CN109745150A - A kind of bone implant and its preparation method and application having immunoloregulation function - Google Patents
A kind of bone implant and its preparation method and application having immunoloregulation function Download PDFInfo
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- CN109745150A CN109745150A CN201711080460.XA CN201711080460A CN109745150A CN 109745150 A CN109745150 A CN 109745150A CN 201711080460 A CN201711080460 A CN 201711080460A CN 109745150 A CN109745150 A CN 109745150A
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Abstract
The present invention relates to a kind of bone implants and its preparation method and application for having immunoloregulation function.The surface of the bone implant has the protrusion having a size of submicron order.This submicrometre structural surface is able to suppress the inflammatory reaction of immunocyte, promotes immunocyte to the direction polarization for pressing down inflammation, facilitating knitting, with the excellent performance for adjusting the immune response of bone implant surface.
Description
Technical field
It is specifically to relate to the present invention relates to a kind of bone implant and its preparation method and application for having immunoloregulation function
And a kind of surface has the bone implant of submicrometer structure, has and promotes immunocyte to suppression inflammation, facilitates knitting direction
Polarized ability can slow down orthopaedics implant inflammatory reaction, belong to bio-medical technical field.
Background technique
Gradually with bone defect caused by the acceleration and orthopaedic disease of aging of population process, wound and traffic accident etc.
Increase, the demand of bone implant material is increasing.After implant enters human body, from recipient immune reaction is during its Integrated implant
To important function, lasting immune response will lead to serious foreign body reaction, granuloma and fiber package encapsulated, finally make chronic
Inflammation occurs and implant failure.If can regulate and control to implant surfaces immunocyte behavior, inhibit its inflammatory reaction,
Regulate and control it to be immunoreacted to facilitating knitting direction to convert, then can be improved the osseointegration character of implant.Therefore, one kind is studied
The surface that immune response can be adjusted is of great significance.
Natural inorganic/organic complex that bone is made of osteocyte, collagenous fiber and bone matrix has Asia abundant
Topological structure on micro-meter scale.
Summary of the invention
The present invention in order to solve the existing defects in the prior art plant by problem, the bone for providing a kind of tool immunoloregulation function
Enter body and its preparation method and application.
The inventors discovered that controlling from bionical angle by surface topography, sub-micron knot is constructed in implant surfaces
Structure is expected to adjust the immune response on surface, improves its osseointegration character.
On the one hand, the application provides a kind of bone implant, and the surface of the bone implant has having a size of submicron order
Protrusion.
This submicrometre structural surface is able to suppress the inflammatory reaction of immunocyte, promotes immunocyte to suppression inflammation, promotees
At the direction polarization of knitting, with the excellent performance for adjusting the immune response of bone implant surface.
Preferably, the protrusion is in regularly arranged, preferably Hexagonal array or square arrangement.
Preferably, the size of the protrusion is 50~800nm, preferably 50~600nm.
Preferably, the height of the protrusion is 100~1000nm, the spacing of the protrusion is 100~1600nm.
Preferably, the section of the protrusion is round, rectangular, triangle or irregular polygon.
Second aspect, the application provides a kind of method of adjusting bone implant surface immune response, in the table of bone implant
Face forms the protrusion having a size of submicron order.
The third aspect, the application provide a kind of preparation method of bone implant, are being etched with having a size of the recessed of submicron order
The silicon face in hole is poured bone implant material, removes after solidification, obtains bone implant.
Fourth aspect, the application provide application of the above-mentioned bone implant in the reparation and alternate material of preparation sclerous tissues.
Detailed description of the invention
Fig. 1 is respectively the stereoscan photograph under comparative example and embodiment 1,2 different amplifications；
Fig. 2 is respectively the contact angle test photo of comparative example and embodiment 1,2, wherein (a) is the flat PDMS table of comparative example preparation
Face, (b) surface PDMS with diameter 780nm cylindrical projections prepared for embodiment 2, (c) has for prepared by embodiment 1
The surface PDMS of diameter 290nm cylindrical projections；
Fig. 3 is respectively comparative example and embodiment 1,2 surface macrophage adhesion patterns；
Fig. 4 is respectively comparative example and embodiment 1, the inflammation of 2 surface macrophages and bone photo is facilitated to close factor expression situation, wherein
TNF-α, IL-6, IL-1 β are proinflammatory inflammation factor；IL-10 and IL-1ra is suppression inflammatory factor；BMP-2 is to facilitate bone correlation factor；
Fig. 5 is respectively the polarization phenotype of comparative example and embodiment 1,2 surface macrophages, and wherein CD11c is proinflammatory disease polarized meter
The marker of type；CD206 is the marker for pressing down inflammation polarization phenotype.
Specific embodiment
The present invention is further illustrated below by way of following embodiments, it should be appreciated that following embodiments are merely to illustrate this
Invention, is not intended to limit the present invention.
An embodiment of the present invention provides a kind of bone implant, and surface topography is submicrometer structure.Preferably, the sub-micro
Rice structure is size in the other protrusion of submicron order.
The protrusion is preferably in regularly arranged, such as Hexagonal array or square arrangement.
The section of protrusion can be the various shapes such as round, rectangular, triangle or irregular polygon.
The size (size on section) of protrusion can be within the scope of 50~800nm.Preferably, raised size 50~
Within the scope of 600nm, the stretching, extension of surface immunocyte can be advantageously promoted in the range, promote surface immunocyte scorching to suppression
Disease, the direction polarization for facilitating knitting.It is highly preferred that the size of protrusion is within the scope of 250~350nm.
The height of protrusion can be within the scope of 100~1000nm, preferably 200~800nm.
The spacing of protrusion can within the scope of 100~1600nm, preferably within the scope of 100~1200nm, more preferably 400~
Within the scope of 800nm.
The material of bone implant is unlimited, may be, for example, titanium alloy, magnesium alloy, stainless steel, polyether-ether-ketone etc..
In present embodiment, submicrometer structure can promote the stretching, extension of surface immunocyte.In addition, submicrometer structure can
Promote surface immunocyte to the direction polarization for pressing down inflammation, facilitating knitting.Specifically, experiments have shown that: RAW264.7 macrophage
Cell can stretch well in submicrometre structural surface, the decline of the rush inflammatory factor gene expression of cell, suppression inflammatory factor and
The expression of bone correlation factor is facilitated to rise；Streaming result confirms the positive in the rush marker of inflammation of submicrometre structural surface cell
Expression ratio is low, and the positive expression ratio for pressing down marker of inflammation is high.This shows that submicrometer structure can regulate and control macrophage to suppression
Inflammation facilitates knitting direction polarization.
An embodiment of the present invention provides the method that can adjust the immune response of bone implant surface, that is, in bone implant
Surface prepares a kind of submicron order structure.The submicrometer structure can be as described above.
Above-mentioned submicrometer structure can be used the methods of photoengraving, ion beam etching, electron beam lithography, template overmolded and be prepared into
It arrives.Wherein, it is preferred to use template overmolded method.Compared with other preparation methods, the advantages of this method, is that its is easy to operate, fast
It is fast, low in cost, and the auxiliary of large scale equipment is not needed, environmental condition requires low.
In one embodiment, it is poured bone implant material in the silicon face for being etched with the pit having a size of submicron order,
It is removed after solidification, obtains the bone implant.Curing mode can be heating etc..In one example, poly- two are carried out using silicon template
Methylsiloxane rubber (PDMS) overmolded, obtains the surface PDMS with submicrometer structure.Specifically, using sub-micro is etched with
The silicon of rice pattern is as template, in its surface casting polydimethylsiloxane rubber (PDMS), by being shelled after being heating and curing
From the pattern on silicon wafer is transferred on the surface PDMS, to obtain the PDMS overmolded that surface has submicrometer structure.Asia on silicon
Micron pattern is complementary with the submicrometer structure on bone implant surface.By adjusting the size of the submicron-pattem on the silicon on silicon,
The size of the submicrometer structure on adjustable bone implant surface.Submicron-pattem can pass through photoengraving, ion beam etching or electricity
The methods of beamlet etching is formed.
The invention has the following beneficial effects: preparation method 1) of the invention have it is easy to operate, quick, low in cost,
Environmental condition requires low advantage；2) submicrometer structure provided by the invention can promote immunocyte to suppression inflammation, facilitate bone
The immune response of bone implant can be effectively suppressed in the direction polarization of healing, promotes quick whole between implant and receptor bone tissue
It closes, accelerates the reparation of bone tissue function.
Enumerate embodiment further below with the present invention will be described in detail.It will similarly be understood that following embodiment is served only for this
Invention is further described, and should not be understood as limiting the scope of the invention, those skilled in the art is according to this hair
Some nonessential modifications and adaptations that bright above content is made all belong to the scope of protection of the present invention.Following examples are specific
Technological parameter etc. is also only an example in OK range, i.e. those skilled in the art can be done properly by the explanation of this paper
In the range of select, and do not really want to be defined in hereafter exemplary specific value.
Embodiment 1
A: Φ 290nm cylindrical projection submicrometer structure preparation and representation
There is the silicon wafer of diameter 290nm, the vertical pit of depth 500nm as template using surface, pit is in Hexagonal array, between pit
Away from for 600nm.Sylgard 184PDMS and its curing agent (Dow Corning company) are uniformly mixed with mass ratio 10: 1, put
Enter and vacuumize 30min in vacuum oven, completely remove in mixture after bubble, mixture is poured into Si template.In room
After the lower standing 2h of temperature, it is placed in baking oven in 60 DEG C of solidification 3h.The PDMS having been cured is taken off after being cooled to room temperature, on silicon wafer
Pattern just has been transferred to the surface PDMS, obtains the PDMS that surface has Φ 290nm cylindrical projection submicrometer structure, spare.
Fig. 1 is the SEM with Φ 290nm cylindrical projection submicrometer structure PDMS (embodiment 1) and comparative example, embodiment 2
Pattern photo, it can be seen that embodiment surface has the cylindrical projection of Hexagonal array, and arrangement is uniform, it was demonstrated that the pattern in silicon template
The surface PDMS is preferably copied to.
Fig. 2 is the contact with Φ 290nm cylindrical projection submicrometer structure PDMS (embodiment 1) and comparative example, embodiment 2
Photo is tested at angle, it can be seen that three kinds of surfaces are in hydrophobic state, hydrophilic similar temperament.
B: submicrometre structural surface immunocyte performance testing
It is tested using Turnover of Mouse Peritoneal Macrophages RAW264.7 cell.
1) cell adherence morphology observation
RAW264.7 cell is pressed 3 × 103The density in/hole, which is inoculated in storing, to be had in 24 orifice plates of each group sample, and culture is for 24 hours.It arrives
After time point, culture solution is abandoned, PBS is cleaned twice.Sample overnight is handled at 4 DEG C using 2% glutaraldehyde, then PBS rinses two
Time.Respectively with 30%, 50%, 70%, 90%, 100%, 100% alcohol serial dehydration, each concentration is dehydrated 10min.Metal spraying
After processing, cell morphology is observed using scanning electron microscope.
As seen from Figure 3, spheroidal is presented in comparative example superficial cell, fails to stretch around；2 superficial cell of embodiment
Slightly stretch, it is elongated in shape；1 superficial cell of embodiment is sprawled the most, shows that embodiment superficial cell has better cytochrome oxidase isozymes.
2) cellular inflammation and facilitate bone photo close factor gene detection of expression
Cell is pressed 5 × 105The density in/hole, which is inoculated in storing, to be had in 6 well culture plates of each group sample, is cultivated 3 days.Use qRT-
PCR method detects the gene expression of cell factor, and concrete operations are as follows:
Ribonucleic acid (RNA) is extracted: abandoning culture solution, PBS is cleaned twice, and every hole is blown repeatedly after 1ml Trizol is added
It makes a call to 20 times, stands 10min.Trizol liquid is transferred in the RNase-freeEP pipe of 1.5ml, 5min is stood.Every pipe is added
Chloroform 0.2ml acutely shakes 15s and is allowed to uniformly mixed, sample is centrifuged (12000rpm, 4 DEG C, 15min) after placement 5min.It is small
The heart draws upper strata aqueous phase into another EP pipe, and the isopropanol that equal volume is added mixes, and stands 10min.It is centrifuged again
(2000rpm, 4 DEG C, 10min) abandons supernatant afterwards, and 75% ethyl alcohol of 1ml is added in every pipe.It is centrifuged (7500rpm, 4 DEG C, 5min) again,
EP pipe is dried after abandoning supernatant, the whiteness of tube bottom is the total serum IgE extracted.30-50ul RNase-free is added in every pipe
Water, after completely dissolution, using the concentration of UV detector A260/280 measurement institute's extract RNA, the RNA of acquisition can be frozen
In -80 DEG C.
Reverse transcription: the RNA of 1 μ g, the 5 × buffer and 1 μ of the oligo (dT) of 1 μ l, 4 μ l are added in every 20 μ l reaction system
The reverse transcriptase of l, then 20 μ l are adjusted to RNase-free water.It is reversed with gene-amplificative instrament (Biometra, Germany)
Record, condition are as follows: after 37 DEG C of incubation 45min, 85 DEG C of incubation 5min；After reaction, sample is put in -20 DEG C of preservations.
Quantitative Real-time PCR detection: it negates the cDNA that transcription obtains and examines skeletonization related as template
The expression of gene, using SYBR Premix Ex Taq system (TakaRa, Japan).10 μ l reaction systems are as follows: 5 μ l
+ 4.2 μ l bis- of+0.5 μ l cDNA template of positive+0.2 μ l Rox dyestuff of anti-primer of SYBRPremix Ex Taq Mix (2 ×)+0.2 μ l
Secondary distilled water.Reaction condition: 95 DEG C, 30s- (95 DEG C, 5s-60 DEG C, 34s, 40 circulations) -4 DEG C of-solubility curve.Using opposite
Ct(2-ΔΔCt) method result is analyzed, and with Glyceraldehyde-3-phosphatedehydrogenase
(GAPDH) it is internal reference, while the value of the value of all experimental groups and control group is normalized.All experiments repeat three
It is secondary.
The primer of institute's cls gene (TNF-α, IL-6, IL-1 β, IL-10, IL-1ra, BMP-2) is referring to following table
Fig. 4 is respectively the inflammation of embodiment 1,2 and comparative example surface macrophage and bone photo is facilitated to close factor expression situation,
Wherein TNF-α, IL-6, IL-1 β are proinflammatory inflammation factor, and IL-10 and IL-1ra are suppression inflammatory factor, and BMP-2 is that bone photo is facilitated to close
The factor.As can be seen that the gene expression of the proinflammatory inflammation factor of Φ 290nm cylindrical projection submicrometre structural surface is significantly lower, suppression is scorching
Inflammation factor and facilitate the expression of bone correlation factor higher, this shows that Φ 290nm cylindrical projection submicrometer structure can effectively press down
Inflammatory reaction processed promotes into knitting.
3) polarization Phenotypic examination
RAW264.7 cell is pressed 5 × 105The density in/hole, which is inoculated in storing, to be had in 6 well culture plates of each group sample, is cultivated 3 days
Afterwards, culture solution is abandoned, pancreatin digestion, which is added, separates cell with material surface, while cleaning cell repeatedly using PBS, and centrifugation is resuspended
Three times.CD206-PE and CD11c-FITC antibody is used to handle (Biolegend& respectively in the cell cleaned
EBioscience), 30min is reacted under dark surrounds, is then detected using FCM analysis instrument
(BDBiosciences, USA), and analyzed using FlowJo software (Tree Star, USA).
As seen from Figure 5, RAW264.7 1 surface of embodiment promote marker of inflammation CD11c positive expression ratio compared with
It is low, only 44.6%, much smaller than the 61.0% of 66.2% and the embodiment 2 of comparative example；Meanwhile 1 surface macrophage of embodiment
The more suppression marker of inflammation CD206 of expression, positive expression ratio reaches 60.9%, more than 31.2% and embodiment of comparative example
The 37.8% of 2.This shows that Φ 290nm cylindrical projection submicrometer structure can preferably regulate and control macrophage to suppression inflammation direction pole
Change.
Comparative example
A: the flat surface PDMS preparation
Template, method and the same embodiment of parameter are used as using flat silicon wafer (Ra < 0.5nm), obtain surface without the flat of micrometer structure
PDMS, it is spare.It is flat to can be seen that comparative example surface for the SEM pattern photo of comparative example from Fig. 1, has high molecular pattern
Feature.Its surface is characterized with atomic force microscope, as a result confirm flat PDMS surface roughness be less than 10nm (Ra <
10nm)。
B: immunocyte performance testing
1) cell adherence morphology observation
Method is the same as embodiment 1；
As seen from Figure 3, comparative example surface cellular contraction is presented spheroidal, does not stretch around, also almost without pseudo- foot-shape
At.
2) cellular inflammation and facilitate bone photo close factor gene detection of expression
Method is the same as embodiment 1；
As seen from Figure 4, the proinflammatory inflammation factor TNF-α in comparative example surface, IL-6, the gene expression of IL-1 β are significantly higher, and suppression is scorching
Inflammation factor IL-10, IL-1ra and the expression for facilitating bone photo to close factor B MP-2 are then lower, this illustrates that flat PDMS makes macrophage
Change towards the direction of proinflammatory disease, generates harmful immune response, be unfavorable for into knitting.
3) polarization Phenotypic examination
Method is the same as embodiment 1；
As seen from Figure 5, RAW264.7 cell is up in the positive expression ratio of the proinflammatory disease marker CD11c in comparative example surface
66.2%, and the positive expression ratio for pressing down marker of inflammation CD206 is 31.2%.Combination cell factor gene expression as a result,
Confirm that flat PDMS causes macrophage to proinflammatory disease direction polarization.
Embodiment 2
The preparation of A: φ 780nm cylindrical projection submicrometer structure
2 preparation method of embodiment and parameter are with embodiment 1, the difference is that having diameter 780nm, depth 500nm to hang down using surface
For the silicon wafer of straight pit as template, pit is in Hexagonal array, and the spacing of pit is 1500nm.Pattern in silicon template is transferred to
Behind the surface PDMS, the PDMS that surface has Φ 780nm cylindrical projection submicrometer structure is obtained, it is spare；
It can be seen that the cylindrical projection that 2 surface of embodiment has Hexagonal array from Fig. 1 stereoscan photograph, arrangement is uniform, it was demonstrated that
Pattern in silicon template has preferably been copied to the surface PDMS.
B: immunocyte performance testing
1) cell adherence morphology observation
Method is the same as embodiment 1；
As seen from Figure 3,2 superficial cell of embodiment slightly stretches, elongated in shape, has lamellipodia to be formed, it was demonstrated that 2 table of embodiment
Face can promote cytochrome oxidase isozymes compared with comparative example, but cell spreading area is less than embodiment 1.
2) cellular inflammation and facilitate bone photo close factor gene detection of expression
Method is the same as embodiment 1；
Fig. 4 is respectively the inflammatory factor expression situation of embodiment 1,2 and comparative example surface macrophage.It can be seen that embodiment 2
The gene expression of the proinflammatory inflammation factor TNF-α in surface and IL-1 β are lower than comparative example, but are higher than embodiment 1；Press down inflammatory factor IL-10
Gene expression with IL-1ra is higher than comparative example.
3) polarization Phenotypic examination
Method is the same as embodiment 1；
As seen from Figure 5, RAW264.7 promotees marker of inflammation CD11c on 2 surface of embodiment and presses down marker of inflammation CD206's
Positive expression ratio is respectively 61.0% and 37.8%, between comparative example and embodiment 1.The expression of combination cell factor gene
Result, it was demonstrated that Φ 780nm cylindrical projection submicrometer structure also has certain suppression Inflammatory effects compared with flat PDMS, but cylinder
The smaller embodiment of diameter (Φ 290nm cylindrical projection submicrometer structure) can preferably regulate and control macrophage to suppression inflammation, rush
Skeletonization direction polarization.
Claims (8)
1. a kind of bone implant with immunoloregulation function, which is characterized in that the surface of the bone implant have having a size of
The protrusion of submicron order.
2. bone implant according to claim 1, which is characterized in that the protrusion is in regularly arranged, preferably six sides row
Column or square arrangement.
3. bone implant according to claim 1 or 2, which is characterized in that the size of the protrusion is 50~800 nm, excellent
It is selected as 50~600 nm.
4. bone implant according to any one of claim 1 to 3, which is characterized in that the height of the protrusion be 100~
1000 nm, the spacing of the protrusion are 100~1600 nm, preferably 100~1200 nm.
5. bone implant according to any one of claim 1 to 4, which is characterized in that the section of the protrusion be it is round,
Rectangular, triangle or irregular polygon.
6. a kind of method for adjusting the immune response of bone implant surface, which is characterized in that form size on the surface of bone implant
For the protrusion of submicron order.
7. a kind of preparation method of bone implant described in any one of claims 1 to 5, which is characterized in that be etched with ruler
The silicon face of the very little pit for submicron order is poured bone implant material, removes after solidification, obtains the bone implant.
8. bone implant described in a kind of any one of claims 1 to 5 is in the reparation and alternate material of preparation sclerous tissues
Using.
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Cited By (1)
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Application publication date: 20190514 |