The preparation method of Nano pearl powder/C-HA compound restsTechnical field
The present invention relates to bioengineering field, and in particular to a kind of preparation method of Nano pearl powder/C-HA compound rests.
Background technology
Cranial defect turns into global health problem.With aging population and the development of society, the reparation of Cranial defect intoFor an important clinical problem and social demand.At present, the treatment clinically for Cranial defect depends on allogeneicBone collection or autologous bone transplanting, but all there are many limitations in both therapeutic modalities.For a long time, autologous bone transplanting is all publicThink the goldstandard of bone collection, but there is the shortcomings of desirable bone amount is limited, bone piece for area's complication, transplanting easily absorbs；AllosomeThen there is immunological rejection and propagate the risk of communicable disease in bone collection.
With the development of medical science, biology and material science, metal material, inorganic non-metallic material and organic material also byReparation applied to Cranial defect.However, research more stainless steel, titanium alloy, aluminium oxide ceramics etc. belong to biologically inert materialMaterial, it is impossible to combined well with tissue, limit its clinical practice.Therefore, it is clinical to promoting bone tissue regeneration, and haveThe demand sustainable growth of the synthetic material of excellent mechanical performances and biocompatibility.
At present, bone tissue engineer has turned into a kind of effective ways that osteanagenesis is treated.It is different from conventional method, bone tissue workJourney is intended to evade the limitation of existing clinical treatment, and the biological substitution product of individuation are prepared to impaired tissue or organ.Bone tissue engineer typically refers to utilize porous bioabsorbable support, guides histiocytic adhesion, propagation and breaks up, so as to promoteEnter regeneration.Support can simulate the function of nature bone, be most important part in bone tissue engineer.Preferable support should possessFollowing characteristics：1. there is good bioactivity, biocompatibility and biological degradability；2. there is suitable pore structure, haveThe growth and the transport of nutriment of propagation, blood vessel beneficial to cell；3. there is good configuration of surface and physicochemical property, canThe release of induced intracellular signal；4. the given shape of Cranial defect is adapted to.
Tissue scaffold design should have appropriate mechanical performance, the complicated ambient stress of human skeletal system be adapted to, with new lifeBone tissue is combined.If the modulus of elasticity of support is excessive, it can cause stress shielding after implantation a period of time, eventually result in bone and repairMultiple failure.The processing conditions and application environment of support depend greatly on the physical property and mechanicalness of timbering materialEnergy.Bioactive ceramics can promote the growth and differentiation of Gegenbaur's cell, however, because its fragility is big, it is difficult to be molded, and degrade non-Often slow the shortcomings of, its clinical practice is restricted.In addition, the accuracy of bioactive ceramics is poor, and porous ceramics scaffoldThe new bone of upper formation can not maintain the mechanical load needed for heavy burden bone.Therefore, its clinical practice is also by more limitation.
Support is except needing appropriate mechanical performance, in addition it is also necessary to obtain loose structure, and the adhesion and propagation for cell are providedSpace, this is also the key point of support.Nature bone has multi-level three dimensional pore structures, and pore diameter is from several nanometers to hundreds ofMicron, can meet the different requirements of tissue growth.Under normal circumstances, the aperture of 150-800 μm of diameter can allow bone tissueWith growing into for big blood vessel；The hole of 10-100 μm of diameter is conducive to the growth of capillary, and the exchange and metabolism of nutriment are uselessThe discharge of thing；Nano level hole can provide bigger specific surface area, be conducive to formation and the Gegenbaur's cell albumen of apatiteThe adhesion of matter, the adhesion and propagation to Gegenbaur's cell is significant.
In recent years, researchers have found the method that some prepare the support with control pore structure and shape, such as moltenThaw collapse product modeling, stereolithography and 3D printing technique.Guo etc. is prepared for good mechanical properties by Templated FDM methodsWith the support of topological property, they have found, with the increase of matrix modulus, and the aperture of support reduces, and marrow stem can be promoted thinThe Osteoblast Differentiation of born of the same parents.Be prepared for calcium phosphate cement bracket material using indirect SL technologies, the inside of support have high opening,The channel design of interconnection.Hydroxyapatite and poly- (ethene) ethanol are prepared into tissue scaffold design using 3D printing technique,The structure of support can be pre-designed, and ensure most interconnectivity.However, due to the limitation of hot spot or nozzle diameter,These technologies are difficult the support for preparing pore diameter range from several nanometers to several microns.Therefore, it can not meet different tissues growthDemand.
Freeze-drying is also known as lyophilization method, and its principle is first to be chilled to aqueous material below freezing, makes liquidThe water of state is frozen into solid-state, then under the conditions of high vacuum, and ice is directly distilled and removed for steam.Left after water sublimedHole just constitute the pore structure of support, therefore, freeze-drying can be by controlling the size of solution concentration to control holeRate.Drying process is carried out at a lower temperature, it is to avoid harmful effect to albumen in timbering material.Due to these advantages,Scholar selects freeze-drying to carry out the preparation of tissue scaffold design more and more.
Someone is prepared for porous hydroxyapatite/chitosan sugar-sodium alginate compound rest using freeze-drying, with heightSpend interrelated between porous, hole.Tested and found by MTT, prepared hydroxyapatite/chitosan-sodium alginateCompound rest does not produce toxicity to MG-63 cells, shows that compound rest has good cell compatibility.Further by hydroxyl phosphorusLime stone/chitin-sodium alginate compound rest is implanted into the skull of mouse, it is found that compound rest can promote repairing for internal Cranial defectIt is multiple.Also pearl powder is combined by someone using freeze-drying with PLA, is successfully prepared with good biocompatibilityPLLA/ aragonitic pearl powder supports and PLLA/ shell pearl layer powder supports, and find that both supports can promote mouse bone marrow cellsThe propagation of mescenchymal stem cell and differentiation.Oyster shell whiting is added in alginates by somebody using freeze-drying, is prepared into threePorous support is tieed up, discovery can promote adhesion and the propagation of cell, there is good application prospect in bone tissue engineer.
The phosphate biological ceramics such as hydroxyapatite, bata-tricalcium phosphate are applied to bone tissue engineer, and existing more than 30 years goes throughHistory.But, hydroxyapatite is not degradable in vivo, and the degradation speed of tricalcium phosphate can not regulate and control again, and natural biologic materialWith good biocompatibility and biodegradability, and by inducing the release of the bio signal factor skeletonization can be promoted thinAdhesion, propagation and the differentiation of born of the same parents.Also, with the development of tissue engineering technique, natural biologic material is modified, one can be enteredThe ability of its promotion osteanagenesis of step enhancing, is that the reparation of Cranial defect brings Gospel.
For a long time, pearl powder is widely used in the multiple fields such as Chinese medicine, cosmetics and health products in China.Pearl powderThere is very high medical value, be made up of aragonite calcium carbonate crystal and the organic substrate being embedded, and can promote containing a variety ofEnter the signaling molecule of bon e formation, be a kind of very promising bone renovating material with good biocompatibility.Pearl powder containsThere is abundant mineral matter, especially containing high-quality calcium and " signal protein ", cell can be controlled by the secretion signal factorGrowth and reparation.Signal protein contained by pearl powder, can promote skin regeneration and tissue repair.Research shows, pearl powderThe insoluble matter that water-soluble base and molecular weight are more than 14kDa can be obviously promoted fibroblastic propagation, collagen depositionWith TIMP-1 generation, so as to promote wound healing, and its main active is probably the insoluble matter that quality is more than 14kDa.In addition, these signal proteins also play an important role in terms of bone health is promoted；They can promote the differentiation of osteocyte, accelerateIts mineralising, so as to increase the density of existing bone.Therefore, if can apply pearl powder on tissue scaffold design, then Jiang HuiThree-dimensional cell culture, tissue engineering bracket and regenerative medicine field are with a wide range of applications.
The content of the invention
The technical problem to be solved in the present invention is to provide one kind have suitable pore structure, be conducive to cell propagation,The growth of blood vessel and the transport of nutriment, with good bioactivity, biocompatibility and biological degradability, adapt to boneThe Nano pearl powder of the given shape of defect/C-HA compound rest preparation methods.
In order to solve the above-mentioned technical problem, the present invention provides following technical scheme：Nano pearl powder C-HA compound restsPreparation method, its operating method is：
First, chitosan and hyaluronic acid are dissolved into the acetum that concentration is 1% respectively, stirred with 2000rpm speedMix and place 24h under 5min, room temperature condition, obtain chitosan-acetic acid solution and hyaluronic acid acetum；
2nd, chitosan-acetic acid solution and hyaluronic acid acetum are mixed after 24h, stirred with 2000rpm speed5min；
3rd, the Nano pearl powder of addition 1~25%, 5min is stirred with 2000rpm speed；
4th, -20 DEG C of refrigerator 24h, then 24h is freeze-dried, obtain compound rest.
The Nano pearl powder prepared using technical solution of the present invention /C-HA compound rests, according to experimental study, of the invention answersThe good wetability of support, compression strength are closed, and is conducive to the propagation of cell, and with good bioactivity, bio-compatibleProperty and biological degradability, adapt to Nano pearl powder/C-HA compound rests and its application of the given shape of Cranial defect.
Further, described hyaluronic acid, the ratio of chitosan are：Hyaluronic acid 1%, chitosan 4%.
Further, the ratio of contained pearl powder is 1~20% in described raw material.
Further, the ratio of contained pearl powder is 5~20% in described raw material.
Further, the compound rest being prepared from, it is shaped as a diameter of 10~20mm, the highly cylinder for 2~10mmBody, with multiple equally distributed pore structures.
Further, the compound rest being prepared from, the porosity of its pore structure is between 89~93%.
Further, the compound rest being prepared from, its crystal formation includes aragonite calcium carbonate crystal formation and calcite crystal formation.
Further, a diameter of 15mm of the described compound rest being made, is highly 5mm.
The compound rest being prepared from by the preparation method of Nano pearl powder C-HA compound rests of the present invention, passes through experimentThe properties and its Osteogenic Mechanism of its compound rest are discussed, conclusion is：(1) it is multiple with the increase of Nano pearl powder ratioWetability and the compression strength increase of support are closed, but the pore structure of support is interfered, porosity slightly declines.It was combinedCheng Zhong, the crystal formation of Nano pearl powder does not change.(2) mouse MC3T3-E1 cells well-grown on compound rest, in shuttleShape, distribution uniform.The increase of Nano pearl powder promotes propagation and the differentiation of MC3T3-E1 cells, to the apoptosis of cell without brightDevelopment rings, and the cell compatibility of compound rest is good.(3) RT-qPCR experiments find that nano-pearl powder content is 10% and 25%Support group Col α tetra- genes of I, OCN, OPN and Runx2 expression be higher than control group.In Western blotting experiments,Nano-pearl powder content is higher than control for the synthesis of 10% and 25% support group Col α tetra- albumen of I, OCN, OPN and Runx2Group；But nano-pearl powder content is less than control group for the synthesis of 0% support group Partial Protein.A high proportion of Nano pearl powder/C-HA compound rests may be by raising I-type collagen, BGP, bone bridge element and the expression and the conjunction of albumen of Runx2 genesInto promotion skeletonization.As can be seen here, Nano pearl powder/C-HA compound rests have good application prospect in terms of bone tissue engineer.
Brief description of the drawings
Fig. 1 is the substantially aspect graph of each group compound rest of present invention experiment one；
Fig. 2 is that the present invention tests hole shape of the one Nano pearl powder concentration under ESEM for 0% compound restState figure；
Fig. 3 is that the present invention tests hole shape of the one Nano pearl powder concentration under ESEM for 1% compound restState figure；
Fig. 4 is that the present invention tests hole of the one Nano pearl powder concentration under ESEM for 2.5% compound restAspect graph；
Fig. 5 is that the present invention tests hole shape of the one Nano pearl powder concentration under ESEM for 5% compound restState figure；
Fig. 6 is that the present invention tests hole shape of the one Nano pearl powder concentration under ESEM for 10% compound restState figure；
Fig. 7 is that the present invention tests hole shape of the one Nano pearl powder concentration under ESEM for 25% compound restState figure；
Fig. 8 is the XRD spectra of each group compound rest of present invention experiment five；
Fig. 9 is the Fourier transform infrared spectroscopy figure of Nano pearl powder in present invention experiment six；
Figure 10 is the Fourier transform infrared spectroscopy figure of chitosan in present invention experiment six；
Figure 11 is the Fourier transform infrared spectroscopy figure of hyaluronic acid in present invention experiment six；
Figure 12 is the Fourier transform infrared spectroscopy that the present invention tests the compound rest that Nano pearl powder concentration in six is 0%Figure；
Figure 13 is the Fourier transform infrared spectroscopy that the present invention tests the compound rest that Nano pearl powder concentration in six is 1%Figure；
Figure 14 is the Fourier transform infrared light that the present invention tests the compound rest that Nano pearl powder concentration in six is 2.5%Spectrogram；
Figure 15 is the Fourier transform infrared spectroscopy that the present invention tests the compound rest that Nano pearl powder concentration in six is 5%Figure；
Figure 16 is the Fourier transform infrared light that the present invention tests the compound rest that Nano pearl powder concentration in six is 10%Spectrogram；
Figure 17 is the Fourier transform infrared light that the present invention tests the compound rest that Nano pearl powder concentration in six is 25%Spectrogram；
Figure 18 is that Nano pearl powder in six, chitosan, hyaluronic acid, the ratio of Nano pearl powder of the invention of testing is 0%The Fourier transform infrared spectroscopy of~25% compound rest compares figure；
Figure 19 is the aspect graph of MC3T3-E1 cells under the ESEM in present invention experiment seven；
Figure 20 is distribution map of the MC3T3-E1 cells on compound rest in present invention experiment seven.
Embodiment
First, embodiment：
Nano pearl powder of the present invention/C-HA compound rests, the ratio containing pearl powder is as shown in table 1, single wherein in raw materialPosition wt%：
Table 1
| Embodiment | The ratio wt% of contained Nano pearl powder in raw material |
| Embodiment one | 1 |
| Embodiment two | 2.5 |
| Embodiment three | 5 |
| Example IV | 10 |
| Embodiment five | 25 |
Now by taking embodiment three as an example, the preparation method of Nano pearl powder of the present invention/C-HA compound rests is illustrated；
Embodiment three：
1st, reagent, as shown in table 2
Table 2
| Reagent | Company and model |
| Micron pearl powder | Hainan Prov Beijing Run pearl Biotechnology Co., Ltd |
| Sodium Hyaluronate | H107141, Aladdin |
| Chitosan | C105803, Aladdin |
| Acetic acid | Analyze pure, Aladdin |
2nd, instrument is as shown in table 3
Table 3
3rd, operating procedure
1) dispersion machine is sanded by nano ceramics uses mechanical attrition method that micron pearl powder is ground to form into Nano pearl powder, doesDry sterilization；
2) 4wt% chitosans and 1wt% hyaluronic acids are dissolved into 1% acetum respectively, in Thinky stirringsStirred in machine with 2000rpm speed and place 24h under 5min, room temperature condition, obtain chitosan-acetic acid solution and hyaluronic acid acetic acidSolution；
3) chitosan-acetic acid solution and hyaluronic acid acetum are mixed after 24h, in Thinky mixers with2000rpm speed stirring 5min；
4) and then 2.5wt% Nano pearl powder is added, 5min is stirred with 2000rpm speed；
5) with syringe by solution-cast in 24 orifice plates, per hole 0.5ml, in -20 DEG C of refrigerator 24h, be freeze-dried 24h,Obtain Nano pearl powder/C-HA compound rests.
Two：The confirming performance of Nano pearl powder/C-HA compound rests
1st, experimental group：
Experimental group one：Compared with embodiment three, the ratio for differing only in contained Nano pearl powder in raw material is 0wt%；
One~embodiment of embodiment five
2nd, experiment reagent and instrument
It is identical with instrument with the reagent of embodiment
One) one, is tested：Determination to compound rest surface topography
1st, the surface topography of compound rest is observed by SEM
Sample is cut into the wide thin slices of 3mm, makes sample surfaces as far as possible smooth.By sample after being dried with vacuum freeze drierIt is bonded at successively on conducting resinl, mark is carried out in the upper left corner of conducting resinl, by sample surface metal spraying 60s.Then scanning electron is openedThe sample room of microscope (MIRA3, Czech), the sample to be observed is fixed, vacuumized, section is directly observed with ESEMPattern.
2nd, conclusion
As shown in figure 1, support has highly porous, hole is uniformly distributed, and connection is good.Discovery is further looked at, whenWhen nano-pearl powder content is relatively low, pore structure and the experimental group one of compound rest：Simple Chitosan-Hyaluronic Acid support phaseSeemingly.However, as shown in Figure 2 to 7, as nano-pearl powder content is gradually stepped up from 0%, when reaching 25%, the surface of supportGradually coarse, pore structure tends to be disorderly, is locally broken.
The ratio for illustrating contained Nano pearl powder in raw material is 1~25wt%, and its pattern can meet the outer of compound restShape requirement.
Two) two, are tested：Determination to compound rest porosity
1st, method
The porosity (%) of compound rest, always enter amount (mL/g) and total hole area (m2/ g) mercury is automatically pressed by high-performanceInstrument is measured.The compound rest sample prepared by one~embodiment of embodiment five, experimental group one is placed in sample box, adjustedPressure, makes mercury enter holes all in material, the weight before and after determination sample infiltration.
2nd, conclusion
As shown in table 4：With the increase of Nano pearl powder ratio, the porosity of support is reduced with amount is always entered, and total hole faceProduct increase, but experimental group one and one~embodiment of embodiment five all have higher porosity, are between 89% to 93%, to implementSupport of the porosity of example one less than experimental group one and embodiment two.
Table 4
The porosity of one~embodiment of embodiment five is higher as can be seen here.
Three) three, are tested：The determination of compound rest wetability
1st, method
Wetability is mainly by water droplet in the contact angle that its surface is formed come what is characterized, and the present invention passes through JC2000C typesStatic Contact angle measuring instrument (Powereach, Shanghai Zhongchen digital technology equipment Co., Ltd, China) carries out composite membrane contact angleMeasure.
2nd, conclusion
The occurrence of each group compound rest contact angle is as shown in table 5, it is seen then that with the increase of Nano pearl powder ratio,The contact angle of compound rest diminishes, i.e. the wetability increase of compound rest, and the support wetability of embodiment one is worst, embodiment4th, five support wetability is optimal.
Table 5, unit：Degree
Four) four, are tested：The determination of compound rest compression strength
1st, method
Present invention use CTM8050 microcomputer controlled electronic universal testers (strong instrument Science and Technology Ltd. of upper Taiwan Strait Exchange Association, inState) carry out compound rest compression strength measurement.
2nd, conclusion
As shown in table 6, the compression strength of compound rest is dramatically increased with the increase of Nano pearl powder ratio.
Table 6
| Group | Ratio | Sample 1 | Sample 2 | Sample 3 |
| Experimental group one | 0% | 0.172 | 0.164 | 0.155 |
| Embodiment one | 1% | 0.211 | 0.219 | 0.225 |
| Embodiment two | 2.5% | 0.292 | 0.309 | 0.295 |
| Embodiment three | 5% | 0.312 | 0.348 | 0.348 |
| Example IV | 10% | 0.339 | 0.399 | 0.4 |
| Embodiment five | 25% | 0.575 | 0.575 | 0.585 |
Five) five, are tested：The determination of compound rest crystal formation
1st, method
Research shows that aragonitic pearl powder can advantageously promote propagation and the differentiation of cell than the pearl powder of ball aragonitic.In the present invention, the compound raw material of support is aragonitic pearl powder, and compound process includes mixing, stirring, dries and sterilize.ForWhether the crystal formation of pearl powder changes in detection recombination process, to compound rest carries out X-ray diffraction with X-ray diffractometerDetection.
2nd, conclusion
As shown in Figure 8：When Nano pearl powder ratio than it is relatively low when, its characteristic peak is not obvious.When Nano pearl powderWhen content is more than 5%, its characteristic peak occurs.In the other compound rest of each group, aragonite calcium carbonate is main crystal formation, onlyThere is a small amount of calcite crystal formation.
Six) six, are tested：The composition of compound rest is determined
1st, method
Infrared spectrum is a kind of spectrum of molecule absorption, also known as vibration-rotation spectrum.Its principle is, when molecule is by redThe radiation of outer light, produces the transition of vibration level.When having dipole moment change when in vibration, infrared photon is just absorbed, so that shapeInto infrared absorption spectroscopy.Infra-red sepectrometry can be from the molecular structure of the characteristic absorption peak authenticating compound of molecule, so as to carry out thingThe qualitative and quantitative analysis of matter, based on qualitative analysis.
The present invention is by fourier transform infrared spectroscopy, and detection and analysis Nano pearl powder, chitosan, hyaluronic acid are differentInteraction between composition molecule.
2nd, conclusion
Detect each in one~embodiment of embodiment five, the compound rest recombination process of experimental group one by FTIR spectrumReaction between composition.The FTIR spectrograms of Nano pearl powder/C-HA compound rests, from spectrogram can be absorbed peak position, inhaleReceive the intensity at peak and the shape of absworption peak.
As shown in Fig. 9~Figure 18：
In spectrogram, 3600 to 3200cm-1Peak be O-H stretching vibration absworption peak.The spectrogram of Nano pearl powder (NPP)In, 1488cm-1For C-O stretching vibration absworption peaks, 860cm-1For CO32-Flexural vibrations absworption peak.
In the spectrogram of chitosan (C), 2879cm-1For C-H stretching vibration absworption peaks, 1650cm-1And 1589cm-1Place's ownershipFor the overlapping of acid amides I and the peaks of acid amides II two.1155cm-1For C-O-C antisymmetric stretching vibration absworption peaks, 1085cm-1For sugared skeletonC-O stretching vibration absworption peaks.
In hyaluronic acid (H) spectrogram, 2921cm-1For C-H stretching vibration absworption peaks, 1623cm-1And 1413cm-1PointCOO- asymmetric and symmetrical stretching vibration absworption peak is not belonged to.1558cm-1For acid amides peak, 1155cm-1Oppose for C-O-CClaim stretching vibration absworption peak, 1044cm-1Locate C-H the and C-O-C elastic vibration absorption that adjacent two peak is attributed on sugared skeleton respectivelyPeak.
As can be seen here：Influence of the hyaluronic acid to compound rest spectrogram is less than chitosan.Compared with the spectrogram of chitosan, respectivelyAmide I peaks and acid amides II peaks there occurs that blue shift, i.e. hypsochromic shift are moved in ratio compound rest.With the spectrogram phase of hyaluronic acidThan 1700 to 900cm in each ratio compound rest-1Peak intensity weaken.In the spectrogram of each ratio compound rest, nanometer is preciousPearl powder, chitosan, the peak of three kinds of compositions of hyaluronic acid are superimposed, and with the increase of Nano pearl powder ratio, its characteristic peak(1488cm-1And 860cm-1) gradually obvious.Show that pearl powder can be effectively embedded in polymer.
3rd, the cell compatibility experiment of Nano pearl powder/C-HA compound rests
In the research of bone tissue engineer, the selection to timbering material, cell compatibility is one of primary index.AlthoughPearl powder, hyaluronic acid, chitosan three have good biocompatibility, but three is combined into the bio-compatible after supportProperty is unclear.In the present invention, the form of cell and distribution on the compound rest of each embodiment and experimental group are observed；Pass throughThe proliferative conditions of CCK-8 testing inspection each group cells；Pass through the differentiation of ALP testing inspection each group cells；Pass through flow cytometryDetect the apoptosis of each group cell；So as to assess the cell compatibility of Nano pearl powder/C-HA compound rests.
1st, experimental group
Experimental group one：Compared with embodiment three, the ratio for differing only in contained Nano pearl powder in raw material is 0wt%
One~embodiment of embodiment five
2nd, experiment reagent, as shown in table 7
Table 7
3rd, laboratory apparatus, as shown in table 8
Table 8
One) seven, are tested：Cellular morphology, distribution in cell culture, compound rest
1st, method
The compound rest of the one~embodiment of embodiment five prepared, experimental group one is carried out disinfection with Co 60, agent is irradiatedMeasure as 15kGy.Compound rest is immersed in α-MEM basal mediums (HyClone, the U.S.) after 12h and carries out cell culture.WillSupport is placed in 24 orifice plates, and the 50 μ l complete mediums containing 50,000 cell are injected per hole, 37 DEG C are placed on, containing 5%CO2TrainingSupport in case and cultivate 1h, cell culture is carried out after adding 1ml complete mediums after cell attachment per hole, the next day change liquid.
Cell is cultivated after 7d on support, support is transferred in 24 new orifice plates, with 2.5% glutaraldehyde solution 4DEG C fixation is stayed overnight, and is dehydrated with graded ethanol solutions (0%, 30%, 50%, 75%, 95%, 100%), each concentration dehydration twoIt is secondary, finally with gradient t-butanol solution (25%, 50%, 100%)) wash away ethanol.Support is freeze-dried 4h, in ESEMThe form of cell on support is observed under (MIRA3, Czech).
Sample is cut into the wide thin slices of 3mm, makes sample surfaces as far as possible smooth.Sample is bonded at conducting resinl successively after dryingOn, surface metal spraying 60s.Then the sample room of ESEM is opened, the sample to be observed is fixed, vacuumized, with scanning electricityThe form of sem observation cell.
2nd, conclusion
By mouse MC3T3-E1 cell culture on compound rest, the next day change liquid, cell under observation three-dimensional cultivation conditionForm.Figure 19 is the form of MC3T3-E1 cells under ESEM, and arrow show MC3T3-E1 cells, and in fusiformis, cell is stretchedGo out pseudopodium to be attached on support.Figure 20 shows that blue-fluorescence (white point) is in distribution of the MC3T3-E1 cells on support, figureThe nucleus of MC3T3-E1 cells, light blue floccule (canescence floccule) is timbering material, distribution of the cell on supportIt is more uniform.
MC3T3-E1 cells on compound rest are in fusiformis, situation are sprawled preferably, with the cell under two-dimentional cultivation conditionsForm is similar, shows that compound rest has good mechanical performance, is conducive to the adhesion and growth of cell.MC3T3-E1 cells existDistribution uniform on support, illustrate MC3T3-E1 cells can on compound rest well-grown, show that support has good thinBorn of the same parents' compatibility.
Two) eight, are tested：The detection of cell proliferative conditions on compound rest
1st, method
Using CCK-8 kits respectively at the proliferative conditions of cell on culture 1d, 3d, 5d, 7d detection support, each sample3 multiple holes of this setting, with spectrophotometer (Bio-Rad xMarkTM, the U.S.) detection 450nm at absorbance.
2nd, conclusion
The proliferative conditions for assessing each group cell are tested by CCK-8 in cell culture 1d, 3d, 5d and 7d, such as the institute of table 9Show.Increase over time, the MC3T3-E1 cells of each support group are bred substantially.Cell concentration between 1d each groups is without substantially poorIt is different.After 3d, the cell of each group rapidly increases, and with the increase of Nano pearl powder ratio, cell breeds more obvious, cellThe most support group for being 25% for Nano pearl powder ratio of propagation.
Increase over time, each support group MC3T3-E1 cells are bred substantially, show that compound support frame material has goodGood cell compatibility.With the increase of Nano pearl powder ratio, cell propagation is more obvious, and this shows that Nano pearl powder can haveEffect ground promotes the propagation of MC3T3-E1 cells.Cell concentration no significant difference between 1d each groups, this is probably due to MC3T3-E1Cell be substantially carried out in initial 24h it is adherent, not yet carry out cell propagation.
Table 9
Three) nine, are tested：The detection of cell differentiation on compound rest
1st, method
Alkaline phosphatase can effectively assess the differentiation situation of MC3T3-E1 cells.By MC3T3-E1 cells with 5 × 105Individual/The density kind of support, with osteogenic culture 28d, is tested in 0d, 7d, 14d, 28d with alkaline phosphatase respectively on supportBox (Bioengineering Research Institute is built up in A059-1, Nanjing) is detected.
2nd, conclusion
It is thin by the Activity Assessment each group for detecting the alkaline phosphatase of MC3T3-E1 cells in cell culture 1d, 7d, 14dThe differentiation situation of born of the same parents.Cell suspension in each group timbering material sample, is computed the alkaline phosphatase activity of gained each group cell(mg/gprot) result is as shown in table 10.Increase over time, the activity increase of alkaline phosphatase；With Nano pearl powderIncrease, the activity of alkaline phosphatase gradually increases, and the difference between each group is most obvious in 7d.
Illustrate that compound rest improves the activity of Cellular alkaline phosphatase in early stage, promote the differentiation of cell.
Table 10
Four) ten, are tested：The detection of Apoptosis on compound rest
1st, method
Annexin V are to detect one of sensitive indexes of Apoptosis.It is a kind of cardiolipin binding protein, can be with early stageThe after birth of apoptotic cell is combined, and one of the change of the change of cytoplasma membrane when being apoptosis earliest.By MC3T3-E1Cell is with 5 × 105The density kind of individual/support is collected after being digested after culture 3d, 7d, 10d with pancreatin, passed through on supportThe apoptosis situation of Annexin V Flow cytometry cells.
2nd, conclusion
The apoptosis situation of Flow cytometry each group cell is used in cell culture 3d, 7d, 10d.By each group cellApoptosis rate statistics is as shown in table 11, and the apoptosis rate of each group shows compound rest not in normal range (NR), and without notable differencePromote the apoptosis of cell, and do not influenceed by Nano pearl powder ratio.
As a result show, Nano pearl powder/C-HA compound rests do not promote the apoptosis of cell, support has nontoxicity, and notInfluenceed by Nano pearl powder ratio.
Table 11
In summary：Mouse MC3T3-E1 cell successful growths are on support, and within the specific limits, a high proportion of compoundSupport promotes propagation and the differentiation of cell, and cell compatibility is good.
4th, the influence that Nano pearl powder/C-HA compound rests are expressed Bone formation-related gene, albumen is synthesized
Bone is considered as a kind of structure organ always.Classical theory thinks that bone plays structure stand in animal body, bagCell containing three types：Gegenbaur's cell, osteocyte and osteoclast.Gegenbaur's cell derives from mesenchymal stem cells MSCs, energyIt is divided into osteocyte.Gegenbaur's cell is the chief functional cells of bon e formation, and its activity can pass through a variety of ways such as Wnt signal pathsFootpath is adjusted.Osteoclast can absorb bone tissue, from the monocyte of Monocytes/Macrophages system.Osteocyte and Gegenbaur's cellSome signal factors, such as type i collagen, BGP, bone bridge element and Runt associated transcription factors 2, to adjust Gegenbaur's cell can be secretedWith the interaction of osteoclast, so as to carry out the regeneration and reconstruction of bone.
Type i collagen be bone extracellular matrix in one of main albumen.Collagen can provide cell surfaceThe binding site of integrin receptor, such as RGD motif, therefore to cell adherence, cell survival, migration and maintain tissue and organPhysical stability is all essential.
BGP is a kind of Osteoblast Specific albumen, is a kind of main non-collagen in extracellular matrixMatter.BGP is synthesized and secreted by Gegenbaur's cell system, such as Gegenbaur's cell, osteocyte.Most BGP is present in bone matrixIn, only it is present on a small quantity in blood, because its affinity to bone matrix is stronger.It has the work(for facilitating bone and bon e formationEnergy.Gegenbaur's cell, which can secrete BGP, stimulates osteoblast differentiation and the maturation of osteocyte.
Since bone bridge element was described for the first time from before more than 20 years, played an important role in many physiology and pathologic process,Including biomineralization, tissue remodeling and inflammation.As extracellular matrix protein and pro-inflammatory cytokine, OPN is considered as to promoteEnter the recruitment of monocyte and macrophage, and mediated leucocytes secrete cytokines.
A large amount of in vivo and in vitro are overexpressed using Runt associated transcription factors 2 to be carried out into come inducing bone mesenchymal stem cellBone breaks up, and induces the activity of some Bone formation-related genes, including BGP, type i collagen, bone bridge element and sialoprotein.GrindStudy carefully proof, C2C12 cells are in osteogenic induction early stage high expression Runx2 genes；In ripe Gegenbaur's cell, Runx2 expressionSignificantly reduce；During osteoblast differentiation is osteocyte, Runx2 can not then be detected.Therefore, Runx2 may be participated inActive cell is divided into Gegenbaur's cell, and it is expressed in later stage reduction, so maintenance of the Runx2 low expression to function of osteoblastIt is vital.
In the present invention, by the way that unsupported blank control group and one~embodiment of embodiment five, experimental group one are answeredSupport group cell culture 0d, 3d, 7d and 14d are closed, real time fluorescent quantitative polynucleotide chain reaction experiment is carried out, detects each groupCompound rest is expressed Bone formation-related gene；Western blotting experiments are carried out in culture 0d, 3d, 7d, 14d, are discussedThe influence that Nano pearl powder/C-HA compound rests are synthesized to skeletonization GAP-associated protein GAP.
1st, reagent, as shown in table 12：
Table 12
2nd, instrument is as shown in table 13
Table 13
3rd, experimental program
Each group cell culture is cultivated in Osteogenic Induction Medium, cell RNA is extracted in culture 0d, 3d, 7d, 14dReal time fluorescent quantitative polynucleotide chain reaction (RT-qPCR) experiment is carried out using SYBR methods, Nano pearl powder/C-HA is detectedThe influence that compound rest is expressed Bone formation-related gene；Western blotting examinations are carried out in culture 0d, 3d, 7d, 14dTest, the influence that detection Nano pearl powder/C-HA compound rests are synthesized to skeletonization GAP-associated protein GAP.
One) 11, are tested：Real time fluorescent quantitative polynucleotide chain reaction
1st, the extraction of cell RNA
2nd, RNA reverse transcriptions
3rd, RT-qPCR operation
1) target gene Col α I, OCN, OPN and Runx2 sequence are searched on NCBI, design of primers is as shown in table 14：
Table 14
2) system composition is as shown in Table 15
Table 15
3) quantitative pcr amplification program：
System is mixed, is placed in quantitative real time PCR Instrument.After 95 DEG C × 10min pre-degenerations, then (become with 95 DEG C × 15sProperty), 60 DEG C × 1min (annealing and extensions) are a circulation, carry out 40 circulations.
4th, conclusion
Shown in target gene Col α I, OCN, OPN and Runx2 relative expression's scale 16,17,18,19：
On the day of cell culture as shown in table 16, the differential expression of each pack support target gene is not statistically significant.
Table 16
As shown in table 17, nano-pearl powder content is high for 25% support group OPN and Runx2 expression by cell culture 3dIn blank control group (p<0.05), remaining each group is not statistically significant.
Table 17
As shown in table 18, nano-pearl powder content is 10% and 25% support group Col α I and Runx2 to cell culture 7dExpression be higher than control group (p<0.05)；Nano-pearl powder content is 25% support group OCN and OPN expression also above controlGroup (p<0.05).
Table 18
As shown in table 19, nano-pearl powder content is 10% and 25% support group OCN and Runx2 to cell culture 14dExpression be higher than control group (p<0.05).
Table 19
The RT-qPCR experiments of the present invention use SYBR Green methods, using β-Actin as reference gene, find nano-pearlPowder content is generally higher than control group for the expression of 10% and 25% support group Col α tetra- genes of I, OCN, OPN and Runx2；But expression and control group no difference of science of statistics of the nano-pearl powder content for 0% support group gene, illustrate a high proportion of nanometerPearl powder/C-HA compound rests may be by raising I-type collagen, BGP, bone bridge element and the expression promotion of Runx2 genesSkeletonization.
Two) 12, are tested：Western blotting immunoblottings are tested
The relative synthetic quantity of destination protein Col α I, OCN, OPN and Runx2 albumen is as shown in table 20,21,22,23.
On the day of cell culture as shown in table 20, nano-pearl powder content is 25% support group Col α I, OCN and Runx2 eggsWhite synthesis is higher than blank control group (p<0.05), nano-pearl powder content is high for the synthesis of 10% support group Runx2 albumenIn blank control group (p<0.05), nano-pearl powder content is higher than blank for the synthesis of 0% support group OCN and Runx2 albumenControl group (p<0.05).
Table 20
As shown in table 21, nano-pearl powder content is 25% support group Col α I and Runx2 albumen to cell culture 3dSynthesis is higher than blank control group (p<0.05), nano-pearl powder content is the synthesis of 10% support group Runx2 albumen higher than skyWhite control group (p<0.05).And on OCN albumen, the synthesis of Nano pearl powder/C-HA compound rest groups of three ratios is lowIn blank control group (p<0.05), and nano-pearl powder content for 0% support group Col α I synthesis be less than blank control group(p<0.05)。
Table 21
Cell culture 7d as shown in table 22, nano-pearl powder content for 25% support group Col α I, OCN, OPN andThe synthesis of tetra- albumen of Runx2 is above blank control group (p<0.05), nano-pearl powder content is 10% support group Col α ISynthesis with Runx2 albumen is higher than blank control group (p<0.05), nano-pearl powder content is the OCN of 0% support group conjunctionInto higher than blank control group, but OPN synthesis is less than blank control group (p<0.05).
Table 22
As shown in table 23, nano-pearl powder content is higher than cell culture 14d for the synthesis of 25% support group OCN albumenBlank control group (p<0.05), nano-pearl powder content is higher than for the synthesis of 10% support group Col α I, OCN and Runx2 albumenBlank control group (p<0.05), nano-pearl powder content is higher than blank control group (p for the OCN of 0% support group synthesis<0.05)。
Table 23
Cell culture 3d of the present invention, the synthesis of Nano pearl powder/C-HA compound rest group OCN albumen of three ratios is equalLess than blank control group (P<0.05), but cell culture 7d, nano-pearl powder content be 0% and 25% support group OCNThe synthesis of albumen is above blank control group (p<0.05), cell culture 14d, Nano pearl powder/C-HA of three ratios is multipleThe synthesis for closing support group OCN albumen is above blank control group (p<0.05).OCN albumen becomes in 3d resulting anomalies, but totallyGesture is that the synthesis of compound rest group is higher than blank control group.
A high proportion of Nano pearl powder/C-HA supports may promote cell by raising type i collagen (Col α I) expressionThe formation of epimatrix.The binding site of the integrin receptor provided by collagen, promotes the adhesion, migration and maintenance of cellThe physical stability of tissue and organ.
A high proportion of Nano pearl powder/C-HA supports may promote the shape of bone matrix by raising BGP OCN expressionInto providing suitable extracellular environment for mouse MC3T3-E1 growth.
A high proportion of Nano pearl powder/C-HA supports may by raising bone bridge element OPN expression, regulation Gegenbaur's cell andThe interaction of osteoclast, and the various factors expression, suppress Apoptosis.
Runx2/Cbf α 1 are also referred to as the core-binding factor of polyoma enhancer associated proteins 2/ or acute myeloid is whiteBlood cause of disease.A high proportion of Nano pearl powder/C-HA supports may be by raising Runx2 expression, inducing mouse MC3T3-E1Cell carries out Osteoblast Differentiation, induces the activity of the Bone formation-related genes such as Col α I, OCN, OPN.
In summary：Compound rest promote the expression of Osteoblast Differentiation related gene such as Col α I, OCN, OPN and Runx2 andAlbumen is synthesized.Consider that compound rest of the content of Nano pearl powder between 1~20 its morphological stability is good and performance compared withIt is excellent, in terms of bone tissue engineer can be widely used in.
The readable carrier content of the nucleotides sequence list is:
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For those skilled in the art, on the premise of technical solution of the present invention is not departed from, if can also makeDry modification and improvement, these should also be considered as protection scope of the present invention, these effects implemented all without the influence present invention andPractical applicability.
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