kind of hydrogel ink of 3D biometric print and preparation method thereof
CN110772669A (en) * 2019-11-04 2020-02-11 中南大学湘雅三医院 Biological ink for 3D printing of artificial skin
CN112870452A (en) * 2020-03-12 2021-06-01 深圳市第二人民医院(深圳市转化医学研究院) Manufacturing method of 3D printing gelatin-hydroxyapatite composite hydrogel scaffold
CN114366856A (en) * 2021-12-17 2022-04-19 同济大学 Method for compounding gelatin and collagen with 3D printing biological scaffold

Also Published As

Publication number Publication date
WO2020206799A1 (en) 2020-10-15

Similar Documents

Publication Publication Date Title
CN109876190A (en) The preparation method and applications of three dimensional biological marking ink
EP1566186B1 (en) Apatite/collagen crosslinked porous material containing self-organized apatite/collagen composite and process for producing the same
ES2280969T3 (en) SUBSTITUTE MATERIAL FOR INORGANIC AND RESTORABLE BONES.
US6521246B2 (en) Inorganic shaped bodies and methods for their production and use
US8734822B2 (en) Composite shaped bodies and methods for their production and use
CN101444646B (en) Gelatin/calcium phosphate composite porous microsphere for bone repair and preparation method and usage of the porous microsphere
CN101564553B (en) Humanization active forging bone and preparation method thereof
CN111097068B (en) Bionic hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support and preparation method thereof
US20180093009A1 (en) Dimensionally Stable Molded Bone Replacement Element with Residual Hydraulic Activity
WO2007025698A2 (en) Osteoinductive material and method for the production thereof
CN100591365C (en) Inorganic resorbable bone substitute material
Suruagy et al. Physico-chemical and histomorphometric evaluation of zinc-containing hydroxyapatite in rabbits calvaria
JP2010531704A (en) Porous composite material, production method thereof and use thereof in regenerative medical devices
CN113350573A (en) Porous microsphere adhesive with osteoinductive capacity and preparation method thereof
US10512709B2 (en) Porous composite and bone regeneration material
RU2804689C2 (en) Method for producing a composite hydrogel formed in situ to replace osteochondral defects
JP2003210567A (en) Functionally graded composite material of absorptive calcium phosphate derived from living body cell and manufacturing method thereof
CN1621097A (en) Porous bone repairing material and manufacturing method thereof
KR20120113374A (en) The method of preparing ca/p mole ratio control of bone graft bioactive biphasic calcium phosphate compositions
CN109675099A (en) Tricalcium phosphate and calcium octahate phosphate composite bioceramic material and preparation method thereof
IL144448A (en) Inorganic shaped bodies and methods for their production and use

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20190614

RJ01 Rejection of invention patent application after publication