CN108295303A - A kind of titanium implantation material and its preparation method and application - Google Patents
A kind of titanium implantation material and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of titanium implantation material, there is the titanium implantation material nano-structured titanium surface, Decorin to be incorporated into the nano-structured titanium surface by medium.Titanium implantation material provided by the invention has high sensibility and specificity to osteosarcoma cell, the proliferation of osteosarcoma cell can not only be pressed down, it can also inhibit the invasive ability of osteosarcoma cell while not influence osteoblast and the Integrated implant of metal prostheses, it is expected to solve defect of the current osteosarcoma field metal body implantation material without antitumor activity energy, improves the postoperative survival rate of osteosarcoma patient.
Description
Technical field
The present invention relates to a kind of titanium implantation materials and its preparation method and application.
Background technology
The malignant tumour generated in the tissues such as bone, cartilage and muscle is known as sarcoma.Osteosarcoma (osteosarcoma,
OS), it is most common Primary Malignant Bone Tumor, osteoid or prematurity bone is mainly generated by the pernicious mesenchymal cell occurred
And it is formed.It accounts for the 30%-80% of primary bone sarcoma, the main children for influencing 10-30 Sui, teenager and young man, and
Male is more than women.The incidence peak of most common OS types (i.e. advanced center OS) is at second ten of pubertal growth
Occur during year.OS can be with operative treatment, radiation and chemotherapy.In general, the treatment of osteosarcoma is first ocal resection tissue,
Then exogenous prosthese is implanted into the limb function of reconstruction patients.However, sometimes tumour cell but cannot be fully erased.Cause
This, needs a kind of exogenous prosthese that can prevent osteosarcoma and occur or recur and promote normal bone tissues to grow.
However, the current exogenous prosthese current material as implantation material does not have antitumor activity energy.For example, titanium and its conjunction
Gold, since its mechanical strength is good, proportion is low, and corrosion resistance and biocompatibility are excellent, and allergic reaction and and bon e formation is not present
Good boundary strength, it is considered to be the preferred material of biomedical applications.And titanium is as excellent implant material,
It is widely used in various prostheses and bone contact application.And the modification for the surface topography that titanium surface is carried out, biology strengthening surface
(biology) Chemical modification, (biology) Chemical modification of medication coat all significantly improves its performance.But titanium table
The modified main application of these three aspects of face still concentrates on reducing healing time and accelerates to be integrated into host tissue, or touches
Cell selective reaction is sent out, antibacterium attachment reduces inflammation risk, improves reliability of material and long-term behaviour etc..And it is right
Occur or recur not illustrate but in pre- preventing tumor, it is therefore desirable to a kind of titanium of (biology) Chemical modification of medication coat
Prosthese is implanted into prevent cancer.
Decorin (DCN) is one of the important member of the little albumen polysaccharide family rich in leucine, mainly by 44-kD cores
Heart protein and dermatan sulfate side chain composition.It has been found that DCN has significant antitumor action (such as lung cancer, breast cancer, liver
Cancer, cancer of pancreas etc.).Due to DCN in the normal and microenvironment of tumor tissues wide expression, it is thus possible to pass through influence matrix knot
Structure and adjusting influence the bioactivity of tumour cell with cell Proliferation and the relevant various receptors of survival, to further play
Antitumor action.Although having had extensively studied expression of the DCN in various tumor types and its suppression to tumor cell proliferation
It makes and uses, but the research of the influence about Binding Capacity DCN to growth of cancers is seldom.Therefore, DCN can be applied to titanium
Surface is modified, and to obtain a kind of anti-osteosarcoma prosthese, being future studies is worth the direction explored.
Invention content
For overcome current joint surgery titanium prothesis implant body material do not have anti-osteosarcoma function, and individually
The ineffective defect of the anti-osteosarcoma of Decorin, the present invention provide a kind of titanium implantation material, and also this provides titanium plant
Enter the preparation method and purposes of object.
To achieve the above object, the technical solution used in the present invention：A kind of titanium implantation material, the titanium implantation
There is object nano-structured titanium surface, Decorin to be incorporated into the nano-structured titanium surface by medium.
Preferably, the medium is poly-dopamine.
Preferably, on the nano-structured titanium surface Decorin a concentration of 3.78 ± 0.02~15.2 ± 0.01 μ g/
cm2。
Most preferably, on the nano-structured titanium surface Decorin a concentration of 7.56 ± 0.02 μ g/cm2。
The present invention provides the preparation methods of titanium implantation material described above, include the following steps：
(1) by the method for chemical oxidation on titanium implantation material Nanostructure fabrication titanium surface；
(2) add dopamine in the nano-structured titanium surface that step (1) obtains, dopamine auto polymerization react to be formed it is poly- more
Bar amine is simultaneously incorporated into the nano-structured titanium surface that the step (1) obtains；
(3) the nano-structured titanium surface that step (2) obtains add Decorin, Decorin with it is described nano-structured
The poly-dopamine on titanium surface combines, and obtains the titanium implantation material.
The present invention provides the purposes of titanium implantation material described above in the implantation instrument for preparing anti-osteosarcoma.
The present invention provides titanium implantation materials described above to prepare the implantation instrument for inhibiting human osteosarcoma cell proliferation
In purposes.
The present invention provides titanium implantation materials described above to prepare the plant for inhibiting osteosarcoma cell invasion or migration
Enter the purposes in instrument.
The present invention provides titanium implantation materials described above to prepare the implantation instrument for promoting apoptosis in osteosarcoma cells
In purposes.
The present invention provides titanium implantation materials described above in preparing the implantation instrument for promoting bone cell proliferation
Purposes.
The beneficial effects of the present invention are：The present invention passes through the method for chemical oxidation using titanium implantation material as substrate
On the nano-structured titanium surface of submicron structured titanium surface construction, reacted on nano-structured titanium surface using dopamine auto polymerization
Titanium-poly-dopamine-Decorin composite constructions are built, a kind of titanium prosthese plant with anti-osteosarcoma function is thus provided
Enter object.Titanium implantation material provided by the invention has high sensibility and specificity to osteosarcoma cell, can not only press down
The proliferation of osteosarcoma cell, moreover it is possible to inhibit the invasive ability of osteosarcoma cell while not influence osteoblast and metal prostheses
Integrated implant is expected to solve defect of the current osteosarcoma field metal body implantation material without antitumor activity energy, improves osteosarcoma
The postoperative survival rate of patient.
Description of the drawings
Fig. 1 is that CCK-8 methods detect proliferation results of two kinds of cells after the surfaces various concentration Ti-DOPA-DCN are cultivated 3 days.
A:SAOS-2, B:MC3T3-E1.Initial seeding density is 1 × 104cells/cm2.Error line indicates average value ± mark when n=3
Poor, the * p of standard<0.05, * * * p<0.001.
Fig. 2 is that CCK-8 methods detection cell is respectively seeded in CONTROL, DCN, Ti, Ti-DOPA, the surfaces Ti-DOPA-DCN
Proliferation results after 1,3,5,7 day.A:SAOS-2,B:MC3T3-E1.Initial seeding density is 1 × 105cells/cm2.Error line
Indicate mean+SD when n=5, * * * p<0.001.
Fig. 3 is that Transwell methods detect cell SAOS-2 cell invasion and migration energy after different substrate surface cultures 3 days
The change result of power.a：CONTROL, b:DCN, c:Ti, d:Ti-DOPA,e:Ti-DOPA-DCN.(A and B) Transwell methods are examined
Survey different substrate surfaces treated cell invasion and transfer ability (violet staining is the cell invaded and migrated)；C:It invades
Attack the cell number by cell.D:Migrate across the cell number of cell；Initial seeding density is 4 × 105cells/cm2.Accidentally
Poor line indicates mean+SD when n=3, * p<0.05, * * p<0.01, * * * p<0.001, scale:200μm.
Fig. 4 is fluorescence microscopy microscopic observation osteoblast MC3T3-E1 and osteosarcoma cell SAOS-2 in different titanium sheet substrates
It is upper co-culture 24,48,72,96 hours after growth result.MC3T3-E1 green fluorescent labels, SAOS-2 red fluorescence marks
Note.A:The fluorescence picture of the pure surfaces Ti co-cultured cell, B:The fluorescence picture of the surfaces Ti-DOPA-DCN co-cultured cell, C:It is pure
Cell count after osteoblast MC3T3-E1 and osteosarcoma cell SAOS-2 is co-cultured 24,48,72,96 hours on Ti substrates,
D:Cell after osteoblast MC3T3-E1 and osteosarcoma cell co-culture 24,48,72,96 hours on Ti-DOPA-DCN substrates
It is 4 × 10 to count Initial seeding densities3cells/cm2.Error line indicates mean+SD when n=3, * p<0.05,**
p<0.01, scale:200μm.
Fig. 5 is the karyomorphism that DAPI decoration methods detect cell SAOS-2 apoptosis after different substrate surface cultures 3 days
Change result.a：CONTROL, b:DCN, c:Ti, d:Ti-DOPA, e:Ti-DOPA-DCN.What white arrow schematic form changed
Karyon, scale:200μm.
Fig. 6 is Flow cytometry cell SAOS-2 Apoptosis and period point after different substrate surface cultures 3 days
The result that cloth changes.a：CONTROL,b:DCN, c:Ti, d:Ti-DOPA,e:Ti-DOPA-DCN.A:Pass through Flow Cytometry
Analysis determines the cell number of apoptosis, by the cell of Annexin V-FITC and PI dyeing is thought that apoptosis (Fig. 6 A) has occurred.B:It withers
Die the percentage (Fig. 6 B) that Index Definition is apoptotic cell.C:Flow cytometry detects the distribution of cell cycle, is cell with PI
Nuclear staining counts the number (Fig. 6 C) of each cycling cells.D:The percentage (Fig. 6 D) of cell cycle distribution.Initial seeding density is 3
×105cells/cm2.Error line indicates mean+SD when n=3, * p<0.05, * * p<0.01.
Specific implementation mode
Purpose, technical solution and advantageous effect in order to better illustrate the present invention, with reference to specific embodiment to this
Invention is described further.
Embodiment 1
100 μ L are seeded in 24 hole cell culture with the osteosarcoma cell SAOS-2 that the high glucose medium of 10%FBS suspends
Two orifice plates (the Decorin albumen that 100ul (100ug/ml) is added in one of hole) of plate, gather at the pure titanium sheet of 10x10mm
The Decorin of the amine-modified titanium sheet of DOPA and 100ug/ml protein modified poly-dopamine titanium sheet (Ti-DOPA-DCN, i.e. sheet
Invention titanium implantation material) on, 24 well culture plates are then put into standard culture case and are incubated 4 hours, wait for cell in each group titanium sheet
After sticking, suitable high glucose medium containing 10%FBS is added in above-mentioned each hole, so that the volume per hole solution reaches 1000ul,
Cell is placed in standard culture case culture again.The experiment required control, DCN, Ti, Ti-DOPA, Ti- are formed with this
Five groupings of DOPA-DCN100.Each experimental group includes 3 multiple holes.SAOS-2 and osteoblast MEC3T3-E1 are detected with this
The DAPI of cell Proliferation vigor and SAOS-2 cells after above-mentioned packet transaction when separation culture is dyed, streaming apoptosis is all
Phase, invasion/migration Function detection.And co-culture osteosarcoma SAOS-2 cells and osteoblast MEC3T3-E1 in titanium sheet,
Cell viability when being co-cultured with two kinds of cell contacts of detection.It is as follows：
1, CCK-8 experiments detection SAOS-2 and MEC3T3-E1 cell viabilities
The determination of nano-structured titanium surface Decorin optimal concentrations：CCK-8 methods detect osteosarcoma SAOS-2 cells and at
Osteocyte MEC3T3-E1 various concentration Ti-DOPA-DCN (DCN initial concentrations be respectively 0ug/ml, 25ug/ml, 50ug/ml,
Each 0.1ml kinds of 100ug/ml and 200ug/ml are planted in titanium plate surface) surface cultivate 3 days after proliferative conditions, from the result table of Fig. 1
Bright, the cell viability of osteosarcoma SAOS-2 is suppressed since Ti-DOPA-DCN50, inhibits effect in Ti-DOPA-DCN100
Fruit reaches most strong, and Ti-DOPA-DCN200 and Ti-DOPA-DCN100 does not have significant difference to the inhibiting effect of osteosarcoma SAOS-2
(Figure 1A).And the Ti-DOPA-DCN of various various concentration DCN does not have apparent shadow to the cell viability of osteoblast MC3T3-E1
It rings (Fig. 1).Accordingly, Ti-DOPA-DCN50, Ti-DOPA-DCN100, Ti-DOPA-DCN200 have the function of anti-osteosarcoma, wherein
Ti-DOPA-DCN100 is optimal concentration.Pass through the anchoring concentration of the nano-structured titanium surface Decorin of ELISA method detection, tool
It is 3.78 ± 0.02~15.2 ± 0.01 μ g/cm to have the preferred scope of anti-osteosarcoma function concentration2, wherein optium concentration is 7.56
±0.02μg/cm2。
Control, Decorin, Ti, Ti-DOPA are planted respectively in five experimental groups of Ti-DOPA-Decorin100
10000 SAOS-2 cells are put into incubator and cultivate 1.3.5.7 days for 37 DEG C.2) CCK-8 kits evaluation cell is used to increase
Grow/vigor.Original culture medium is replaced with the 500ul complete mediums for containing 10%CCK-8 at time point to be measured, then will
Cell is further incubated for 4 hours at 37 DEG C.Suspension obtained by 100 μ L is transferred in 96 orifice plates, and is existed using microplate reader
Absorbance is measured under 450nm.MEC3T3-E1 cells are handled with above-mentioned same method.Experiment is repeated 3 times.Experimental result such as Fig. 2 institutes
Show, after culture 1,3,5 and 7 day, the SAOS-2 cells cultivated on Ti-DOPA-DCN substrates show more apparent than pure Ti substrates
Lower cell activity (Fig. 2A).The SAOS-2 cells being grown on Ti-DOPA substrates only show similar with pure Ti substrates
Cell activity.And the SAOS-2 cells cultivated on Ti-DOPA-DCN substrates show it is more apparent than DCN groups and CONTROL groups more
Low cell activity.The result shows that the DCN being fixed in titanium sheet has cytotoxicity, and this toxic effect ratio DCN albumen
The simple effect dissipated in the solution becomes apparent from.It observes and is connect on Ti, Ti-DOPA and Ti-DOPA-DCN substrates in our current research
The cell viability of the osteoblast MC3T3-E1 of kind is not substantially change (Fig. 2 B).Titanium-poly-dopamine-Decorin composite constructions
The proliferation that osteosarcoma cell SAOS-2 can be significantly inhibited, the anti-osteosarcoma for being also secured to the DCN in titanium sheet are acted on than DCN albumen
The simple effect dissipated in the solution becomes apparent from, and does not influence the cell viability of normal osteoblast MEC3T3.
2, cell invasion/migration experiment detection osteosarcoma cell SAOS-2 invasion/transfer ability
Control, Decorin, Ti, Ti-DOPA are planted respectively in five experimental groups of Ti-DOPA-Decorin100
50000 SAOS-2 cells are cultivated 2 days for 37 DEG C in incubator.Invasion cell is made, 150ul Matregel glue is drawn first and presses
1：3 dilutions, that is, be added serum free medium 450ul, obtain the diluted matrigels of 500ul.Each cell upper layer (24 holes
The hole transwell, 8um radial cellule) paving dilution glue 100ul, 5 invasion cells are made altogether.Then 24 hole transwell plates are put
Being placed in 37 DEG C of incubators makes gelling consolidate in 2 hours.Then small indoor residual liquid is sucked out.The training of 100ul serum-frees is added in each cell
Base is supported, 37 DEG C of incubator 30min aquations basilar memebranes (the step for migration experiment does not need) are placed in.Pancreatin digestion has handled 2
It cell is 1 × 10 with serum free medium adjustment cell density5/ ml draws 200ul and is added to the small interior in upper layer, every group
3 cells of cell kind.Complete high glucose mediums of the 600ul containing 10%FBS is added in lower room.Routine culture for 24 hours after, fixed with methanol
10min, small cotton swab wipe small indoor profile cell, then violet staining liquid are used to dye 15min, and then tap water cleans cell
It is dried afterwards in room temperature, by cell, room is placed under microscope and randomly selects 5 visuals field and take pictures downward, and calculates invasion cell number.
Experiment is in triplicate.Experimental result is handled as shown in figure 3, the results show that compared with pure Ti substrates with Ti-DOPA-DCN substrates
SAOS-2 cells, invasive ability inhibited by highly significant.And more equally have with CONTROL and DCN groups bright
Aobvious effect (Fig. 3 A).In migration is tested, the transfer ability of cell is similarly subjected to obviously inhibit on Ti-DOPA-DCN substrates,
There is the result (Fig. 3 B) similar with Matrigel.That poly-dopamine-Decorin composite constructions can significantly inhibit osteosarcoma to titanium-is thin
Invasion/transfer ability of born of the same parents SAOS-2.
3, SAOS-2 and MEC3T3-E1 co-culture experiments
Osteoblast MEC3T3-E1 and osteosarcoma cell SAOS-2 are used on fluorescent dye DiO and DiD by specification respectively
Method be its pre-staining, the cell for then having marked two kinds respectively is by cell number 1：1 mixing, is seeded in Ti and Ti- respectively
On DOPA-decorin100, after being put into standard cell incubator incubation 4h, the high glucose medium containing 10%FBS is added, is put into mark
Quasi- cell incubator continues to cultivate, then respectively for 24 hours, tetra- time points of 48h, 72h, 96h in fluorescence microscopy under the microscope it is thin
Born of the same parents, and 5 fields is taken to take pictures at random, carry out fluorescence sxemiquantitative counting with image j softwares.Experiment is in triplicate.Fig. 4 is co-cultured
The results show that on pure Ti substrates, the density of osteosarcoma cell SAOS-2 increases with the time of culture and is increased, and preceding skeletonization is thin
The density of born of the same parents MC3T3-E1 is in different time points without significant changes (Fig. 4 A).On the contrary, being co-cultured on Ti-DOPA-DCN substrates
After 72 and 96 hours, compared with osteosarcoma cell SAOS-2, observes that the density of osteoblast MC3T3-E1 dramatically increases, show
Ti-DOPA-DCN substrates provide more favorable environment (Fig. 4 B) for osteoblast MC3T3-E1.Importantly, at 72 hours
Afterwards, significant change occurs for the cellular morphology of the osteosarcoma cell SAO-2 on Ti-DOPA-DCN.SAOS-2 is trained altogether with osteoblast
Experiment prompt is supported to can inhibit SAOS-2 proliferation while promoting osteoblastic proliferation.
4, nucleus DAPI is dyed
Control, Decorin, Ti, Ti-DOPA are planted respectively in five experimental groups of Ti-DOPA-Decorin100
10000 SAOS-2 cells are cultivated 2 days for 37 DEG C in incubator.The cells rinsed with PBS of culture two days later 3 times, and at room temperature
10 minutes are fixed with 4% paraformaldehyde.Fixed cell is used into 500ul DAPI working solutions rinse one time again, then at 37 DEG C
Further DAPI working solutions is used to dye 10 minutes.With PBS liquid rinse one time after the completion of dyeing, 500ul binding are used in combination
Buffer is fixed, and karyomorphism is then observed under inverted fluorescence microscope, and randomly select 3 regions take pictures calculate apoptosis
The cell nuclei of cell.Experiment is in triplicate.As shown in figure 5, compared with the cell on pure Ti and Ti-DOPA substrates, inoculation
Cell on Ti-DOPA-DCN substrates has obviously nuclear chromatin condensation and fragmentation.In morphological examination, find
The SAOS-2 cells shows cultivated on Ti-DOPA-DCN substrates go out typical apoptosis metamorphosis, such as cell shrinkage, karyorrhexis
It is formed with apoptotic body.The SAOS-2 cells cultivated on Ti-DOPA-DCN are shown shows apparent cell than CONTROL groups
Nuclear morphology changes, and the SAOS-2 cells cultivated on Ti-DOPA-DCN substrates are shown than DCN groups and CONTROL groups obviously
More cell karyorrhexis and apoptotic body are formed.These results demonstrate that Ti-DOPA-DCN substrates can significantly induce people's bone
The apoptosis of sarcoma SAOS-2 cells, and the effect that this apoptosis-induced effect dissipates merely in the solution than DCN albumen becomes apparent from.
Titanium-poly-dopamine-Decorin composite constructions can induce osteosarcoma cell SAOS-2 and apoptosis occur, it may be possible to which the coating inhibits bone
One of mechanism of sarcoma.
5, flow cytometry SAOS-2 Apoptosis and cycle experimental
Control, Decorin, Ti, Ti-DOPA are planted respectively in five experimental groups of Ti-DOPA-Decorin100
30000 SAOS-2 cells are cultivated 2 days for 37 DEG C in incubator.Suspension cell：With the trypsin digestion cell 3min without EDTA,
Centrifugation.PBS is washed twice, while ensureing cell quantity 100,000 or more.The binding buffer suspension cells of 500 μ l are added.
After the Annexin V-FITC mixings of 5 μ l are added, the propidium lodidie of 5 μ l, mixing, upper machine examination in 1 hour are added
It surveys.FCM analyses are carried out to detect cell cycle distribution.
Control, Decorin, Ti, Ti-DOPA, five experimental groups of Ti-DOPA-Decorin100 plant 30000 respectively
A SAOS-2 cells are cultivated 2 days for 37 DEG C in incubator.Culture medium is removed, with trypsin digestion cell 3min and collects, then uses
PBS cleans twice (2000 turns, 5 minutes), is then fixed with 70% ethyl alcohol 500ul of precooling, 4 DEG C of preservations.It is removed before dyeing solid
Determine liquid.Then 100ul RNase A37 DEG C water-baths 30min is added.400ul PI dyeing mixings are added, 4 DEG C are protected from light 30min,
Then cell week is analyzed by FACSCalibur TM flow cytometers (BD Biosciences, Franklin Lakes, US)
Phase tests in triplicate.The results are shown in Figure 6, and Annexin-V-FITC/PI is dyed in CONTROL group cells as shown in Figure 6A
FACS scatter plots show and detect only few dead or apoptotic cell cell mass.Compared with pure Ti substrates, in Ti-
The significant increase of apoptotic cell is found on DOPA-DCN substrates, and is apparently higher than CONTROL and DCN groups.This shows Ti-DOPA-
DCN has the function of significantly inducing SAOS-2 Apoptosis.As shown in Figure 6 C, with the cell on pure Ti substrates in the G0/G1 phases
Cell quantity is compared, and cell is obviously accumulated in the G0/G1 phases on Ti-DOPA-DCN substrates, and compared with CONTROL and DCN groups
It equally has a significant effect, shows the SAOS-2 cells being inoculated on Ti-DOPA-DCN substrates in G0/G1 phase growth retardations.
As a result prompt titanium-poly-dopamine-Decorin composite biological coatings can dramatically increase SAOS-2 Apoptosis and make SAOS-2 cells
Proliferation stays in the G1 phases.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of titanium implantation material, which is characterized in that the titanium implantation material has nano-structured titanium surface,
Decorin is incorporated into the nano-structured titanium surface by medium.
2. titanium implantation material according to claim 1, which is characterized in that the medium is poly-dopamine.
3. titanium implantation material according to claim 1, which is characterized in that on the nano-structured titanium surface
A concentration of 3.78 ± 0.02~15.2 ± 0.01 μ g/cm of Decorin2。
4. titanium implantation material according to claim 1, which is characterized in that on the nano-structured titanium surface
A concentration of 7.56 ± 0.02 μ g/cm of Decorin2。
5. the preparation method of titanium implantation material as described in claim 1, which is characterized in that include the following steps：
(1) by the method for chemical oxidation on titanium implantation material Nanostructure fabrication titanium surface；
(2) dopamine is added in the nano-structured titanium surface that step (1) obtains, dopamine auto polymerization reacts to form poly-dopamine
And it is incorporated into the nano-structured titanium surface that the step (1) obtains；
(3) Decorin, Decorin and the nano-structured titanium table are added in the nano-structured titanium surface that step (2) obtains
The poly-dopamine in face combines, and obtains the titanium implantation material.
6. purposes of the titanium implantation material as described in claim 1 in the implantation instrument for preparing anti-osteosarcoma.
7. the titanium implantation material as described in claim 1-4 is any is in preparing the implantation instrument for inhibiting human osteosarcoma cell proliferation
Purposes.
8. the titanium implantation material as described in claim 1-4 is any is preparing the implantation for inhibiting osteosarcoma cell invasion or migration
Purposes in instrument.
9. the titanium implantation material as described in claim 1-4 is any is in preparing the implantation instrument for promoting apoptosis in osteosarcoma cells
Purposes.
10. the titanium implantation material as described in claim 1-4 is any is in preparing the implantation instrument for promoting bone cell proliferation
Purposes.
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