CN106729972A - The composition of bone filler, reserve and their preparation method and application - Google Patents
The composition of bone filler, reserve and their preparation method and application Download PDFInfo
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- CN106729972A CN106729972A CN201611102957.2A CN201611102957A CN106729972A CN 106729972 A CN106729972 A CN 106729972A CN 201611102957 A CN201611102957 A CN 201611102957A CN 106729972 A CN106729972 A CN 106729972A
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- 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
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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- 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
- A61L27/222—Gelatin
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- 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
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- 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
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- 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 belongs to medical biomaterial technical field, and in particular to the composition of bone filler, reserve and their preparation method and application.The bone filler composition, including bone meal, gelatin and crosslinking agent, have the good advantage of inducing action, elasticity, plasticity concurrently.Bone filler reserve be bone filler composition prepare solution it is freeze-dried after product.The method for preparing bone filler, including the bone filler composition or reserve are mixed with medically acceptable solvent.Raw material sources of the present invention extensively, are prepared simply, in clinical practice, can in time provide the bone filler that can be filled.Bone filler of the invention can be widely used for scientific research field or be used as medical material.
Description
Technical field
The invention belongs to medical biomaterial technical field, and in particular to the composition of bone filler, reserve and
Their preparation method and application.
Background technology
Human body Bone Defect Repari technology have passed through very long developing period, is mainly made that in terms of the biocompatibility of material
It is extremely improved, from original wicker to metal, then to medical high polymer, nowadays bone renovating material has come into biomimetic material
Stage.But either which kind of material, all without great improvement in clinical use means, substantially still continues to use
These are difficult moulding material for grain type or bar shaped, very high to Doctors' skill requirement in Clinical practice, especially for bone split or
The sutura of disunion, operates and is inconvenient.Due to the bone wound that wound, tumour, pathological conditions or other forms are caused
And in department of stomatology bone implantation, shape at Cranial defect usually very irregular, or wound site is deeper.And commonly used in bone grafting operation
Filler shape fix regular (or dissipate into graininess shapeless), be difficult arbitrarily moulding, cause in the filling process in defect
Place's residual dead space, influences skeletonization effect, extends healing time.To improve bone grafting effect, if film, finger shaping, note can be used
The modes such as emitter injection carry out moulding to bone filler, can necessarily obtain more preferable repairing effect, but this just fills material to bone
The plasticity of material proposes requirements at the higher level.
Chinese patent literature CN102755668 discloses a kind of medical plastic bone cement, and it is main by polysaccharide or albumen
The pureed composite of glue or organic bond and bone meal composition.The bone filler of this bone mud form, because it is soft easily
The characteristic of modeling, in addition to still having certain shortcoming in the filling in tiny sutura gap, can meet the moulding of most bone grafting operation will
Ask, but in the application of filling material of bone, however it remains problems with.
For bone meal, by taking Bio-Oss bone meal as an example, Bio-Oss bone meal is general to be extracted from ox bone, by technique plus
Work, all of organic principle is thoroughly removed from the cancellous bone of ox, and fine trabecular bone structure and internal voids is saved
Get off, so that growing into there is provided support for Gegenbaur's cell, and ensure that the regeneration of the stabilization and blood vessel of blood clot, be a kind of day
Right, no antigen, the graft materials with bone conduction effect, are clinically widely used at present.Bio-Oss bones
Efflorescence studies tap person of modern times's body bone inorganic structure (the low natural apatite of crystal), macroscopic view and microphysics structure and human body cancellous bone
Also it is very much like.But had the following disadvantages in terms of Clinical practice and osteogenic induction ability：(1) elastic modelling quantity of bone meal particle
About 1GPa, significantly larger than generally acknowledged can induce stem cell bone to the elastic substrates hardness (about 0.1MPa) of differentiation；(2) simple nothing
Machine bone material can not promote osteogenic action under chemistry or biotic factor inductive condition；(3) make as graininess bone meal at those
In situation, graininess bone meal also haves the shortcomings that displacement, migration, is difficult to shape, is difficult to place, and causes clinical manipulation not
Just, bone grafting effect is difficult to ensure that.
At present, clinically conventional bone filler is in addition to Bio-Oss bone meal, also Bio-Oss osseins, refer to by
The collagen composition of 90% Bio-Oss and 10%, is loose and porous structure, spatially rectangular block shape (3mm*5mm*7mm).The material
The elastic modelling quantity of material decreases (about 1MPa) compared with Bio-Oss bone meal, but still higher than induction stem cell bone to the ideal broken up
Scope, and it is expensive, bring financial burden to patient.
To sum up, existing bone filler is difficult to solve problems with simultaneously at present：
(1) clinically conventional bone filler (bone- xenograft or artificial synthesized bone material) hardness is excessive, is filled between being not suitable for
The bone of matter stem cell is to differentiation and the maintenance of bone cell function；
(2) material lacks the inducing action of growth factor only for defect provides physical support；
(3) material plasticity is poor, it is impossible to meets the Defect operation of large area or specific form requirement, is not suitable for facing
The individual demand of bone defect healing on bed.
Additionally, the finished commercial prod of part bone filler, is also present expensive, it is difficult to wide variety of problem.
The content of the invention
In view of this, the present invention provides a kind of bone filler composition, including bone meal, gelatin and crosslinking agent.
The present invention also provides a kind of bone filler reserve, and it is solution warp prepared by the bone filler composition
The freeze-drying prods obtained after freeze-drying process.
The present invention also provides a kind of method for preparing bone filler reserve, including to the bone filler composition
The solution of preparation implements freeze-drying process.
The present invention further also provides a kind of method for preparing bone filler, including by the bone filler composition
Or bone filler reserve mixes with medically acceptable solvent.
Additionally, the present invention also provides application and conduct doctor of the bone filler of the inventive method preparation in scientific research field
With the application of material.
Bone filler composition of the present invention can be obtained the filling material of bone of suitable resilient modulus, and because bone fills material
Material hardness is suitable, can further promote Bone Marrow Mesenchymal Stem Cells to differentiation and function of osteoblast.Freeze-dried treatment
Bone filler composition, form the bone meal supporting structure with a large amount of spaces, more flexible and toughness, it is easy to moulding,
And more inducing action.It can be used with powder, fluid, semisolid form, thus plasticity is good.Therefore, bone of the invention
Filler have concurrently elasticity, inducing action, plasticity it is good the characteristics of.Methods described raw material sources extensively, are prepared simply, are being faced
When bed is applied, the bone filler that can be filled can be in time provided, be suitable for the moulding of various Bone Defect Repari situations.
Brief description of the drawings
The outside drawing of bone meal-gelatin film that Fig. 1 is prepared for the experimental group 4 of embodiment 1；
Fig. 2 is prepared in gelatin/Geniposide-bone meal stent procedures for embodiment 2, the outside drawing of product after first time pre-freeze；
Fig. 3 is prepared in gelatin/Geniposide-bone meal stent procedures for embodiment 2, for the first time lyophilized rear product Buddhist nun's par in Beijing
The outside drawing soaked in solution；
Fig. 4 is prepared in gelatin/Geniposide-bone meal stent procedures for embodiment 2, second lyophilized rear product Buddhist nun's par in Beijing
Outside drawing after being soaked in solution；
Fig. 5 is the environmental scanning electronic microscope figure of gelatin/Geniposide-bone meal support prepared by embodiment 2；
Fig. 6 is the form X100 of BMSCs under inverted microscope；
Fig. 7 is immune-blotting method into bone seeker protein expression figure；
Fig. 8 is Real-time fluorescence quantitative PCR detection skeletonization marker mRNA horizontal expression figures；
Fig. 9 detects into the distribution of bone seeker for immunofluorescence dyeing --- Col-I under laser confocal microscope
The cell dyeing result of Osteoblast Differentiation marker protein mark；
Figure 10 detects into the distribution of bone seeker for immunofluorescence dyeing --- under laser confocal microscope OCN into
The cell dyeing result of bone differentiation marker protein mark；
Figure 11 detects into the distribution of bone seeker for immunofluorescence dyeing --- under laser confocal microscope OPN into
The cell dyeing result of bone differentiation marker protein mark；
Figure 12 detects into the statistical results chart of the distribution of bone seeker for immunofluorescence dyeing；
Figure 13 be determination of alkaline phosphatase activity culture dish in cell dyeing visually observe situation (on) and inversion it is micro-
Microscopic observation situation (under)；
Figure 14 is the statistical results chart of alkaline phosphatase (ALP) determination of activity.
Specific embodiment
In the present invention, unless otherwise instructed, all operations are implemented in room temperature, condition of normal pressure, and it is quality to own " % "
Percentage.
Bone filler composition of the invention, including bone meal, gelatin and crosslinking agent.
The gelatin that the present invention is used, is one of Osteoinductive Factor as degradable biological macromolecular, can be effectively facilitated
The new life of sclerotin around bone graft area；Gelatin also reduces production cost compared with collagen.With finished product bone meal as raw material, retain
Material chemical characteristic in itself, by the mixing with gelatin gel, has rebuild the porous stent structure of material.Gelatin and bone meal
Ratio can adjust the elastic modelling quantity of material, the bone filler of dual extension-compression modulus can be prepared, using physical environment
Inducing action promotes the skeletonization effect after bone collection, and the bone of suitable mescenchymal stem cell is to differentiation and the dimension of bone cell function
Hold.Gelatin has an effect for bonding solid, it is crosslinked after form semi-solid, after adding bone meal particle, material become prone to it is moulding,
Do not limited by Cranial defect form and size, prepared by the material that can as needed carry out personalization.
We, it is known that the Gegenbaur's cell of source for mesenchymal stem cells quantity and function to promote new bone formation to close weight
Will.In recent years, research finds that extracellular matrix (extracellular matrix, ECM) elasticity is the material that cell is perceived
Elasticity, can influence induce stem cell to osteoblast differentiation process.We have found stem cell Osteoblast Differentiation by exploring
It is about 10 in elasticity5Decided advantage in the matrix of Pa, excessively soft or really up to the mark extracellular matrix is all to the dry thin of in vitro culture
Born of the same parents' Osteoblast Differentiation, function of osteoblast remain unfavorable.Clinically the elastic modelling quantity about 1GPa of conventional bone meal particle, clinically single
It is pure to use this kind of material, may not reach optimal rush osteogenic action in physical environment angle.That is, traditional bone filling material
Material (including most of bone mud) hardness is very high, and is shown through research, and substrate (substrate that i.e. bone filler is formed) really up to the mark is no
Sharp stem cell bone is to differentiation, and suitable stiff material can promote osteocyte vigor and skeletonization.Bone filler combination of the present invention
Thing can make material hardness controllable by adjusting its component ratio, for stem cell to osteoblast differentiation provides good physical rings
Border.
The bone filler composition of the present invention, may also include medically acceptable solvent.Specifically as product
During packaging, the component can be separated and packed, or partly or entirely packaging after component mixing.
Collagen is the main component of extracellular matrix, is the most abundant a kind of structural proteins of content in human body, accounts for human body egg
More than the 30% of white total amount.Collagen have wide material sources, be readily available, biological safety is good, the low feature of immunogenicity, in body
Interior to be degraded by organism, catabolite can be absorbed by organisms and degradation rate is controllable, beneficial to the sticking of stem cell, breed, point
Change, and be conducive to the holding of phenotype.But, collagen is small and high-purity due to the rack mechanical strength that degradation rate is too fast, prepare
Degree collagen acquisition cost is high to wait not enough, is restricted its application.Gelatin is the catabolite of collagen, is that a kind of natural polypeptides gathers
Compound.Using upper, gelatin has the advantages that correctability, is easy to process plastotype.Gelatin is similar to collagen, strong with bioactivity,
The advantages of growing multiplication of cell can be promoted.Aqueous gelatin solution can form the gel of thermal reversibility in temperature less than 35 DEG C, in temperature
It is gel state when degree is less than 35 DEG C, is solution during higher than 35 DEG C.The heat endurance and mechanical stability of gelatin hydrogel be not high,
The general method by being chemically crosslinked improves.
The gelatin that the present invention is used has the effect that biotic factor is induced, while also reducing cost compared with NTx.
More it is surprising that using cooperatively for gelatin and bone meal, can also adjust the elastic modelling quantity of bone filler, promote bone collection
Skeletonization effect afterwards, while being also easy to moulding.
In a preferred embodiment, the bone meal is that hydroxyapatite is the bone alternate material of main component；It is excellent
Select Bio-Oss bone meal.Bio-oss bone meal main components are hydroxyapatite (Hydroxyapatite, HA), and HA is human body and moves
The main inorganic composition of thing bone, good biocompatibility, hardness are big, with good bearing, also with osteoinductive, and can be with bone
Tissue strong bonded.But HA fatigue resistances are not good, fragility big, low intensity, limit its direct application in biomaterial.
Bone meal of the present invention is used with cooperateing with for gelatin, it is to avoid the problem.
The bone meal is shaped as standard with microscopic particles size and required bone filler, finished product bone meal can be carried out into difference
The grinding of degree.In a preferred embodiment, the granular size of the bone meal is 10um-200um.
In a preferred embodiment, the crosslinking agent be Geniposide, glutaraldehyde, 1- (3- dimethylamino-propyls)-
One or more in 3- ethyl-carbodiimide hydrochlorides (EDC HCl) etc.；It is preferred that Geniposide.
Geniposide (genipin) is that geniposide is extracted from cape jasmine, then the ring by being obtained after beta-glucosidase enzyme hydrolysis
Alkene ether terpenoid, can make biomaterial with the crosslinking such as protein, collagen, gelatin and shitosan, be a kind of excellent
Natural biological crosslinking agent, its toxicity is far below traditional chemical crosslinking agents such as formaldehyde, glutaraldehydes.Research shows, the cell of genipin
1/10 of toxicity less than glutaraldehyde4, and its ability for promoting cell proliferation is then the 5 × 10 of glutaraldehyde3Times.
The bone meal is with the use magnitude relation of gelatin：Under identical crosslinker concentration, bone meal ratio is bigger, and gelatin concentration is got over
Greatly, material is harder.In a preferred embodiment, the gelatin and the mass ratio of bone meal are 0.05-0.4:1, more preferably
0.15-0.4:1。
The consumption of the crosslinking agent, with skilled artisan understands that can reach crosslinking purpose amount ranges be
Limit.In a preferred embodiment, the consumption of the crosslinking agent is the 0.05-0.5% of bone filler gross weight, preferably
0.2%.
The present invention also provides a kind of bone filler reserve, is the solution for preparing of the bone filler composition through cold
Freeze the freeze-drying prods obtained after dried process.The reserve is prepared by freeze drying process, forms the bone with a large amount of spaces
Powder supporting structure, more flexible and toughness, it is easy to moulding.
The present invention also provides a kind of method for preparing bone filler reserve in addition, including to the bone filler group
Solution prepared by compound implements freeze-drying process.
In a preferred embodiment, the freeze-drying process is carried out in two steps：
The first step, prepares gelatin solution, and the mixture of gelatin solution and bone meal, freeze-drying gelatin are mixed into bone meal
The mixture of solution and bone meal, obtains gelatin-bone meal support；
Second step, prepares cross-linking agent solution, and be mixed into cross-linking agent solution and gelatin-bone meal branch with gelatin-bone meal support
The mixture of the mixture of frame, freeze-drying cross-linking agent solution and gelatin-bone meal support, obtains gelatin/crosslinking agent-bone meal support,
Obtain the bone filler reserve.
In above-mentioned steps, the common solvent of solution is prepared to adapt to the conventional use of solvent of freeze drying process, typically
It is physiological saline.
Gelatin/crosslinking agent-bone meal support prepared by freeze-drying, except the chemical property and biological nature that make use of material
Outward, the three dimensional scaffold structure for being formed can in analogue body Grafting Cancellous Bone Bolt girder structure, for Cranial defect area vascularization after transplanting and
The attachment of mescenchymal stem cell provides good space structure.And, method that can be by mixing with Bio-oss bone meal, to material
The mechanical property of material is improved and regulates and controls.The freeze-drying for using herein, is risen under vacuo using the solvent of cryogenic refrigeration
The principle of China is prepared for porous support.Using secondary lyophilized operation, can obtain being more uniformly distributed the bone meal support with a large amount of spaces, with
Beneficial to skeletonization；And, the cross-linked structure being achieved in that is more flexible and toughness, it is easy to moulding.
Additionally, when desivac prepares three-dimensional rack, can also be by the size that changes the concentration of mixture to change hole.More
In preferred embodiment, during first step freeze-drying, the mass fraction of institute's gelatine solution is 5-10%.It is another more preferably
Embodiment in, during second step freeze-drying, the mass fraction of the cross-linking agent solution is 0.3%-1%, preferably 0.3%.
The present invention also provides a kind of method for preparing bone filler in addition, including：By the bone filler composition
Or the bone filler reserve mixes with medically acceptable solvent.
The medically acceptable solvent, for example, blood, physiological saline etc..The consumption of solvent, is easy to face to be formed
The semisolid filling material of bone of bed bone grafting operation operation is limited.The filling material of bone of semisolid is that can be used for various bone filling situations
It is moulding.Specifically in clinical practice, after can also mixing the bone meal, gelatin and crosslinking agent, the bone collection of patient is directly applied
Area, body fluid, blood of patient etc. and the mixture are acted on and realize the filling of bone.
In a preferred embodiment, the elastic modelling quantity of the bone filler of acquisition is 0.05-0.8MPa, more preferably
0.05-0.3MPa, most preferably 0.1-0.2MPa.
Present invention additionally comprises the bone filler composition and bone filler reserve and their preparation method
Between be combined after formed embodiment.
Bone filler prepared by the above method can be widely used in scientific research field as experiment material, for example, set up dynamic
Thing model, experimental analysis etc..
Bone filler prepared by the above method as medical material, in can be widely used for field of medicaments, such as bone
Repair materials of bone or tooth etc..
Referring to embodiment and accompanying drawing, the invention will be further described, wherein the experiment side of unreceipted actual conditions
Method, is carried out according to normal condition.
Embodiment 1
(1) aseptically finished product Bio-Oss bone meal (being purchased from GeistlichPharmaAG) is ground into powder,
Powder particle after grinding is observed under environmental scanning electron microscope, granular size about 10um-200um.This granular size is just
Suitably mix and make the uniform timbering material of elastic hardness, and form the space of suitable size, be beneficial to cell and grow into, skeletonization.
(2) aqueous gelatin solution of 5% and 10% concentration is configured, 50 DEG C of stirring in water bath dissolution solutions are recovered stand-by to room temperature；Match somebody with somebody
Put 5% glutaraldehyde water solution.
(3) according to the consumption of table 1, as steps described below, the bone meal-gelatin film of different ratio is prepared respectively.
Operating procedure：
A. the bone meal powder after grinding and gelatin solution, glutaraldehyde solution are mixed rapidly on the glass sheet, uses another glass
Glass piece lid colloid surface upon mixing, and suitably pressurize, form mixed colloid complete uniform membranaceous, thickness is about
1mm。
B. after being stored at room temperature 10 minutes, the bone meal-gelatin film that will be stamped sheet glass is put into pure water, carefully throws off glass
Piece, it is ensured that film it is complete, note avoiding prolonged exposure in air, preventing gelatin water loss from causing film to deform.
C. soaked and eluted repeatedly for several times, at least 24 hours with 1% glycine solution after suction filtration, to neutralize free penta
Dialdehyde.
D. film is cleaned 3 times with PBS solution, and 30-40min is irradiated with ultraviolet, material is prepared and finished.
As shown in table 1, the outward appearance of bone meal-gelatin film prepared by experimental group 4 is such as the elastic modelling quantity of obtained bone meal-gelatin film
Shown in Fig. 1.
The preparation of 1 bone meal of table-gelatin film and elastic modelling quantity result
Remarks：Above-mentioned elastic modelling quantity is measured to base material by AFM and measures that (method of testing is specific
See experimental sections), the measuring method is generally satisfactory and applies in the experiment of biomethanics elasticity measurement.
As shown in Table 1, when the concentration of gelatin solution is 5%, the mass ratio that we gradually increase gelatin in composition comes
The elastic modelling quantity of regulation composition, in identical gelatin solution concentration, increases gelatin quality than simultaneous solvent (water)
Increase, the elastic modelling quantity of composition can be made as the mass ratio of gelatin increases and declined, now regulation composition elastic modelling quantity
Key substance is solvent --- water；But the addition of the material with bioactivity is original intention of the invention and advantage, then we
Trial increased the concentration of gelatin solution, find under 10% gelatin solution concentration, obtain compared to 5% gelatin solution concentration
When more small range Flexible change, and solution-bone meal mass ratio be 4：When 1, resulting elastic mould value meets at present
The ideal grade scope of known stem cell Osteoblast Differentiation.In addition, when being prepared to the material, it has been found that 10% gelatin
Solution is resulting because with good semi-solid property, good plasticity being shown in the preparation of bone meal-gelatin film
Bone meal-gelatin film has good toughness, it is easy to preserve, and such as Fig. 1 shows.
Embodiment 2
(1) aseptically finished product Bio-Oss bone meal (being purchased from GeistlichPharmaAG) is ground into powder,
Granular size about 10um-200um is observed under powder particle environmental scanning electron microscope after grinding.This granular size is just suitable
Mix and make the uniform timbering material of elastic hardness, be beneficial to cell and grow into, skeletonization.
(2) aqueous gelatin solution of 10% concentration is configured, 50 DEG C of stirring in water bath dissolution solutions are recovered stand-by to room temperature；Configuration 1%
Geniposide aqueous solution liquid storage (Geniposide is purchased from Shanghai Shi Feng bio tech ltd).
(3) according to the consumption of table 2, as steps described below, gelatin/Geniposide-bone meal support is prepared.What is prepared is bright
The elastic modelling quantity of glue/Geniposide-bone meal support is shown in Table 2.
Operating procedure：
A. operated on ice, aqueous gelatin solution slow solidification at low temperature is stirred continuously in process of setting, makes bone meal uniform
It is distributed in gel, after about 5min stabilizations, is immediately placed in -20 DEG C, pre-freeze is overnight.Obtain gelatin-bone meal as shown in Figure 2.
B. after sample takes out from -20 DEG C of refrigerators, vacuum freeze drier, freeze-drying 24h are put into.
C. the sample after freezing soaks, 37 in spongy in the Geniposide aqueous solution that configuration good quality fraction is 0.3%
DEG C constant temperature, 24h, now because of cross-linking reaction, gel is in navy blue, as shown in Figure 3.
D. pre-freeze and freeze-drying process are carried out again, obtain gelatin/Geniposide-bone meal support, in navy blue loose structure,
As shown in Figure 4.
The preparation of 2 gelatin of table/Geniposide-bone meal support and elastic modelling quantity result
(4) electron microscopic observation
Gelatin/Geniposide-bone meal the brace aperture of acquisition is evenly distributed, pore size between 20-100 microns, such as Fig. 5
It is shown.A and b show the surface topography of material in figure, and multiplication factor is respectively 500X and 2000X, and it is bone meal that arrow is signified
Grain, * meanings are the pore structure of gelatin support.Visible material surface is in uniform loose structure, and bone meal particle is uniformly alternate
In gelatin porous structure.
Tested more than and result, using the method for freeze-drying, can obtain hole be uniformly distributed, suitable size
Supporting structure, pore size is attachment of the stem cell in stock support and sprawls there is provided good between 20-100 microns
Good space structure；Bone meal after grinding is uniformly inlayed in supporting structure, makes the biological active matter played a role in the support
Matter well exposes, so as to play effect of its induction stem cell bone to differentiation；By the mixing of gelatin and Bio-oss bone meal, make
The elastic modelling quantity of resulting composition is controllable, has reached ideal grade scope of the suitable stem cell bone to differentiation；Resulting composition
Form, size can all be adjusted in above steps, can meet the space requirement of different defects.So, side of the invention
Method dexterously by combined factors influential on stem cell skeletonization such as space structure, rack elasticity, biological inductions together, it is maximum
The skeletonization effect that ensure that after bone grafting of degree.
Experimental example Osteoblast Differentiation Induction experiments
(1) culture of rat bone marrow mesenchymal stem cellses
Rat bone marrow mesenchymal stem cellses (BMSCs) can be stablized and pass on, and sprawl thin is seen under inverted phase contrast microscope
Born of the same parents are in fusiformis (such as Fig. 6), reach 90% density within three days or so, carry out 1:2 passages, this experiment takes 3-6 and carries out skeletonization point for cell
Change Induction experiments observation.
(2) preparation of base material
Matched using the crosslinking agent of table 3, experiment substrate is prepared by the following method：
A. Elastic forming board material dimethyl silicone polymer (Polydimethylsiloxane, PDMS) predecessor A agent (bases are taken
This component) in centrifuge tube, weigh its quality.
B. in mass ratio 10:1、20:1、35:1、60:1、80:1 ratio is separately added into B agent (crosslinking agent), is sufficiently mixed
Even, 5000rpm is centrifuged 5 minutes after trim, the bubble that the internal cause stirring of removal colloid is produced.
C. the PDMS mixed liquors that will be mixed are poured into culture dish, horizontal rest 1h, treat that colloid spreads in ware bottom completely
Afterwards, holding level inserts 70 DEG C of baking 24h in air dry oven.
D. it is placed in super-clean bench, under ultraviolet light after 2h, then one layer of type i collagen of 0.2mg/mL is covered in gel surface
(I-type collagen is purchased from protein solution：Sigma Ltd), overnight incubation.
E. before inoculating cell, with PBS gel 3 times, and 30~40min is irradiated with ultraviolet, treats that cell is inoculated with.
(3) base material hardness measurement
Elasticity measurement is carried out to base material using AFM (Atomic Force Microscope, AFM),
This experiment instrument is AFM (NT-MDT, NTEGRA).Rectangular pyramid needle point is used in experiment, AFM probe is positioned at substrate table
Face, measures under AFM force curve patterns, and curve obtained is fitted using hertz-Si Neideng models, obtains Young mould
Value.The dynamics measurement method is used for the mechanical performance of research material, using AFM power-distance Curve measuring system in identical
Measurement obtains the curve of material surface power under speed under load, and the material of each hardness measures at least 10 points, obtains at least respectively
10 force curves.Measured value such as table 3, it is known that, based elastic hardness declines with the reduction of crosslinking agent, and the difference of the order of magnitude is presented
It is different.
The base material of table 3 is with when Hardness Measurement Results
(4) immune-blotting method (Western blot) is into bone seeker protein expression
The extracellular matrix and a blank that rat bone marrow mesenchymal stem cellses are incubated at above-mentioned 5 different hardness respectively are trained
Support on ware, extract the total protein of cell after being inoculated with 7 days, Diagnosis of Sghistosomiasis document is carried out with the specific antibody of Col-I, OPN, BMP2
Test, GAPDH is used as internal reference.
Result such as Fig. 7, in figure, A is the slice result of immunoblotting analysis, and B, C, D are the data statistics to A results.The experiment
Result comes from 8 independent experiments, and the numerical value of its statistics is the result averaged with normalizing after standard deviation in each group, each group
Value and 1:10 groups are compared, and " * " represents P<0.05.
(5) Real-time fluorescence quantitative PCR detections skeletonization marker mRNA horizontal expression
The extracellular matrix and a blank that rat bone marrow mesenchymal stem cellses are incubated at above-mentioned 5 different hardness respectively are trained
Support on ware, extract the total serum IgE for collecting cell after inoculation 7 days, reverse transcription is cDNA, with the promoter region of Col-I, OPN, BMP2
Primer carries out real-time fluorescence quantitative PCR and goes to detect the amount of the DNA fragmentation being enriched to that GAPDH is used as internal reference.
Result such as Fig. 8, A, B, C, D are data statistics of the result of real-time quantitative PCR to experiment gained CT values in figure.The reality
Result is tested from 4 independent experiments, the numerical value of its statistics is the result averaged with normalizing after standard deviation in each group, respectively
Class value and 1:10 groups are compared, and " * " represents P<0.05, " * * " represents P<0.01.
(6) immunofluorescence dyeing detects into the distribution of bone seeker
After 6 groups of cells are cultivated 7 days in above-mentioned different base, cell is collected, using anti-Col-I, OCN, OPN antibody,
FITC, rhodamine and DAPI dyeing, laser confocal microscope observation (20 × oil mirror).
Result is shown in Fig. 9-12.Fig. 9-11 is the cell dyeing result under laser confocal microscope；At random 5 are selected to regard
Open country, calculates cell fluorescence intensity, amounts to cell concentration and is more than 50, statistics such as D figures.The experimental result is independent real from 3 times
Test, the numerical value of its statistics is the result averaged with normalizing after standard deviation in each group, each class value and 1:10 groups are compared
Compared with as a result such as Figure 12, " * " represents P<0.05, " * * " represents P<0.01.Green：Show Col-I and OPN, it is red：Show OCN, it is blue：
Show nucleus.
(7) alkaline phosphatase (ALP) determination of activity
After 6 groups of cells cultivate 7 days on different substrates, alkaline phosphatase activities detection is carried out with the method for dyeing, dyeed
Visually observed respectively afterwards and Microscopic observation.
Result is shown in Figure 13-14.Figure 13 show cell dyeing in culture dish visually observe situation (on) and inverted microscope under
Observation situation (under)；Lower 5 visuals field of mirror are randomly selected, the statistics of stain density, each class value and 1 is carried out:10 groups of (high resiliency moulds
Amount) it is compared, gained statistics such as Figure 14, " * " represents P<0.05.
Conclusion：By above description of test, using different detection methods, on different detection levels, rat marrow mesenchyma
It is poor to breaking up that stem cell (BMSC) shows roughly the same bone under the induction of dimethyl silicone polymer (PDMS) elastic substrates
Different, compared with harder substrate induction condition, its differentiation peak is appeared in the range of 0.354 ± 0.04MPa of based elastic modulus,
The result shows the bone filler of high elastic modulus may not as Bio-oss bone meal (elastic modelling quantity about 1GPa)
For stem cell provides optimal physical environment to Osteoblast Differentiation, and the present invention is using the bone meal and low elasticity mould of high elastic modulus
The gelatin of amount is mixed, and reduces the elastic modelling quantity of material, and by attempting, realize specific material preparation process,
Simulate the structure of suitable stem cell growth differentiation.Most of all, under this theoretical direction tested, having obtained close to reality
Test the preferred gelatin-bone meal ratio and elastic modelling quantity numerical value of conclusion.Under the guidance of the numerical value, can be filled out by bone of the invention
Fill material compositions and prepare suitable stiff/elasticity, have bone guided and the good filling material of bone of bone inductive effect, plasticity concurrently.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted.Art technology
Personnel in the case of the objective and scope for not departing from technical solution of the present invention, the modification that is carried out to technical scheme or
Person's equivalent, all should cover in the middle of scope of the presently claimed invention.
Claims (10)
1. a kind of bone filler composition, it is characterised in that including bone meal, gelatin and crosslinking agent.
2. bone filler composition according to claim 1, it is characterised in that the bone meal is based on hydroxyapatite
Want the bone alternate material of composition；It is preferred that Bio-Oss bone meal.
3. bone filler composition according to claim 1 and 2, it is characterised in that the crosslinking agent is Geniposide, penta
One or more in dialdehyde and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC HCl)；It is preferred that capital Buddhist nun
It is flat.
4. the bone filler composition according to claim any one of 1-3, it is characterised in that
The gelatin is 0.05-0.4 with the mass ratio of bone meal:1, preferably 0.15-0.4:1；And/or
The consumption of the crosslinking agent is that can reach the amount for being crosslinked purpose, the preferably 0.05-0.5% of bone filler gross weight,
It is preferred that 0.2%.
5. a kind of bone filler reserve, it is characterised in that the bone filler composition described in claim any one of 1-4
The freeze-drying prods obtained after the freeze-dried treatment of solution of preparation.
6. a kind of method for preparing bone filler reserve, it is characterised in that including to bone described in claim any one of 1-4
Solution prepared by filler composition implements freeze-drying process.
7. method according to claim 6, it is characterised in that the freeze-drying process is carried out in two steps：
The first step, prepares gelatin solution, and the mixture of gelatin solution and bone meal, freeze-drying gelatin solution are mixed into bone meal
With the mixture of bone meal, gelatin-bone meal support is obtained；
Second step, prepares cross-linking agent solution, and is mixed into cross-linking agent solution and gelatin-bone meal support with gelatin-bone meal support
The mixture of mixture, freeze-drying cross-linking agent solution and gelatin-bone meal support, obtains gelatin/crosslinking agent-bone meal support, obtains institute
State bone filler reserve.
8. method according to claim 7, it is characterised in that
During first step freeze-drying, the mass fraction of institute's gelatine solution is 5-10%；And/or
During second step freeze-drying, the mass fraction of the cross-linking agent solution is 0.3%-1%, preferably 0.3%.
9. a kind of method for preparing bone filler, it is characterised in that including：
Bone filler composition described in claim any one of 1-4 is mixed with medically acceptable solvent；Or
Bone filler reserve prepared by claim any one of 6-8 is mixed with medically acceptable solvent.
10. the bone filler that prepared by claim 9 methods described is in scientific research field and the application as medical material.
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| CN110947031A (en) * | 2019-09-25 | 2020-04-03 | 中山大学附属第八医院（深圳福田） | Bone tissue engineering scaffold material with high biological activity and preparation method and application thereof |
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