CN105797211A - Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel - Google Patents
Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel Download PDFInfo
- Publication number
- CN105797211A CN105797211A CN201610202115.8A CN201610202115A CN105797211A CN 105797211 A CN105797211 A CN 105797211A CN 201610202115 A CN201610202115 A CN 201610202115A CN 105797211 A CN105797211 A CN 105797211A
- Authority
- CN
- China
- Prior art keywords
- hydrogel
- osteoblast
- rgd
- dialdehyde
- hyaluronic acid
- 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
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
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
-
- 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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
-
- 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
- A61L27/56—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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The invention provides a preparation method of hydrogel, osteoblast containing hydrogel and a preparation method of the osteoblast containing hydrogel.The preparation method of the hydrogel includes the following steps that S1, dialdehyde hyaluronic acid and RGD are provided; S2, the objective ratio needed by dialdehyde hyaluronic acid and RGD is obtained through the high-throughput screening technology; S3, dialdehyde hyaluronic acid and RGD in S2 are crosslinked to form the needed hydrogel with a three-dimensional porous structure.The concentrations needed by the dialdehyde hyaluronic acid water solution and the RGD water solution and the objective ratio for mixing of the dialdehyde hyaluronic acid water solution and the RGD water solution are rapidly obtained through the high-throughput screening technology, and therefore the hydrogel material with the needed hole diameter size and porosity can be obtained, the hydrogel is beneficial for cell growth, especially, osteoblast can obtain better growth, differentiation and propagation conditions in the hydrogel material, the cell survival rate is effectively increased accordingly, and the bone repair effect is effectively improved accordingly.
Description
[technical field]
The present invention relates to bioengineered tissue technical field, particularly relate to the preparation method of a kind of hydrogel, containing osteoblast hydrogel and preparation method thereof.
[background technology]
Bone is one of most important tissue of human body, is responsible for support, the storage function such as calcium, hemopoietic.As the support of human body, bone injury is always up a big problem of the puzzlement mankind.Current clinical treatment bone injury mainly adopts the means such as autologous bone transplanting, allogenic bone transplantation and artificial bone's transplanting, but these methods are subject to source less and serious immunologic rejection effect etc. affects, and clinical practice is limited.In recent years because of the three-dimensional porous structure of its uniqueness, the research utilizing hydrogel material treatment bone injury receives the extensive concern of people, it is hydrogel prepared by raw material in particular by natural polymers such as alginic acid, collagen protein, fibroin albumens, because it has abundance, good biocompatibility and the advantage such as biodegradable, treat field in bone injury and have broad application prospects.
Hydrogel is hydrophilic polymer network, and hydrogel can absorb substantial amounts of moisture, but owing to the physical crosslinking between polymer chain and chemical crosslinking effect are without being dissolved in the water, it remains to the shape that maintenance is certain after swelling.In regenerative medicine field, injectable hydrogel can be used for bioactive molecule controlled release, the embedding of cell and is used as the aspect such as tissue stent material.Hydrogel original position in vivo is formed, and thus can avoid that the height in surgical procedures is traumatic, also can make corrective surgery wound mouth healing acceleration, to reduce patient painful and reduce medical expense etc..Mostly existing hydrogel material is that the inorganic constituents that hydroxyapatite, calcium ion etc. can promote mineralization of skeleton is incorporated in gel three-dimensional network structure by covalent bond or the mode such as blended, utilizes the slow releasing of gel or as tissue engineering bracket material for Bone Defect Repari.Prior art is generally adopted after hydrogel material surface modification grafting, inoculates osteoblast in hydrogel surface, but the method for this inoculation has certain limitation, such as: the material Bone Defect Repari obtained is limited in one's ability, hydrogel material is poor for the adhesion of cell, and the porosity of hydrogel, pore size and mechanical balance cannot meet.It would therefore be highly desirable to the preparation method that a kind of novel hydrogel is provided.
[summary of the invention]
For overcoming the porosity of current hydrogel and the problem that pore size is not good, the present invention provides the preparation method of a kind of hydrogel, containing osteoblast hydrogel and preparation method thereof.
The present invention solves the preparation method that a technical scheme of above-mentioned technical problem is to provide a kind of hydrogel, it comprises the following steps that step S1, dialdehyde hyaluronic acid and RGD (peptidesarginine-glycine-asparticacid, arginine-glycine-aspartic acid polypeptide) are provided；Step S2, utilizes High Throughput Screening Assay to obtain the target proportioning needed for dialdehyde hyaluronic acid and RGD；And step S3, there is needed for being cross-linked to form by the step S2 target proportioning obtained with RGD by dialdehyde hyaluronic acid the hydrogel of three-dimensional porous structure.
Preferably, above-mentioned steps S1 farther includes: by soluble in water for 5g-15g hyaluronate sodium, and be added thereto to 2.5g-7.5g sodium metaperiodate, obtains reactant liquor, and place reaction liquid into lucifuge reaction 2-24h under room temperature after it dissolves；And in reactant liquor, add the termination lucifuge reaction of 8-12ml ethylene glycol, by product after ethanol precipitates and washs, dry under 38 DEG C of-42 DEG C of conditions, it is thus achieved that required dialdehyde hyaluronic acid.
Preferably, in above-mentioned steps S2, High Throughput Screening Assay is utilized to form dialdehyde hyaluronic acid and RGD hydrogel micro-array chip.
Preferably, above-mentioned steps S3 farther includes: dialdehyde hyaluronic acid and RGD are each configured to the dialdehyde hyaluronic acid aqueous solution that mass concentration is 8-12mg/ml and the RGD aqueous solution that mass concentration is 0.5-10.5mg/ml；And utilize High Throughput Screening Assay that with 1: 9-9: 1 volume ratio, dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution are mixed to form mixed solution respectively；Controlling reaction temperature is 36.5 DEG C-37.5 DEG C, makes dialdehyde hyaluronic acid aqueous solution prepare the hydrogel material with three-dimensional porous structure after reacting 0.3h-0.7h with RGD aqueous solution.
The present invention solves that the another technical scheme of above-mentioned technical problem is a kind of preparation method containing osteoblast hydrogel, it comprises the following steps that step T1, it is provided that dialdehyde hyaluronic acid and RGD；Step T2, utilizes High Throughput Screening Assay to obtain the target proportioning needed for dialdehyde hyaluronic acid and RGD；And step T3, after dialdehyde hyaluronic acid is mixed by the target proportioning of the acquisition of step T2 with RGD, add needed for osteoblast suspension is mixed to form and there is three-dimensional porous structure containing osteoblastic hydrogel.
Preferably, above-mentioned steps T3 farther includes: configuration cell concentration is 5.0 × 105-6.0×107The osteoblast suspension of individual/ml is with standby；Configuration quality concentration is the dialdehyde hyaluronic acid aqueous solution of 8-12mg/ml and RGD aqueous solution that mass concentration is 0.5-10.5mg/ml, and after described dialdehyde hyaluronic acid aqueous solution is mixed with 1: 9-9: 1 volume ratio with described RGD aqueous solution, join and described osteoblast suspension carries out mix and after cross-linking reaction, prepare and there is three-dimensional porous structure containing osteoblastic hydrogel.
Preferably, described step T3 farther includes: the reaction temperature of described cross-linking reaction is 36.5 DEG C-37.5 DEG C, and the response time is 0.3h-0.7h.
The present invention solves that the another technical scheme of above-mentioned technical problem is a kind of containing osteoblast hydrogel, the described employing containing the osteoblast hydrogel preparation method containing osteoblast hydrogel described above is prepared from.
Preferably, the described maximum elastance containing osteoblast hydrogel is 0.5 × 102Pa-1×104Pa, in the medium that pH value is 7.4, the described equilibrium swelling ratio containing osteoblast hydrogel is 330%-810%, and in the medium that pH value is 1.2, the described equilibrium swelling ratio containing osteoblast hydrogel is 120%-230%.
Preferably, the described porosity containing osteoblast hydrogel is 79%-93%, and the described pore size containing osteoblast hydrogel is 80-200 μm.
Relative to prior art, in the preparation method of hydrogel provided by the present invention and the preparation method containing osteoblast hydrogel, by utilizing the High Throughput Screening Assay can according to different purposes, quickly obtain the target proportioning to the concentration needed for dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution and mixing thereof, it is hereby achieved that have the hydrogel material of more excellent pore size and porosity, it is conducive to Growth of Cells, especially osteoblast can obtain more excellent growth at hydrogel material, differentiation and proliferation conditions, thus being effectively improved cell survival rate and Bone Defect Repari effect, the treatment cycle of Bone Defect Repari and the cure rate of Bone Defect Repari treatment can also be shortened further.
[accompanying drawing explanation]
Fig. 1 is the preparation method schematic flow sheet of first embodiment of the invention hydrogel.
Fig. 2 is the second embodiment of the invention preparation method schematic flow sheet containing osteoblast hydrogel.
Fig. 3 is that the present invention utilizes High Throughput Screening Assay to prepare the hydrogel stereoscan photograph that the method for the hydrogel that can be used for Oesteoblast growth prepares.
Fig. 4 utilizes what obtain prepared by different RGD concentration to cultivate the cell survival rate block diagram after 24h containing osteoblast hydrogel in the present invention preparation method containing osteoblast hydrogel.
Fig. 5 is the cell survival rate block diagram after utilizing the prepared hydrogel containing osteoblast obtained of different RGD concentration to co-culture 7d (day, sky) shown in Fig. 4.
[detailed description of the invention]
In order to make the purpose of the present invention, technical scheme and advantage are clearly understood, below in conjunction with accompanying drawing and embodiment, the present invention are further elaborated.Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
Referring to Fig. 1, the preparation method that first embodiment of the invention provides a kind of hydrogel, it comprises the following steps that
Step S1, it is provided that dialdehyde hyaluronic acid and RGD；Step S2, utilizes High Throughput Screening Assay to obtain the target proportioning needed for dialdehyde hyaluronic acid and RGD；And
Step S3, has the hydrogel of three-dimensional porous structure with RGD needed for being cross-linked to form by the step S2 target proportioning obtained by dialdehyde hyaluronic acid.
The present invention some preferably in embodiment, above-mentioned steps S1 includes utilizing sodium metaperiodate and hyaluronate sodium to prepare dialdehyde hyaluronic acid: be specially soluble in water for 5g-15g hyaluronate sodium, and it is added thereto to 2.5g-7.5g sodium metaperiodate, reactant liquor is obtained after it dissolves, and place reaction liquid into lucifuge reaction 2-24h under room temperature, after completing lucifuge reaction, reactant liquor adds 8-12ml ethylene glycol and terminates reaction, by product after ethanol precipitates and washs 2-5 time, insert and 38 DEG C of-42 DEG C of vacuum drying ovens are dried to obtain dialdehyde hyaluronic acid powder.
After above-mentioned step S1 can farther including be dissolved in the deionized water of 500ml by 8g-12g hyaluronic acid, then to adding 4g-6g sodium metaperiodate in it.
Hyaluronic acid and sodium metaperiodate react when lucifuge, and sodium metaperiodate can promote hyaluronic C-C bond fission and generate new functional group aldehyde radical in C-C bond fission place, and aldehyde radical can to hyaluronic modifying surface.
In the present invention, the time that hyaluronic acid and sodium metaperiodate lucifuge are reacted can be specially 2h, 4.5h, 6h, 7h, 7.5h, 10.5h, 11.8h, 12h, 16h, 22.5h or 24h, difference according to the response time can obtain the dialdehyde hyaluronic acid of different oxidizability, wherein, it can be 16%-82% that hyaluronic acid and the lucifuge of sodium metaperiodate react the hyaluronic oxidizability of the dialdehyde obtained, the present invention some preferably in embodiment, the hyaluronic oxidizability of described dialdehyde can be also 16%-21%, 21%-47%, 47%-73%, 47%-61% or 73%-82% etc..
In some preferably embodiments, in above-mentioned step S1, lucifuge at hyaluronic acid with sodium metaperiodate is reacted the ethylene glycol that can pass through to add 9.6ml-10.8ml and is terminated reaction, by product after ethanol precipitates and washs 3-4 time, insert drying in 38 DEG C of-42 DEG C of vacuum drying ovens and obtain dialdehyde hyaluronic acid powder.
In some embodiments that the present invention is other, described dialdehyde hyaluronic acid can directly buy acquisition, it is not necessary to adopts above-mentioned preparation process.
In above-mentioned steps S2, target proportioning needed for above-mentioned dialdehyde hyaluronic acid and RGD be applicable to the hydrogel structure making cell (such as osteoblast) realize propagation, differentiation and climbing, and construction features is relevant to the pore size of hydrogel structure and porosity etc. for it.Described target proportioning also can for prepare the dialdehyde hyaluronic acid of hydrogel and the more excellent proportioning of RGD, adopt the hydrogel structure that described target proportioning prepares can require to match to the mechanical property of hydrogel structure, swelling behavior and pH sensitivity etc. with patient affected part, thus obtaining preferably ratio range.
In some preferably embodiments, in above-mentioned steps S2, described High Throughput Screening Assay is specially formation dialdehyde hyaluronic acid and RGD hydrogel micro-array chip, wherein, hydrogel micro-array chip is prepared by inkjet printing methods, mixture will be configured to according to different target proportionings containing dialdehyde hyaluronic acid solution and RGD solution, and mixture printed on substrate and cause cross-linked polymeric to form the micro-point of hydrogel, the micro-point of each hydrogel contains dialdehyde hyaluronic acid and the RGD of the target proportioning of different mixing.After obtaining hydrogel micro-array chip, described High Throughput Screening Assay comprises the steps:
Step P1, osteoblast is placed on hydrogel micro-array chip and cultivates 2d, use automatization's high flux screening platform to nuclear targeting (6-diamidino-2-phenylindole, DAPI) cell counts automatically, thus can determine that the micro-point of 150-300 candidate's hydrogel being conducive to Oesteoblast growth, wherein, the micro-point of hydrogel is prepared from by dialdehyde hyaluronic acid solution and RGD solution crosslinking.And
Step P2, proceed 4d (day, my god), 6d and 10d cultivate after, repeat step P1 to use automatization's high flux screening platform nuclei dyeing cytochrome is counted automatically, according to osteoblastic growing state in the test result analysis micro-point of 150-300 candidate's hydrogel, and choose wherein required candidate's hydrogel, the final target proportioning obtaining this required preferably candidate hydrogel.
In some preferably embodiments, in above-mentioned steps S3, specifically include and dialdehyde hyaluronic acid and RGD are each configured to the aqueous solution that mass concentration is 8-12mg/ml and 0.5-10.5mg/ml, utilize in the different sample cells that two kinds of solution join on substrate with 1: 9-9: 1 volume ratio by High Throughput Screening Assay respectively and form mixed solution, wherein, the volume of mixed solution is 0.2ml-0.7ml；Controlling reaction temperature is 36.5 DEG C-37.5 DEG C, prepares hydrogel material after making two kinds of solution hybrid reaction 0.3h-0.7h.
In described step S3, further dialdehyde hyaluronic acid and RGD can be each configured to the aqueous solution that mass concentration is 9-11mg/ml and 0.8-1.2mg/ml.The concentration of dialdehyde hyaluronic acid aqueous solution is more excellent in 9.3mg/ml, 10.4mg/ml, 10.9mg/ml in the present invention, and the concentration of RGD aqueous solution is more excellent in 0.82mg/ml, 0.97mg/ml, 1.07mg/ml, 1.14mg/ml, 1.2mg/ml, 1.5mg/ml, 2.7mg/ml, 5.5mg/ml, 6.9mg/ml, 8.7mg/ml or 9.6mg/ml, in the present invention, the concentration optimum of described dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution is 10mg/ml and 1mg/ml.
In some preferably embodiments, in above-mentioned steps S3, reaction temperature can be preferably 36.5 DEG C, 36.7 DEG C, 36.8 DEG C, 36.9 DEG C, 37.0 DEG C or 37.1 DEG C, 37.3 DEG C or 37.5 DEG C, prepares hydrogel material after making two kinds of solution hybrid reaction 0.38h, 0.4h, 0.63h, 0.5h, 0.57h or 0.6h.The final hydrogel porosity obtained and pore size thereof may be impacted by different reaction temperatures and response time.
Referring to Fig. 2, second embodiment of the invention provides a kind of preparation method containing osteoblast hydrogel, and it comprises the steps:
Step T1, it is provided that dialdehyde hyaluronic acid and RGD；
Step T2, utilizes High Throughput Screening Assay to obtain the target proportioning needed for dialdehyde hyaluronic acid and RGD；And
Step T3, after being mixed by the target proportioning of the acquisition of step T2 with RGD by dialdehyde hyaluronic acid, adds needed for osteoblast suspension is mixed to form and has three-dimensional porous structure containing osteoblastic hydrogel.
In the present embodiment, the concrete steps of above-mentioned steps T1 and step T2 are identical with the concrete steps of the step S1 of first embodiment of the invention and step S2, do not repeat them here.Wherein, in above-mentioned steps T2, described dialdehyde hyaluronic acid and the target proportioning needed for RGD are the more excellent proportioning of dialdehyde hyaluronic acid and the RGD selected according to different purposes, and described required target proportioning is preferably 1: 9-9: 1 volume ratio.In some preferred embodiments, the volume ratio of described dialdehyde hyaluronic acid aqueous solution and described RGD aqueous solution is more preferably 1.5: 9-9: 2,4: 9-3: 1,5: 9-9: 5,2: 3-3: 2,1: 1-1.5: 1 etc., also may further be 2: 9,4: 9,7: 9,3: 2,4.4: 1,7: 1 or 8.4: 1 etc..
In above-mentioned steps S3, specifically include: take patient bone reparation or the osteoblast at bone collection position, being configured to cell concentration with DMEM in high glucose (DulbeccoModifiedEagleMedium, a kind of culture medium containing each seed amino acid and glucose) culture fluid is 5.0 × 105-6.0×107The suspension of individual/ml, is each configured to the aqueous solution that mass concentration is 8-12mg/ml and 0.5-10.5mg/ml by dialdehyde hyaluronic acid and RGD；Being joined with 1: 9-9: 1 volume ratio respectively by two kinds of solution in the different sample cells on substrate, wherein, the volume of mixed solution is 0.2ml-0.7ml；Controlling reaction temperature is 36.5 DEG C-37.5 DEG C, after osteoblast suspension, dialdehyde hyaluronic acid and RGD solution hybrid reaction 0.3h-0.7h, prepares acquisition and has three-dimensional porous structure, the osteoblastic hydrogel material of embedding in situ.In some preferred embodiments, the cell concentration of described osteoblast suspension can be 5.0 × 105Individual/ml, 6.7 × 105Individual/ml, 8.9 × 105Individual/ml, 1.8 × 106Individual/ml, 3.7 × 106Individual/ml, 6.0 × 106Individual/ml, 6.9 × 106Individual/ml, 9.4 × 106Individual/ml, 1.1 × 107Individual/ml, 2.5 × 107Individual/ml, 4.5 × 107Individual/ml or 6.0 × 107Individual/ml.
In some preferably embodiments, in above-mentioned steps T3, further dialdehyde hyaluronic acid and RGD can be each configured to the aqueous solution that mass concentration is 9-11mg/ml and 0.8-1.2mg/ml.The concentration of dialdehyde hyaluronic acid aqueous solution is more excellent in 9.3mg/ml, 10.4mg/ml, 10.9mg/ml in the present invention, and the concentration of RGD aqueous solution is more excellent in 0.82mg/ml, 0.97mg/ml, 1.07mg/ml, 1.14mg/ml, 1.2mg/ml, 1.5mg/ml, 2.7mg/ml, 5.5mg/ml, 6.9mg/ml, 8.7mg/ml or 9.6mg/ml, in the present invention, the concentration optimum of described dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution is 10mg/ml and 1mg/ml.
Specifically, in one preferably embodiment, in above-mentioned osteoblast suspension, cell concentration is 6.0 × 106Individual/ml.Wherein, concentration respectively 9.3mg/ml and 0.97mg/ml of dialdehyde hyaluronic acid solution and RGD solution.Utilize High Throughput Screening Assay to obtain and be conducive to the preferred target proportioning of Oesteoblast growth, and be 6.0 × 10 by dialdehyde hyaluronic acid solution, RGD solution with cell concentration according to this target proportioning6Individual/ml osteoblast suspension mixing, and at 37 DEG C hybrid reaction 0.5h.
In first embodiment of the invention with the second embodiment, between aldehyde radical and the amido of RGD in dialdehyde hyaluronic acid, functional group interacts, thus forming porous hydrogel material, wherein, osteoblast is embedded among porous hydrogel material, compared with the osteoblast being seeded in hydrogel material surface, osteoblast can directly mix with the component forming hydrogel material, so that osteoblast is embedded in hydrogel material.
Third embodiment of the invention provides a kind of containing osteoblast hydrogel, the described hydrogel containing osteoblast can be used for osteoblastic cultivation and differentiation, described the preparation method containing osteoblast hydrogel provided in second embodiment of the invention can be adopted to be prepared acquisition containing osteoblast hydrogel.
The described maximum elastance containing osteoblast hydrogel is 0.5 × 102Pa-1×104Pa, the present invention some preferably in embodiment, the described maximum elastance containing osteoblast hydrogel is more preferably 1 × 102Pa-6×103Pa。
In the medium that pH value is 7.4, the described equilibrium swelling ratio containing osteoblast hydrogel is 330%-810%, the present invention some preferably in embodiment, the described equilibrium swelling ratio containing osteoblast hydrogel can be 400%-760%, 450%-640%, 500%-740% or 320%-540% etc..
In the medium that pH value is 1.2, the described equilibrium swelling ratio containing osteoblast hydrogel is 120%-230%, the present invention some preferably in embodiment, the described equilibrium swelling ratio containing osteoblast hydrogel can be 130%-220%, 175%-215%, 189%-217% or 197%-209% etc..
Refer to Fig. 3, it is 79%-93% in the porosity containing osteoblast hydrogel adopting described in second embodiment of the invention the preparation method containing osteoblast hydrogel to prepare gained, the present invention some preferably in embodiment, the described porosity containing osteoblast hydrogel is 83%-91% further.The described pore size containing osteoblast hydrogel is 80-200 μm, the present invention some preferably in embodiment, the described pore size containing osteoblast hydrogel is 80-170 μm, 83-117 μm, 87-103 μm, 91-101 μm, 81-93 μm, 83-87 μm, 120-197 μm, 151-197 μm, 157-181 μm or 163-179 μm etc. further, the described pore size containing osteoblast hydrogel concretely 80 μm, 84 μm, 93 μm, 97 μm, 101 μm, 106 μm, 111 μm, 132 μm, 147 μm, 186 μm or 193 μm etc..
In the method utilizing High Throughput Screening Assay to prepare embedding in situ osteoblast hydrogel provided by the present invention, its raw material hyaluronic acid is as the linear mucopolysaccharide of one, it it is the important component of knuckle synovia and cartilage matrix, it is usually used in treatment of arthritis, osseous tissue is had good biocompatibility, and RGD is present in various kinds of cell epimatrix, using RGD as cross-linking agent, modified hyaluronic acid (dialdehyde hyaluronic acid) is cross-linked, form hydrogel material, it is possible to improve hydrogel material to osteoblastic viscosity.
Provided by the present invention by osteoblast embedding in situ in hydrogel material, and osteoblast Growth and Differentiation under optimum conditions can be made containing osteoblast hydrogel, thus playing Bone Defect Repari ability, along with completing of Bone Defect Repari.Degraded can be there is and discharged by body in the hydrogel material adopting the prepared dialdehyde hyaluronic acid obtained of the present invention and RGD.
High flux combined material triage techniques is utilized to filter out the required hydrogel material formula of applicable Oesteoblast growth differentiation rapidly in the present invention.
Below to adopting the preparation method of hydrogel provided by the present invention and the hydrogel material for preparing containing osteoblastic hydrogel preparation method to test accordingly.
Containing osteoblast hydrogel product performance test
Experimental group 1:
Adopting the method utilizing High Throughput Screening Assay to prepare embedding in situ osteoblast hydrogel provided in second embodiment of the invention, it specifically includes:
Step Q1, modified hyaluronic acid sodium: utilize sodium metaperiodate and hyaluronate sodium to prepare dialdehyde hyaluronic acid；It is specially soluble in water for 10g hyaluronate sodium, and it is added thereto to the sodium metaperiodate of 5g, after it dissolves, under room temperature, lucifuge reacts 2.5h, after completing lucifuge reaction, reactant liquor adds 10ml ethylene glycol and terminates reaction, by product after ethanol precipitates and washs 3 times, insert and 38 DEG C of vacuum drying ovens are dried to obtain dialdehyde hyaluronic acid powder.
Step Q2, High Throughput Screening Assay is utilized to obtain the target proportioning needed for combined material: wherein, target proportioning needed for combined material is the more excellent proportioning of dialdehyde hyaluronic acid and RGD (a kind of tripeptides being made up of arginine, glycine, aspartic acid), and proportioning is preferably 5.5: 1.4 volume ratios.And
Step Q3, dialdehyde hyaluronic acid and RGD is adopted to prepare the osteoblastic hydrogel of embedding in situ: being specially the osteoblast taking patient bone reparation or bone collection position, being configured to cell concentration with DMEM in high glucose (a kind of culture medium containing each seed amino acid and glucose) culture fluid is 6.0 × 106The suspension of individual/ml, is each configured to the aqueous solution that mass concentration is 10mg/ml and 1mg/ml by dialdehyde hyaluronic acid and RGD；Being joined with 5.5: 1.4 volume ratios respectively by two kinds of solution in the different sample cells on substrate, wherein, the volume of mixed solution is 0.5ml；Controlling reaction temperature is 37 DEG C, after osteoblast suspension, dialdehyde hyaluronic acid and RGD solution hybrid reaction 0.5h, prepares acquisition and has three-dimensional porous structure, the osteoblastic hydrogel material of embedding in situ.
Experimental group 2:
This experimental group and experimental group 1 are distinctive in that: the concentration of RGD is 2mg/ml.
Experimental group 3:
This experimental group and experimental group 1 are distinctive in that: the concentration of RGD is 5mg/ml.
Experimental group 4:
This experimental group and experimental group 1 are distinctive in that: the concentration of RGD is 10mg/ml.
In following experiments group 5-10, by adjusting the time of hyaluronic acid and the hybrid reaction of sodium metaperiodate, thus obtaining the dialdehyde hyaluronic acid product with different degree of oxidation.
Experimental group 5:
This experimental group and experimental group 1 are distinctive in that: after hyaluronic acid with sodium metaperiodate mixed dissolution, and the two mixed solution is lucifuge reaction 4.5h under room temperature.
Experimental group 6:
This experimental group and experimental group 1 are distinctive in that: after hyaluronic acid with sodium metaperiodate mixed dissolution, and the two mixed solution is lucifuge reaction 7h under room temperature.
Experimental group 7:
This experimental group and experimental group 1 are distinctive in that: after hyaluronic acid with sodium metaperiodate mixed dissolution, and the two mixed solution is lucifuge reaction 10.5h under room temperature.
Experimental group 8:
This experimental group and experimental group 1 are distinctive in that: after hyaluronic acid with sodium metaperiodate mixed dissolution, and the two mixed solution is lucifuge reaction 11.8h under room temperature.
Experimental group 9:
This experimental group and experimental group 1 are distinctive in that: after hyaluronic acid with sodium metaperiodate mixed dissolution, and the two mixed solution is lucifuge reaction 16h under room temperature.
Experimental group 10:
This experimental group and experimental group 1 are distinctive in that: after hyaluronic acid with sodium metaperiodate mixed dissolution, and the two mixed solution is lucifuge reaction 22.5h under room temperature.
On the basis of above-mentioned experimental group 1-10, in the present invention, following contrast groups is set and compares:
Contrast groups 1:
The hydrogel containing osteoblast prepares by the following method, and it specifically comprises the following steps that
(1) in sodium alginate aqueous solution, sodium metaperiodate is added, lucifuge stirring reaction 1-2h → obtain mixed liquor with addition ethylene glycol in sodium metaperiodate mixed aqueous solution to sodium alginate, continue reaction 0-2h → (2) and above-mentioned mixed liquor is carried out the sodium alginate of dialysis 3-7d → acquisitions oxidation → with 1: 1 mass ratio, sodium alginate and the sodium alginate of oxidation are configured to the fluid,matching that total sodium alginate concentration is 1-5%w/v, NaCl (the sodium chloride of 1mol/L is added to fluid,matching, the fluid,matching adding NaCl is added in the hole of culture plate by sodiumchloride) → (3), and after culture plate is placed in-80 DEG C of freezing 10h, it is complete to be placed in dehydrated alcohol by culture plate submergence, it is slowly added into 2%CaCl again2Carrying out cross-linking reaction acquisition cross-linking products → (4) in solution utilizes polypeptide RGD that cross-linking products carries out cross-linking modified (addition EDC/NHS (dichloroethanes/N-hydroxy-succinamide, Dichloroethane/N-hydroxysuccinimide) as cross-linking agent), obtaining Sodium Alginate Hydrogel Films → (5) the Sodium Alginate Hydrogel Films surface seeding osteoblast in surface modification of surface modification, final acquisition has osteoblastic Sodium Alginate Hydrogel Films.
Contrast groups 2:
The hydrogel containing osteoblast prepares by the following method, and it specifically comprises the following steps that
(1) in ice-water bath, type i collagen is dissolved in the hydrochloric acid solution that concentration is 0.003M, it is slowly stirred, it is configured to the collagen solution that concentration is 9mg/ml, the hyaluronic acid aqueous solution of the configuration 10mg/ml that is placed in temperature 4 DEG C stand for standby use → (2) also adds the sodium periodate solution of 20Mm wherein, dialyse 72h with deionized water after lucifuge stirring 8h, obtaining oxidizability is that the DHA of acquisition is configured to DHA solution → (4) of concentration 10.15mg/ml and is mixed and stirred for being formed uniformly mixed liquor by type i collagen solution and DHA solution by DHA → (3) of 60%, put into temperature 4 DEG C and be 1 × 10 to middle standing 12h → (5) by cell concentration8The osteoblast suspension of individual/ml is mixed homogeneously with above-mentioned mixed liquor, and is placed in 37 DEG C of constant incubators and stands 10-25min, obtain having osteoblastic composite.
Contrast groups 3:
Preparing by the following method containing osteoblastic hydrogel, it specifically comprises the following steps that
(1) with the phosphate buffer solution of pH=7.4 prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution → (2) be 9: 1-5: 5 mixing by volume by them, obtain hydrogel solution → (3) adds in this solution 5-10ng/ml concentration somatomedin and be coated with oxidation Na-alginate microsphere → (4) of somatomedin be uniformly mixed rapidly → cell concentration is 6 × 10 by (5)6The osteoblast suspension of individual/ml mixes with oxidized sodium alginate microsphere, prepares and has osteoblastic compound hydrogel material.
Contrast groups 4:
Contrast groups 4 and experimental group 1 are distinctive in that: the concentration of RGD aqueous solution is 0.1mg/ml.
Contrast groups 5:
Contrast groups 5 and experimental group 1 are distinctive in that: the concentration of RGD aqueous solution is 0.45mg/ml.
Contrast groups 6:
Contrast groups 6 and experimental group 1 are distinctive in that: the concentration of RGD aqueous solution is 15mg/ml.
Contrast groups 7:
Contrast groups 7 and experimental group 1 are distinctive in that: the concentration of RGD aqueous solution is 20mg/ml.
Contrast groups 8:
Contrast groups 8 and experimental group 1 are distinctive in that: the hyaluronic concentration of dialdehyde is 0.1mg/ml.
Contrast groups 9:
Contrast groups 9 and experimental group 1 are distinctive in that: the hyaluronic concentration of dialdehyde is 5.6mg/ml.
Contrast groups 10:
Contrast groups 10 and experimental group 1 are distinctive in that: the hyaluronic concentration of dialdehyde is 13.2mg/ml.
Contrast groups 11:
Contrast groups 11 and experimental group 1 are distinctive in that: the hyaluronic concentration of dialdehyde is 18.0mg/ml.
Contrast groups 12:
Contrast groups 12 and experimental group 1 are distinctive in that: the proportioning of dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution is 11: 1.
Contrast groups 13:
Contrast groups 13 and experimental group 1 are distinctive in that: the proportioning of dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution is 15: 1.
Contrast groups 14:
Contrast groups 14 and experimental group 1 are distinctive in that: the proportioning of dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution is 1: 11.
Contrast groups 15:
Contrast groups 15 and experimental group 1 are distinctive in that: the proportioning of dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution is 1: 15.
Contrast groups 16:
Contrast groups 16 and experimental group 1 are distinctive in that: sodium metaperiodate and hyaluronic acid lucifuge response time are 1h；
Contrast groups 17:
Contrast groups 17 and experimental group 1 are distinctive in that: sodium metaperiodate and hyaluronic acid lucifuge response time are 26h；
Contrast groups 18:
Contrast groups 18 and experimental group 1 are distinctive in that: sodium metaperiodate and hyaluronic acid lucifuge response time are 48h；
For in above-mentioned experimental group 1-10 and contrast groups 1-18 containing osteoblastic
Cell survival rate test in hydrogel
Method of testing:
Adopt tetramethyl azo azoles salt (MTT, methylthiazolyltetrazolium) method detection comparative survival rate of cells, will cultivate in culture fluid containing osteoblastic hydrogel material, and within every 3 days, change liquid once, to study osteoblastic proliferative conditions.
Wherein, the computing formula of cell survival rate is as follows:
Cell survival rate=(cell summary-dead cell number)/total cellular score × 100%
The following experimental group of concrete employing and contrast groups are tested:
Adopting experimental group 1-4 to test below, its variable quantity is the change of RGD concentration, RGD concentration of aqueous solution respectively 1mg/ml, 2mg/ml, 5mg/ml and 10mg/ml.
Test result:
Test group 1: adopt the method utilizing High Throughput Screening Assay to prepare embedding in situ osteoblast hydrogel provided by the present invention and other preparation method contrast test group；
Table 1, experimental group 1 of the present invention and osteoclast survival rate synopsis in contrast groups 1-3
Osteoclast survival rate is affected test group by test group 2:RGD concentration change；
Table 2, osteoclast survival rate synopsis in experimental group 1-4 of the present invention and contrast groups 4-7
Refer to Fig. 4 and Fig. 5, for the cell survival rate and the RGD concentration change figure that carry out cultivation 24h and 7d containing osteoblastic hydrogel in experimental group 1-4.
Test group 3: osteoclast survival rate is affected test group by the change of dialdehyde hyaluronic acid；
Table 3, experimental group 1 of the present invention and osteoclast survival rate synopsis in contrast groups 8-11
Test group 4: osteoclast survival rate is affected test group by the proportioning change of dialdehyde hyaluronic acid and RGD；
Table 4, experimental group 1 of the present invention and osteoclast survival rate synopsis in contrast groups 12-15
Test group 5: osteoclast survival rate is affected test group by the response time change of hyaluronic acid and sodium metaperiodate in the present invention；
Table 5, osteoclast survival rate synopsis in experimental group 1 of the present invention, 5-10 and contrast groups 16-18
Test result analysis:
In above-mentioned test group 1, experimental group 1 adopt the hydrogel that the preparation method containing osteoblast hydrogel that provides as second embodiment of the invention in obtains after cultivation 24h and 7d, its osteoblastic survival rate respectively 99% and 110%, osteoblast has had significant proliferation in hydrogel.
Compared with experimental group 1 of the present invention, after contrast groups 1 adopts sodium periodate oxidation sodium alginate, the sodium alginate of oxidation is cross-linked with RGD, obtain hydrogel material, wherein, when sodium alginate cross-links with RGD, need to add cross-linking agent EDC/NHS to cross-link, adopt contrast groups 1 obtain containing in osteoblastic hydrogel material, osteoblast is inoculated in the surface of hydrogel material, and osteoblastic proliferation function is significantly worse than osteoblast embedding in situ in hydrogel material by it.
Contrast groups 2 adopt type i collagen and the hyaluronic acid of oxidation carry out cross-linking the porosity obtaining hydrogel material less compared with experimental group 1-10 of the present invention with pore size, as shown in table 1, the hydrogel material that the preparation method provided in contrast groups 2 obtains is adopted to be unfavorable for osteoblastic propagation.
In contrast groups 3, the N-succinyl-chitosan solution by the oxidized sodium alginate solution of RGD grafting and 2.5% is 9: 1 mixing by volume, obtain hydrogel solution, this solution adds the somatomedin of 10ng/ml concentration and the oxidation Na-alginate microsphere of cladding somatomedin, is uniformly mixed rapidly；It is 6 × 10 by cell concentration6The osteoblast suspension of individual/ml mixes with oxidized sodium alginate microsphere, prepares containing osteoblastic compound hydrogel material.As shown in table 1, adopt contrast groups 3 to be obtained and be worse than experimental group 1-10 containing cell survival rate in osteoblastic compound hydrogel material, be unfavorable for osteoblastic growth.
The part material preparing hydrogel of above-mentioned contrast groups 1-3 employing and method step and experimental group 1 differ, after osteoblast and hydrogel co-culture 24h and 7d, the cell survival rate of its 7d is little with the cell survival rate change of 24h, visible, adopt contrast groups 1-3 obtain containing osteoblastic hydrogel and be unfavorable for osteoblastic propagation.In addition, the preparation process of above-mentioned contrast groups 1-3 is all increasingly complex compared with experimental group 1 of the present invention and the hydrogel of prepared acquisition is to osteoblast toxicity bigger (namely cell survival rate is relatively low), it is seen that experimental group 1 of the present invention has the step more simplified and more excellent osteoblastic proliferation effect.
nullIn above-mentioned test group 2，The concentration of RGD aqueous solution respectively 1mg/ml in experimental group 1-4、2mg/ml、5mg/ml and 10mg/ml，After osteoblast embedding in situ grows in hydrogel and breaks up 24h，The survival rate of cell is followed successively by 99%、96%、94% and 98%，And after osteoblast and hydrogel material co-culture 7d，The survival rate of cell rises to 110%、107%、115% and 110%，Visible co-culture 7d after，Cell survival rate increases when co-culturing 24h relative to osteoblast and hydrogel to some extent，Wherein，Experimental group 1-4 cell all breeds about 110% for germinal cell，Visible，The osteoblast provided in experimental group 1-4 and hydrogel material is adopted to have more excellent biological general character compared with contrast groups 4-7，And can more effectively promote osteoblastic growth and differentiation.And the concentration of RGD aqueous solution respectively 0.1mg/ml, 0.45mg/ml, 15mg/ml and 20mg/ml in contrast groups 4-7, contrast osteoblast co-cultures 24h and 7d with hydrogel, cell survival rate slightly declines, as in contrast groups 4, contrast groups 5 and contrast groups 7, cell survival rate declines bigger, visible when the concentration adopting RGD aqueous solution exceeds limited range of the present invention, the hydrogel of prepared acquisition is unfavorable for osteoblastic growth.
In above-mentioned test group 3, in experimental group 1, the hyaluronic concentration of dialdehyde is 10mg/ml, and in contrast groups 8-11, dialdehyde hyaluronic concentration respectively 0.1mg/ml, 5.6mg/ml, 13.2mg/ml and 18.0mg/ml, from table 3 it is observed that contrast groups 8-11 is compared with experimental group 1, after the hydrogel obtained in contrast groups 8-11 and osteoblast co-culture 24h and 7d, the difference of its cell survival rate is little, it is seen then that contrast groups 8-11 will not make osteoblastic proliferation.
In above-mentioned test group 4, in experimental group 1, dialdehyde hyaluronic acid aqueous solution is 5.5: 1.4 with the ratio of RGD aqueous solution, and in contrast groups 12-15, the ratio of dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution respectively 11: 1,15: 1,1: 11,1: 15, as can be seen from Table 4, contrast groups 12-15 is compared with experimental group 1, after the hydrogel obtained in contrast groups 12-15 and osteoblast co-culture 24h and 7d, the difference of its cell survival rate is little, visible, contrast groups 12-15 will not make osteoblast produce had significant proliferation.
In above-mentioned test group 5, in experimental group 1 and experimental group 5-10, sodium metaperiodate and hyaluronic acid carry out time of lucifuge reaction and are followed successively by 2.5h, 4.5h, 7h, 10.5h, 11.8h, 16h and 22.5h, experimental group 1 and in experimental group 5-10 obtained hydrogel there is preferably osteoblastic proliferation effect.And in contrast groups 16-18, sodium metaperiodate and hyaluronic acid carry out the time of lucifuge reaction and are followed successively by 1h, 26h and 48h, as can be seen from Table 5, contrast groups 16-18 is compared with experimental group 1, after the hydrogel obtained in contrast groups 16-18 and osteoblast co-culture 24h and 7d, the difference of its cell survival rate is little, it is seen then that contrast groups 16-18 will not make osteoblast produce had significant proliferation.
In above-mentioned contrast groups 1-18, the pore size of the three-dimensional porous structure of formed hydrogel material and porosity impact, and the cell survival rate of contrast 24h and 7d is it can be seen that the hydrogel that obtains of contrast groups 1-18 is to skeletonization cultivation effect inconspicuous.
In cell cultivation process, when utilizing the repairing and treating that osteoblast carries out Cranial defect especially clinically, the hydrogel material adopted can play the effect of osteoblast tissue scaffold, and osteoblast adheres in the inside of hydrogel material, breeds and climbs in the present invention is the key of implant site Bone Defect Repari.Wherein, the pore size of hydrogel supporting construction and porosity, osteoblastic growth and differentiation can be impacted.As too small in the aperture when hydrogel material, porosity is too small, osteoblastic propagation and climbing can be made to impact, and less aperture also can be internally formed anoxic zone and moisture at hydrogel material and make the transmission of nutrient substance first be unfavorable for osteoblastic growth and differentiation；And when the aperture of polyester resin material and porosity are excessive, then the osteoblast being unfavorable for, and affect the structure of epimatrix between cell and cell.In the present invention, there is the pore size respectively 98.5 μm, 101.4 μm, 100.3 μm and 101.7 μm of the hydrogel material of three-dimensional porous structure, and its porosity respectively 89.1%, 91.2%, 90.7% and 92.3%, its pore size and porosity are all beneficial to the osteoblast growth in patient bone reparation or bone collection position.
The Mechanics Performance Testing of hydrogel
Test object:
Adopt method as described in experimental group 1-10 and contrast groups 1-18, and save the correlation step that hydrogel material mixes with osteoblast, cultivates, obtaining without osteoblastic hydrogel material, concrete test object is deformation experiment group 1-10 and deformation contrast group 1-18.
Method of testing:
Rheological method is utilized to carry out Mechanics Performance Testing, take gel sample and be placed on flow graph (parallel plate fixtures, diameter 25mm), space (Gap) value is set to 1mm, frequency is set to 1Hz, carry out load-deformation curve mensuration, take maximum stress value and be the maximum elastance of gel sample, and the maximum elastance that test is obtained is recorded.
Test result:
Adopting above-mentioned mechanic property test method respectively above-mentioned deformation experiment group 1-10 and deformation contrast group 1-18 to be tested, test result is as shown in table 6:
Table 6, deformation experiment group 1-10 and deformation contrast group 1-18 mechanical experimental results list
Test result analysis:
The maximum elastance degree of cross-linking of hydrogel material is correlated with, and therefore, can effectively be reflected by the maximum elastance of hydrogel and adopt the mechanical property characteristic of the prepared hydrogel obtained in deformation experiment group 1-10 and deformation contrast group 1-18.
It can be seen that adopt in deformation experiment group 1-10 provided by the present invention from above-mentioned table 6, the elastic modelling quantity of prepared hydrogel material is 1 × 102Pa-6×103Pa.The comparison is as follows compared to deformation contrast group 1-18 for deformation experiment group 1-10 of the present invention:
(1) as shown in table 6, the relatively maximum elastance of deformation experiment group 1 and the prepared hydrogel obtained of deformation contrast group 1-3, known, the preparation method adopting hydrogel provided by the present invention, the hydrogel material with optimal resilience modulus can be obtained, and adopt other method, the maximum elastance of hydrogel material can be made more than or less than optimum range, thus affecting presenting of the tissue effect after hydrogel injects human body.
null(2) as shown in table 6，Relatively deformation experiment group 1-4 and deformation contrast group 4-7、Relatively deformation experiment group 1 and deformation contrast group 8-11、And compare deformation experiment group 1 and deformation contrast group 12-15，Known，The maximum elastance of the hydrogel that the hydrogel preparation method described in employing different distortion experimental group or deformation contrast group obtains，Known，The concentration change of RGD aqueous solution、The amount ratio of the concentration change of dialdehyde hyaluronic acid aqueous solution and dialdehyde hyaluronic acid and RGD，All the maximum elastance of hydrogel can be impacted，Wherein，The concentration of RGD aqueous solution、The concentration of dialdehyde hyaluronic acid aqueous solution and make the concentration of RGD aqueous solution that consumption limits lower than the present invention、During the concentration range of dialdehyde hyaluronic acid aqueous solution，Its maximum elastance is less than the maximum elastance of the hydrogel adopting hydrogel preparation method provided by the present invention to obtain，If the concentration of RGD aqueous solution、Concentration and both consumptions that makes of dialdehyde hyaluronic acid aqueous solution are higher than the RGD concentration that the present invention limits，Then its maximum elastance is higher than the maximum elastance of the hydrogel adopted prepared by the present invention.
(3) as shown in table 6, relatively deformation experiment group 1, deformation experiment group 5-10 and deformation contrast group 16-18, known, by adjusting sodium metaperiodate and hyaluronic lucifuge response time, the hyaluronic degree of oxidation of dialdehyde that the two reaction can be obtained is adjusted, the lucifuge response time is long or too short, all the elastic modelling quantity of the hydrogel material that the crosslinking of dialdehyde hyaluronic acid with RGD generates can be impacted, visible, lucifuge reaction range that the present invention limits and reacting in the concrete response time, can obtain the mechanical property of required hydrogel.
In the present invention, the experimental group 1-10 provided and deformation experiment group 1-10 preferably mechanical property needed for all can obtaining, thus required reactant concentration and proportioning thereof can be realized quickly obtaining, to meet different application targets.The mechanical property of the hydrogel material obtained in described experimental group 1-10 and deformation experiment group 1-10 is superior to the mechanical property of contrast groups 1-18 and the deformation contrast group 1-18 hydrogel material obtained.
The method for testing swelling properties of hydrogel
Test object:
Adopt method as described in experimental group 1-10, and save the correlation step that hydrogel material mixes with osteoblast, cultivates, it is thus achieved that without osteoblastic hydrogel material, concrete test object is deformation experiment group 1-10.
Method of testing: prepare xerogel sample by dry at 50 DEG C for the gel of gained.
Weigh a certain amount of dry glue sample, be dipped in the medium of different pH value (pH value be 1.2 HCl solution and pH value be the PBS solution of 7.4) carry out equilibrium swelling experiments.After sample is fully swelling, blots the moisture on surface with filter paper and weighs weight in the balance, repeatedly for several times until gel constant mass, now it is believed that the swelling ratio of sample has reached balance.Equilibrium swelling ratio can be calculated by below equation:
QES=[(WES-W0)/W0]×100
Wherein, QESIt is equilibrium swelling ratio, WESIt is the quality of sample, W after swelling equilibrium0It it is the quality of dry glue sample.
Table 7, the method provided in deformation experiment group 1-10 prepare containing the swelling of osteoblast hydrogel and pH sensitivity tests the results list
As shown in table 7, adopting the equilibrium swelling ratio in pH7.4 medium of the hydrogel prepared by above-mentioned deformation experiment group 1-10 is 330%-810%, equilibrium swelling ratio in pH1.2 medium is 120%-230%, by contrasting hydrogel at pH7.4 equilibrium swelling ratio under pH1.2, it can be seen that adopt hydrogel material of preparing provided by the present invention to have pH sensitivity.
In above-mentioned experimental group 1-10 and contrast groups 1-18, the prepared hydrogel containing osteoblast obtained is equally applicable to the Mechanics Performance Testing of above-mentioned hydrogel and the swelling behavior test of hydrogel, and there is the test effect close with above-mentioned deformation experiment group 1-10 and deformation contrast group 1-18, visible, adopt method provided by the present invention to prepare, containing osteoblast hydrogel, there is preferably mechanical property and swelling behavior equally.
Adopt the preparation method of hydrogel provided by the present invention and containing, in osteoblastic hydrogel preparation method, adopting High Throughput Screening Assay, can carry out organizing contrast experiment, thus can quickly obtain required target proportioning compared to prior art more simultaneously.
Compared with prior art, the preparation method of the hydrogel provided in the present invention, have the advantage that containing osteoblast hydrogel and preparation method thereof
(1) traditional hydrogel preparation method is adopted, it is impossible to realizing fast accurate treatment, be generally 7 days to 15 days from being prepared into the cycle finally obtaining spendable hydrogel material, manufacturing cycle is longer, is unfavorable for the extensive use that Bone Defect Repari is treated.In the preparation method of hydrogel provided by the present invention and the preparation method containing osteoblast hydrogel, by utilize High Throughput Screening Assay quickly obtain the desired concn to dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution and mixed containing target proportioning, such that it is able to quickly obtain the hydrogel material with required pore size and porosity.Adopting the hydrogel material that method provided by the present invention obtains to be conducive to Growth of Cells, especially osteoblast can obtain required growth, differentiation and proliferation conditions in hydrogel material, thus being effectively improved cell survival rate and Bone Defect Repari effect.Adopt method provided by the present invention, it is possible to quickly obtain the target proportioning of required dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution according to different demand purposes, thus being conducive to hydrogel product widely using in the Bone Defect Repari treatments such as osteoarthritis.
(2) preparation method adopting hydrogel provided by the present invention, scope according to the obtainable required target proportioning of High Throughput Screening Assay is wider, the scope of required target proportioning can be 1: 9-9: 1 volume ratio, therefore, adopt the selection that High Throughput Screening Assay carries out described target proportioning to have the stronger suitability, the hydrogel cultivated for cell and grow can be prepared.Additionally, the preparation method adopting hydrogel provided by the present invention, also quickly in the wider dialdehyde hyaluronic acid ratio range with RGD, preferably target proportioning can be obtained, to obtain the hydrogel structure with the more excellent suitability.
The method obtaining hydrogel is tested different from prior art adopting traditional method carry out, adopt preparation method of the present invention and containing osteoblast hydrogel, can quickly obtain preferably hydrogel structure required in Bone Defect Repari therapeutic process and composition thereof, and can according to carrying out the feature of required different hydrogels of osteocyte repairing and treating needed for patient affected part (such as cell survival rate, cell proliferation effect, mechanical property, swelling behavior and pH sensitivity thereof etc.), by quickly confirming the target proportioning of required dialdehyde hyaluronic acid aqueous solution and RGD aqueous solution, thus quickly obtaining required hydrogel structure, realize fast accurate treatment, reduce patient bone cytothesis treatment required time and improve the cure rate that patient bone is repaired.
(3) in the present invention, osseous tissue is had good biocompatibility by the hydrogel with three-dimensional porous structure prepared by the mixed proportion of described dialdehyde hyaluronic acid and described RGD, by osteoblast embedding in situ in hydrogel material, osteoblastic growth can be promoted, differentiation and increment, thus giving full play to Bone Defect Repari ability, along with osteoblast completing Bone Defect Repari, there is degraded and can be discharged by body in the hydrogel material prepared by method of the present invention, in addition, in the present invention, adopt High Throughput Screening Assay rapid screening can go out the required hydrogel material formula that applicable Oesteoblast growth breaks up.
(4) adopting RGD as cross-linking agent, it is possible to be effectively improved the cell adhesion of hydrogel material, by making RGD and modified hyaluronic acid cross-link, osteoblast being had the more excellent compatibility and adhering containing osteoblast hydrogel material thus being formed.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all any amendments made within the principle of the present invention, equivalent replacement and improvement etc. all should comprise within protection scope of the present invention.
Claims (10)
1. the preparation method of a hydrogel, it is characterised in that: it comprises the following steps that
Step S1, it is provided that dialdehyde hyaluronic acid and RGD；
Step S2, utilizes High Throughput Screening Assay to obtain the target proportioning needed for dialdehyde hyaluronic acid and RGD；And
Step S3, has the hydrogel of three-dimensional porous structure with RGD needed for being cross-linked to form by the step S2 target proportioning obtained by dialdehyde hyaluronic acid.
2. the preparation method of hydrogel as described in the appended claim 1, it is characterised in that: in above-mentioned steps S2, the target ratio range needed for the dialdehyde hyaluronic acid of described acquisition and RGD is 1: 9-9: 1 volume ratio.
3. the preparation method of hydrogel as described in claim 1 or 2, it is characterised in that: in above-mentioned steps S2, utilize High Throughput Screening Assay to form dialdehyde hyaluronic acid and RGD hydrogel micro-array chip.
4. the preparation method of hydrogel as described in the appended claim 1, it is characterised in that: above-mentioned steps S3 farther includes: dialdehyde hyaluronic acid and RGD are each configured to the dialdehyde hyaluronic acid aqueous solution that mass concentration is 8-12mg/ml and the RGD aqueous solution that mass concentration is 0.5-10.5mg/ml；And
Controlling reaction temperature is 36.5 DEG C-37.5 DEG C, makes dialdehyde hyaluronic acid aqueous solution prepare the hydrogel material with three-dimensional porous structure after reacting 0.3h-0.7h with RGD aqueous solution.
5. the preparation method containing osteoblast hydrogel, it is characterised in that: it comprises the following steps that
Step T1, it is provided that dialdehyde hyaluronic acid and RGD；
Step T2, utilizes High Throughput Screening Assay to obtain the target proportioning needed for dialdehyde hyaluronic acid and RGD；And
Step T3, after being mixed by the step T2 target proportioning obtained with RGD by dialdehyde hyaluronic acid, adds needed for osteoblast suspension is mixed to form and has three-dimensional porous structure containing osteoblastic hydrogel.
6. as claimed in claim 5 containing the preparation method of osteoblast hydrogel, it is characterised in that: above-mentioned steps T3 farther includes:
Configuration cell concentration is 5.0 × 105-6.0×107The osteoblast suspension of individual/ml is with standby；Configuration quality concentration is the dialdehyde hyaluronic acid aqueous solution of 8-12mg/ml and RGD aqueous solution that mass concentration is 0.5-10.5mg/ml, and after described dialdehyde hyaluronic acid aqueous solution is mixed with 1: 9-9: 1 volume ratio with described RGD aqueous solution, join and described osteoblast suspension carries out mix and after cross-linking reaction, prepare and there is three-dimensional porous structure containing osteoblastic hydrogel.
7. as recited in claim 6 containing the preparation method of osteoblast hydrogel, it is characterised in that: described step T3 farther includes:
The reaction temperature of described cross-linking reaction is 36.5 DEG C-37.5 DEG C, and the response time is 0.3h-0.7h.
8. one kind containing osteoblast hydrogel, it is characterised in that: described adopt as according to any one of claim 5-7 the preparation method containing osteoblast hydrogel to be prepared from containing osteoblast hydrogel.
9. as claimed in claim 8. containing osteoblast hydrogel, it is characterised in that: the described maximum elastance containing osteoblast hydrogel is 0.5 × 102Pa-1×104Pa, in the medium that pH value is 7.4, the described equilibrium swelling ratio containing osteoblast hydrogel is 330%-810%, and in the medium that pH value is 1.2, the described equilibrium swelling ratio containing osteoblast hydrogel is 120%-230%.
10. as claimed in claim 9 containing osteoblast hydrogel, it is characterised in that: the described porosity containing osteoblast hydrogel is 79%-93%, and the described pore size containing osteoblast hydrogel is 80-200 μm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610202115.8A CN105797211A (en) | 2016-03-31 | 2016-03-31 | Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610202115.8A CN105797211A (en) | 2016-03-31 | 2016-03-31 | Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105797211A true CN105797211A (en) | 2016-07-27 |
Family
ID=56460441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610202115.8A Pending CN105797211A (en) | 2016-03-31 | 2016-03-31 | Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105797211A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109316630A (en) * | 2018-11-19 | 2019-02-12 | 重庆凝骄生物科技有限公司 | A kind of 3D printing ink of bionic matrix and preparation method thereof |
| CN110029084A (en) * | 2019-04-12 | 2019-07-19 | 河海大学常州校区 | A kind of regulatable nonuniformity glucan 3D gel of partial cross-linking intensity, preparation method and applications method |
| CN110478532A (en) * | 2019-08-22 | 2019-11-22 | 上海交通大学医学院附属第九人民医院 | Raw hole aquogel system of a kind of Injectable in-situ and its preparation method and application |
| CN114870092A (en) * | 2022-05-20 | 2022-08-09 | 广州骊贝生物科技有限公司 | Bone substitute complex and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342170A (en) * | 1999-02-05 | 2002-03-27 | 维特罗莱夫英国有限公司 | Process for cross-linking hyaluronic acid to polymers |
| CN1502375A (en) * | 2002-11-26 | 2004-06-09 | hemostatic wound dressing containing aldehyde-modified polysaccharide and hemostatic agents | |
| CN102492158A (en) * | 2011-12-01 | 2012-06-13 | 北京博恩康生物科技有限公司 | Injectable in situ immobilization gel and its preparation method |
| WO2014032748A1 (en) * | 2012-08-31 | 2014-03-06 | Eth Zurich | Process of cartilage repair |
-
2016
- 2016-03-31 CN CN201610202115.8A patent/CN105797211A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342170A (en) * | 1999-02-05 | 2002-03-27 | 维特罗莱夫英国有限公司 | Process for cross-linking hyaluronic acid to polymers |
| CN1502375A (en) * | 2002-11-26 | 2004-06-09 | hemostatic wound dressing containing aldehyde-modified polysaccharide and hemostatic agents | |
| CN102492158A (en) * | 2011-12-01 | 2012-06-13 | 北京博恩康生物科技有限公司 | Injectable in situ immobilization gel and its preparation method |
| WO2014032748A1 (en) * | 2012-08-31 | 2014-03-06 | Eth Zurich | Process of cartilage repair |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109316630A (en) * | 2018-11-19 | 2019-02-12 | 重庆凝骄生物科技有限公司 | A kind of 3D printing ink of bionic matrix and preparation method thereof |
| CN109316630B (en) * | 2018-11-19 | 2021-03-26 | 中国人民解放军陆军军医大学第一附属医院 | 3D printing ink of biological bionic matrix and preparation method thereof |
| CN110029084A (en) * | 2019-04-12 | 2019-07-19 | 河海大学常州校区 | A kind of regulatable nonuniformity glucan 3D gel of partial cross-linking intensity, preparation method and applications method |
| CN110029084B (en) * | 2019-04-12 | 2023-04-07 | 河海大学常州校区 | Heterogeneous glucan 3D gel with adjustable local crosslinking strength, and preparation method and application method thereof |
| CN110478532A (en) * | 2019-08-22 | 2019-11-22 | 上海交通大学医学院附属第九人民医院 | Raw hole aquogel system of a kind of Injectable in-situ and its preparation method and application |
| CN114870092A (en) * | 2022-05-20 | 2022-08-09 | 广州骊贝生物科技有限公司 | Bone substitute complex and preparation method and application thereof |
| CN114870092B (en) * | 2022-05-20 | 2023-08-22 | 广州骊贝生物科技有限公司 | Bone substitute complex, preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Negrini et al. | Tissue-mimicking gelatin scaffolds by alginate sacrificial templates for adipose tissue engineering | |
| Thein-Han et al. | Chitosan as scaffold matrix for tissue engineering | |
| CN101632841B (en) | Tissue engineering scaffold containing alginate, glycosaminoglycan and collagen and preparation method thereof | |
| CN104892962A (en) | Preparation method and application of sulfhydryl/disulfide bond controllable self-crosslinked hyaluronic acid hydrogel | |
| CA2704738A1 (en) | Method for preparing porous scaffold for tissue engineering | |
| CN110818921B (en) | Rapidly-curable double-crosslinked hydrogel and preparation method and application thereof | |
| US20200399431A1 (en) | Covalently Cross-Linked Hyaluronic Acid Aerogel, Hydrogel, and Preparation Method Thereof | |
| CN105797211A (en) | Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel | |
| CN109432496A (en) | It is a kind of can the molding sulfhydrylation polysaccharide based aquagel of in-situ injection and its pharmaceutical carrier preparation method and application | |
| CN102488925A (en) | Injectable articular cartilage tissue repair material and its preparation method | |
| CN102319449A (en) | Poly(lactic-co-glycolic acid)-based growth factor gradient release microsphere stent as well as preparation method and application thereof | |
| do Nascimento et al. | Evaluation of cell interaction with polymeric biomaterials based on hyaluronic acid and chitosan | |
| CN102380129A (en) | Sodium hyaluronate and KGM porous bracket material and method for preparing same | |
| CN102107022B (en) | Natural polymer-hydroxyapatite bone tissue engineering scaffold material with secondary three-dimensional network structure and seed-crystal induction preparation method thereof | |
| CN107638590B (en) | A kind of bionical compound support frame material of chitosan-based gradient and its construction method | |
| Tortorella et al. | Biocompatible pectin-based hybrid hydrogels for tissue engineering applications | |
| Kil’deeva et al. | Biodegradablescaffolds based on chitosan: Preparation, properties, and use for the cultivation of animal cells | |
| Li et al. | Magnetic liquid metal scaffold with dynamically tunable stiffness for bone tissue engineering | |
| CN111617319A (en) | Composite hydrogel, preparation method and application thereof | |
| CN110746548B (en) | Hydrogel for preparing cell scaffold | |
| US6872387B1 (en) | Three-dimensional hydrogel/cell system | |
| Kong et al. | Preparation and characterization of polyacrylamide/silk fibroin/graphene oxide composite hydrogel for peripheral nerve regeneration | |
| CN104672484A (en) | Cross-linked polysaccharide tissue engineering porous scaffold preparation method | |
| TW201016722A (en) | Porous composite biomaterials and production method of the same | |
| KR20220019775A (en) | Means for use in the production of hydrogels based on hydroxyphenyl derivatives of hyaluronan, methods for producing hydrogels and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | 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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160727 |