CN104127913A - Medical composite material with improved osseointegration performance - Google Patents
Medical composite material with improved osseointegration performance Download PDFInfo
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- CN104127913A CN104127913A CN201310163527.1A CN201310163527A CN104127913A CN 104127913 A CN104127913 A CN 104127913A CN 201310163527 A CN201310163527 A CN 201310163527A CN 104127913 A CN104127913 A CN 104127913A
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Abstract
The invention provides a medical composite material with improved osseointegration performance. Specifically speaking, the medical composite material comprises a substrate and a coating which coats the substrate, wherein the coating is a Ta coating. The medical composite material has good mechanical performance and osseointegration performance, so the medical composite material is especially applicable to preparation of medical grafts and has wide application prospects.
Description
Technical field
The present invention relates to field of compound material, particularly, the invention provides a kind of medical composite material and method for making and purposes of bone conformability improvement.
Background technology
The effect of metal prostheses is checked by increasing clinical research and preclinical study.Desirable prosthetic material should have appropriate elastic modelling quantity, good corrosion resistance, good biocompatibility and bone grappling.But most medical prosthesis materials can not meet above-mentioned characteristic simultaneously.So having developed multiple different types of prosthese coating, people improve osseo-compatible and the osteoinductive of load-bearing material.Hydroxyapatite (HA) coating is exactly one of outstanding representative of these coatings.Titanium (Ti) the alloy prosthese of HA coating has biological activity that HA is good and the very good mechanical properties of Ti alloy simultaneously, thereby in bone surgery, is widely used the substitution material into osseous tissue.But the ceramic coating fragility of HA and other kinds is all relatively large, be easy to split away off from load-bearing prosthese, these shortcomings have hindered the broader applications of HA and other ceramic coatings.So medical implant still needs to have good biocompatibility, osteoinductive, chemical stability, and the new coating material of energy and base material strong bonded.
Tantalum (Ta) is the very promising medical prosthesis coating material of tool of one.Ta(atom sequence number 73) be a kind of transition rare metal, there is in vivo high corrosion resistance and inertia.Since the middle of last century, Ta starts to be applied to medical practice and shows good the medical biotechnology compatibility and safety.Due to its good chemical stability, body fluid resistance, biocompatibility and and the stationarity of the bioconjugation of osseous tissue, Ta is considered to the very promising biomaterial of one.But, the obvious high elastic modulus of Ta and and osseous tissue machinery incompatibility make it be not suitable as the medical prosthesis material of monoblock.May improve the medical applications prospect of Ta by manufacturing porous Ta coating in prosthetic surface, these porous materials provide lower elastic modelling quantity, higher skin resistance characteristic and good bone conformability (such as, biological activity, biocompatibility and growing into property of bone).But the relatively high production cost of Ta and its complicated production technology (need to carry out at inert environments, can not weld, the solution temperature that grinding is difficult and higher etc.), limited its extensive use in medical practice.
In sum, this area still lacks a kind of medical composite material with good mechanical performance and bone conformability.
Summary of the invention
The object of this invention is to provide a kind of medical composite material that is applicable to prepare osseous tissue graft.
A first aspect of the present invention, provides a kind of composite, and described composite comprises:
(a) matrix, wherein said matrix is metallic matrix; With
(b) be coated on the coating on matrix, described coating is Ta coating.
In another preference, described composite is medical composite material.
In another preference, all or part of coating of described matrix surface.
In another preference, described coating has the one or more features that are selected from lower group:
Described coating is loose structure; And/or
Described coating layer thickness is 30～500 microns; And/or
Described coating forms by vacuum plasma spray coating.
In another preference, described coating layer thickness is 40～300 microns, is preferably 100～250 microns.
In another preference, described metallic matrix is selected from lower group: Ti metallic matrix, Ti alloy substrate, stainless steel base, Co alloy substrate, or its combination.
In another preference, described matrix is titanium-aluminium alloy matrix.
In another preference, described matrix is Ti-6Al-4V matrix.
In another preference, described matrix is Ti-6Al-7Nb matrix.
Second aspect present invention, provides a kind of purposes of the medical composite material as described in first aspect present invention, for the preparation of medical implant.
In another preference, described composite is for the preparation of the graft that is selected from lower group: artificial bone, artificial joint, internal fixation of spine, dental implant, mandibular bone graft, nail, hone lamella, wound reparation, intramedullary pin, or its combination.
In another preference, described artificial joint comprises: shoulder joint, hip joint, knee joint, elbow joint.
Third aspect present invention, provides a kind of medical implant, and described graft comprises:
(a) matrix, wherein said matrix is metallic matrix; With
(b) be coated on the coating on matrix, described coating is Ta coating.
In another preference, described metallic matrix is selected from lower group: Ti metallic matrix, stainless steel base, Fe metallic matrix, Co alloy substrate, or its combination;
In another preference, all or part of coating of described matrix surface; And/or;
Described coating is positioned at the position that described graft contacts with bone.
In another preference, described coating has the one or more features that are selected from lower group:
Described coating is loose structure; And/or
Described coating layer thickness is 30～500 microns; And/or
Described coating is standby by vacuum plasma spray coating legal system.
In another preference, described coating layer thickness is 40～300 microns, is preferably 100～250 microns.
In another preference, described graft is used for the transplanting with bone contact site.
In another preference, described graft is for the transplanting at sclerotin position.
In another preference, described graft is for being selected from the medical transplanting of lower group: artificial bone, artificial joint, internal fixation of spine, dental implant, mandibular bone kind of plant, nail, hone lamella, wound reparation, intramedullary pin, or its combination.
In another preference, described artificial joint comprises: shoulder joint, hip joint, knee joint, elbow joint.
In another preference, described composite is standby by vacuum plasma spray coating legal system.
In another preference, described preparation method comprises step:
(i) provide a base material;
(ii) by vacuum plasma spray technology, on described base material, spray tantalum coating.
In another preference, described base material is medical metal base material or medical metal alloy base material.
In another preference, described base material is to prepare with the material that is selected from lower group: Ti, Co, Fe, Ti alloy, rustless steel, Co alloy, or its combination.
In another preference, in described step (ii), described plasma gas is Ar, H
2mist with powder carrier gas.
In another preference, the flow of described plasma gas Ar is 35～50SLM(Standard Liter Per Minute); And/or
Described plasma gas H
2flow is 8～18SLM; And/or
Powder carrier gas flux is 1.5～3.5SLM.
In another preference, the spray distance of described plasma gas is 80～380mm.
In another preference, in described vacuum plasma spray technology, spraying current is 500～700A; And/or
Powder feeding rate is 10～30g/min.
A fourth aspect of the present invention, provides a kind of goods, and described goods have composite described in first aspect present invention or made by the composite described in first aspect present invention.
In another preference, described goods comprise medical product.
In another preference, described medical product comprises graft.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.
Brief description of the drawings
Fig. 1 flow cytometer detects the surface marker molecule that separates the hBMSC obtaining, and result shows that separating the hBMSC obtaining is CD29
+, CD34
-, CD105
+, CD44
+, CD45
-, and CD90
+;
Fig. 2 hBMSC pedigree differentiation: osteoblast (ALP dyeing), and becomes fat (Oil Red O dyeing);
The XRD figure spectrum of Fig. 3 Ta coating and Ti coating;
The contact angle of Fig. 4 porous Ta coating surface and porous Ti coating surface
(t inspection, does not suppose variance not etc., error line representative ± SD, n=3 for the contact angle of Fig. 5 porous Ta coating surface and porous Ti coating surface; Ti coating, 131.917 ± 5.10882; Ta coating, 130.455 ± 5.55294; Probability >|t|, 0.7125, indicate without significantly difference);
The configuration of surface figure of Fig. 6 SEM observation porous Ta and Ti coating, enlargement ratio: 300 ×, 500 ×, 1000 ×, and 3000 ×;
Mesenchymal stem cells MSCs feature image multiplying power 500 that two kinds of coating surfaces of Fig. 7 SEM observation are cultivated × and 800 ×;
Fig. 8 hBMSC F-actin cytoskeleton is cultivated the laser scanning co-focusing microscope image (red, F-actin, blueness, the nucleus of DAPI) of 3 hours and 24 hours two kinds of coating surface forms;
The cell concentration that the hBMSCs of 48h is cultivated in Fig. 9 DRAQ5 dyeing on Ta and Ti coating surface detects that (t inspection, does not suppose variance not etc., error line representative ± SD, n=3; Ti coating, 65.377 ± 18.2972; Ta coating, 164.80 ± 46.1212; Probability >|t|, 0.0498*);
Figure 10 PrestoBlue method detects the cell survival rate of hBMSCs on each surface; (t inspection, does not suppose variance not etc., error stick representative ± SD, n=3; In general surface, the cell survival rate of hBMSCs as a control group);
Figure 11 hBMSCs cultivates the life or death cell viability experiment of 48h at coating surface; Living cells is green, and dead cell is red, and black is the coating surface that does not have Growth of Cells;
The hBMSCs of Figure 12 DAPI dyeing cultivates the anti-Ki67 experiment of 48h at coating surface;
Figure 13 hBMSCs cultivates the CFSE fluorescence situation rectangular histogram of 7 days at coating surface; Wherein, the meaning of each curve is as follows:
Yellow represents the 0th day not planting in the fluorescence situation of the hBMSC of the CFSE of coating surface labelling, and hBMSC does not also divide, and the amount of CFSE is not because cell division weakens;
The red fluorescence situation that represents to plant the hBMSC of the CFSE labelling after 5 days in Ta coating surface, the CFSE in hBSMC successively reduces by half and successively decreases due to cell division, and the CFSE fluorescence signal of hBMSC all weakens, and all there is propagation in most of hBMSC, and propagation is active;
The blue fluorescence situation that represents to plant the hBMSC of the CFSE labelling after 5 days in Ti coating surface, do not weaken with respect to the CFSE fluorescence of planting in the more hBMSC of the hBMSC of Ta coating surface, illustrate that the hBMSC of Ta coating surface is more active with respect to the hBMSC propagation of Ti coating surface;
The fluorescence situation of the green hBMSC that represents to plant the CFSE labelling of the culture dish surface that scribbles collagen protein in surface after 5 days, CFSE fluorescent weakening is obvious;
Figure 14 PCR in real time detects the mrna expression of the RUNX2 of the coating surface osteogenic induction liquid inducing culture hBMSCs of 21 days.GAPDH is as with reference to (t inspection, does not suppose variance not etc., error stick representative ± SD, n=3; Ti coating, 1 ± 0.1404; Ta coating, 1.9678 ± 0.2370; Probability >|t|, <0.01*);
Figure 15 immunofluorescence detects coating surface cultivates the ALP protein expression of the hBMSCs of 6 days and 12 days;
Figure 16 ALP staining examine coating surface is cultivated the hBMSCs of 3,7,15,21 days;
The Ta that the new bone of Figure 17 is implanted in vivo and Ti coating surface formation figure, the new bone that Van Gieson ' s Picric – Fuchsin around forms implant dyes; Red 3 months inherent implants new bone of growth around that represents, double fluorescence labeling is used for representing new osteoplastic mineralising deposition; Green and white arrow is illustrated in the fluorescent labeling of the calcein in 3 months;
In Figure 183 month, the new bone amount that body undercoating implant around forms, (t inspection, does not suppose variance not etc., error stick representative ± SD, n=3; Ti coating, 14.0741 ± 6.46293; Ta coating, 32.6501 ± 0.9072; Probability >|t|, 0.0358*).
Detailed description of the invention
The inventor, through long-term and deep research, is surprised to find that, by the medical material surface spraying Ta coating at Ti alloy, can make material keep the general formula of good mechanical attributes, significantly improves its bone conformability.Based on above-mentioned discovery, inventor has completed the present invention.
Term
As used herein, term " medical implant " or " medical kind of plant " (referred to as " graft " or " kind of plant ") refer to clinically for the medical prosthesis in implantable bioartificial body, as artificial skelecton, artificial organ etc., nail etc.
Term " vacuum plasma spray coating method ", " Vacuum Plasma Spray Technology ", " vacuum plasma spray technology " are all used interchangeably, and all refer to the Plasma Spraying Process Using of carrying out in the controlled negative pressure hermetic container of atmosphere.
Term " metallic matrix " refers to the matrix that metal or alloy material is made, as Ti metallic matrix, Ti alloy substrate, ferrum (Fe) metallic matrix, stainless steel base, cobalt (Co) alloy substrate etc.Especially, in the present invention, described metallic matrix is not Ta metallic matrix.
Medical composite material
Medical composite material provided by the invention, comprising:
(a) matrix, wherein said matrix is metallic matrix, comprises that (but being not limited to) is selected from the matrix of lower group: Ti metallic matrix, Ti alloy substrate, ferrum (Fe) metallic matrix, stainless steel base, cobalt (Co) alloy substrate etc.
(b) be coated on the coating on matrix, described coating is preferably Ta coating.
In another preference, described matrix is titanium-aluminium alloy matrix, as Ti-6Al-4V or Ti-6Al-7Nb etc., to improve the mechanical performance of composite.
Described coating can be positioned at all or part of surface of matrix, preferably, is positioned at the part surface of matrix, more preferably, is positioned at the position contacting with osseous tissue, as mortar cup, patellar prosthesis, tibial prosthesis, type femoral bone end prosthesis, cavitas glenoidalis and artificial body of the humerus etc.
Described coating can be loose structure, with the skin resistance characteristic of improved coating, the mechanical performances such as elastic modelling quantity.Described coating layer thickness is not particularly limited, and in preference of the present invention, the composite as described in first aspect present invention, is characterized in that, described coating layer thickness is 30～500 microns, is preferably 40～300 microns, is more preferably 100～250 microns.
Described coating can be prepared by various conventional meanses, as chemical deposition, vapour deposition process etc.Preferably, described coating can be standby by vacuum plasma spray coating legal system.
In preference of the present invention, described medical composite material can be for the preparation of medical implant.
In another preference, described composite is for the preparation of the graft that is selected from lower group: and artificial bone, artificial joint (as shoulder joint, hip joint, knee joint, elbow joint etc.), internal fixation of spine, dental implant, mandibular bone kind of plant, nail, hone lamella, wound reparation, intramedullary pin.
Medical implant
The present invention also provides a kind of medical implant, and described graft comprises:
(a) matrix, wherein said matrix comprises (but being not limited to): Ti matrix, Co matrix, Fe matrix, stainless steel base, Ti alloy substrate, or its combination;
(b) be coated on the coating on matrix, described coating is Ta coating.
In another preference, described matrix is titanium-aluminium alloy matrix, as medical Ti-6Al-4V etc., to improve the mechanical performance of graft.
Wherein, described coating is positioned at all or part of surface of described matrix, preferably, can be positioned at the position contacting with osseous tissue, as mortar cup, patellar prosthesis, tibial prosthesis, type femoral bone end prosthesis, cavitas glenoidalis and artificial body of the humerus etc.
Described coating can be loose structure, with the skin resistance characteristic of improved coating, the mechanical performances such as elastic modelling quantity.Described coating layer thickness is not particularly limited, and in preference of the present invention, described coating layer thickness is 30～500 microns, is preferably 40～300 microns, is more preferably 100～250 microns.
Described coating can be prepared by various conventional meanses, as chemical deposition, vapour deposition process etc.Preferably, described coating can be standby by vacuum plasma spray coating legal system.
Described graft for the transplanting of osteocyte contact site, as nail, jawbone reproduce, thigh bone transplanting etc.
In another preference, described graft is for the transplanting at sclerotin position.
In another preference, described graft is for being selected from the medical transplanting of lower group: artificial bone, artificial joint (shoulder joint, hip joint, knee joint, elbow joint), internal fixation of spine, dental implant, mandibular bone kind of plant, nail, hone lamella, wound reparation, intramedullary pin.
The preparation of medical composite material
The present invention also provides a kind of preparation method of medical composite material, and described composite is standby by vacuum plasma spray coating legal system.
Preferably, described preparation method comprises step:
(i) provide a base material;
(ii) by vacuum plasma spray technology, on described base material, spray tantalum coating.
Wherein, base material can be medical metal or medical metal alloy, as Ti, Co, Fe, Ti alloy; Or rustless steel, Co base alloy etc.
In the present invention, the various process parameters of vacuum plasma spray technology is not particularly limited.A kind of preferred technique comprises following one or more features:
Described plasma gas is Ar, H
2mist with powder carrier gas;
Wherein, the flow of described plasma gas Ar is 35～50SLM(Standard Liter Per Minute); And/or
Described plasma gas H
2flow is 8～18SLM; And/or
Powder carrier gas flux is 1.5～3.5SLM.
The spray distance of described plasma gas is 80～380mm.
In described vacuum plasma spray technology, spraying current is 500～700A; And/or
Powder feeding rate is 10～30g/min.
In another preference, described preparation technology is as follows:
Vacuum plasma spray technology is prepared tantalum coating on base material, plasma gas Ar flow 35～50SLM(Standard Liter Per Minute), plasma gas H
2flow is 8～18, and powder carrier gas flux is 1.5～3.5SLM, and spray distance is 80～380mm, and spraying current is 500～700A, and powder feeding rate is 10～30g/min.
Major advantage of the present invention
(1) provide first a kind of medical composite material with good mechanical performance (elastic modelling quantity is low, skin resistance characteristic is high, corrosion resistance good) and bone conformability (biocompatibility, biological activity, growing into property of bone);
(2) provide first a kind of have good mechanical performance and biocompatibility medical implant;
(3) provide a kind of new medical composite material preparation method.The composite coating of preparing by the inventive method is in conjunction with firm, and fragility is little, difficult drop-off;
(4) preparation method cost of the present invention is low, the more existing technique of production technology is more simple thereby be suitable for very much suitability for industrialized production.
(5) medical composite material of the present invention has good osseo-compatible, compared to the Ti alloy material of prior art, the Ti alloy material of the Ta of having coating provided by the invention can make bone marrow stem cell show the state of better sprawling, and is conducive to osteogenetic process.Cell proliferation rate is fast, apoptosis is few.Bone is integrated in experiment in vivo, has also shown better new bone shaping rate, has great using value.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Ethics statement
This experiment is passed through (human relations [2012] 5 are moved by Shanghai section) by the examination & verification of the attached medical college of Shanghai Communications University the 9th attached medical college of independence Ethics Committee of the People's Hospital and Shanghai Communications University the 9th laboratory animal Ethics Committee of the People's Hospital.Test all rabbit laboratory animals and obtain (SCXK2007-0007) from the attached medical college of Shanghai Communications University the 9th the People's Hospital's Animal House.All the processing of animal is all undertaken by the regulation of making attached medical college the 9th the People's Hospital of Shanghai Communications University.
The preparation of embodiment 1 composite
Ta and Ti coating are prepared by VPS system (Sulzer, Winterthur, Switzerland).Medical Ti-6Al-4V is as the base material of coating.The sample that detects BMSC in-vitro multiplication and differentiation be 10mm × 10mm × 2mm cuboid and
cylinder.Detection bodies internal skeleton is integrated and new osteoplastic sample is
cylinder.
Embodiment 2 material surface patterns detect
Coating surface pattern characteristic is undertaken by following instrument: detect X-ray diffractometer (XRD, D/max2550v, Rigaku, Japan; MDIJade6.5), contact angle analyser (SL200, Kino, Shanghai, P.R.China), scanning electron microscope (SEM, JSM6700F, JEOL, Akishima, Tokyo, Japan), electron probe (EPMA, JXA-8100, JEOL, Akishima, Tokyo, Japan)
The separation and purification of embodiment 3BMSC and skeletonization become fat induction
This tests required reagent preparation and key instrument equipment
(1) phosphate buffer (PBS): 9gNaCl, 1.44gNa
2hPO
47H
2o, 0.25gKH
2pO
42H
2o, with the configuration of 1000ml ultra-pure water, pH value is transferred to 7.2, for subsequent use after high temperature sterilize;
(2) α-MEM culture medium (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA);
(3) hyclone (FBS, Hyclone, Thermo Fisher Scientific, Waltham, MA, USA);
(4) 0.25%Trypsin-EDTA solution (GibcoTM, Invitrogen);
(5)DMSO(Sigma–Aldrich，St.Louis，MO，USA）；
(6) α-MEM culture fluid: α-MEM culture medium enters 10% hyclone, then add 100U/ml penicillin and 100mg/l streptomycin (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA), be put in 4 DEG C for subsequent use;
(7) cells frozen storing liquid: 90%FBS, 10%DMSO, be put in 4 DEG C for subsequent use;
(8) 5% CO2 gas incubator: HerAcell150(Heraeus, Germany);
(9) superclean bench;
(10) pH meter (METTLERTOLEDO320, Switzerland);
(11) electric suction apparatus (YB.DX.23D, Shanghai);
(12) cell counter (Invitrogen, Carlsbad, CA);
(13) desk-top horizontal centrifuge (5810R, Eppendorf, Germany);
(14) electronic analytical balance (METTLERTOLEDO, AB204-ESwitzerland);
(15) demineralizer (MilliporeUS);
(16) liquid nitrogen container (BCBSCRYO SYSTEMS, 47/11US);
(17) CO
2incubator (MCO-18AIC, SAVYO, Panasonic, Kadoma, Osaka, Japan);
(18) six porocyte culture plates (Corning, Corning, NY, USA);
(19) osteogenic induction liquid: α-MEM culture medium, 10%FBS, 50 μ M ascorbic acid/vitamin Cs (L-ascorbicacid2-phosphate, Sigma – Aldrich, St.Louis, MO, USA), 10mM-sodium glycerophosphate energy (β-glycerophosphate, Sigma – Aldrich, St.Louis, MO, USA), 100nM dexamethasone (dexamethasone, Sigma – Aldrich, St.Louis, MO, USA), 100U/mL penicillin, 100mg/L streptomycin
(20) become fat induced liquid: α-MEM culture medium, 10%FBS, 1 μ M dexamethasonephosphate, 200 μ M Indomethin(Sigma – Aldrich, St.Louis, MO, USA), 10 μ g/m Linsulin(Sigma – Aldrich, St.Louis, MO, USA), 0.5mM3-isobutyl-1-methylxanthine(IBMX, Sigma – Aldrich, St.Louis, MO, USA), 100U/mL penicillin, 100mg/L streptomycin
(21) become fat maintenance medium: α-MEM culture medium, 10%FBS, 10 μ g/mL insulin, 100U/mL penicillin, 100mg/L streptomycin
(22) 4% paraformaldehydes;
(23) 0.5% oil red O:0.5g oil red O powder add 100ml isopropyl alcohol, stirring and dissolving, and after filter paper filtering, room temperature is for subsequent use, keeps in Dark Place.
The isolation and purification method of 3.1BMSC
The people's bone marrow aspiration thing obtaining clinically and α-MEM culture fluid (1mL aspirate: 20mL culture fluid) piping and druming are mixed to rear inoculation
culture dish; In 5%CO2 incubator, 37 ° of C are hatched cultivation, and 48h full dose is changed liquid and removed not adherent heteroproteose cell, within later every 3 days, all change liquid once; When Growth of Cells reaches 85% while converging, draw after original culture fluid and clean once with PBS, then with 0.25%Trypsin-EDTA digestion 2min left and right, 500g, the centrifugal cell of collecting of 5min room temperature, resuspended with appropriate 37 DEG C of fresh mediums after, cell counting, according to 2 × 10
5/ cm
2go down to posterity, become first generation cell (P1); Go down to posterity after (general ratio is 1:3) through twice, most cells is frozen in liquid nitrogen container, stays a small amount of cell to test again.
Frozen program is: peptic cell >> uses cryopreserving liquid re-suspended cell >> to adjust the cell concentration >> program freezing storing box-70 ° C >> that spends the night and moves to liquid nitrogen container.
3.2 osteogenic induction
BMSCs is to six orifice plates (5 × 10 in plantation
4cells/well), α-MEM culture fluid is cultivated, and adds osteogenic induction liquid after fusion, within every 3 days, changes liquid once, and 7 days afterwards by BCIP/NBT ALP Color Development Kit(Beyotime, P.R.China) dye.
3.3 one-tenth fat inductions
BMSCs is to six orifice plates (2 × 10 in plantation
5cells/well), α-MEM culture fluid is cultivated, and adds into fat induced liquid 2 days after fusion, then becomes fat maintenance medium to cultivate 1 day.Said process to 14 day carries out oil red O stain repeatedly.
3.4 oil red O stain
Suction is cleaned 2 times with PBS after abandoning culture medium;
With 4% paraformaldehyde fixed cell 20min;
After distilled water rinsing 3 times, air dries;
The oil red O room temperature dyeing of new filtration 0.5% 1 hour;
The cell of Microscopic observation oil red stained positive after 70% ethanol rinsing 3 times.
Embodiment 4 cell viabilities and cytoskeleton
This tests required reagent preparation and key instrument equipment
(1) 24 well culture plate (Corning, Corning, NY, USA);
(2)DRAQ5（Danvers，MA，USA）；
(3)Odyssey?near-infrared?scanner（LI-COR?Biosciences，Millipore，Billerica，MA，USA）；
(4)PrestoBlue?Cell?Viability?Reagent（Invitrogen，Life?Technologies，Carlsbad，CA，USA）；
(5)microplatereader（InfiniteM200pro，TECAN，Switzerland）；
(6)LIVE/DEAD?Cell?Viability?Assays（Invitrogen，Life?Technologies，Carlsbad，CA，USA）；
(7)Phalloidin-TRITC（Sigma–Aldrich，St.Louis，MO，USA）；
(8) Anti-Ki67 antibody (Abcam, Cambridge, UK);
(9)DAPI（Sigma–Aldrich，St.Louis，MO，USA）；
(9) α-MEM culture medium (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA);
(10) α-MEM culture fluid: α-MEM culture medium enters 10% hyclone, then add 100U/ml penicillin and 100mg/l streptomycin (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA), be put in 4 DEG C for subsequent use;
(11) CO
2incubator (MCO-18AIC(UV), SAVYO, Panasonic, Kadoma, Osaka, Japan);
(12) Laser Scanning Confocal Microscope (LSM510, CarlZeissAG, Oberkochen, Germany)
4.1 detect cell viability and cytoskeleton experimental technique
10mm × 10mm × 2mm sample is put into 24 well culture plates and is added BMSCs(2 × 10
4cells/well), α-MEM culture medium culturing 24h, puts into 24 new well culture plates by sample.Carry out the fluorescence staining of DNA with DRAQ5.Sample scans the propagation situation that detects coating surface by Odyssey near-infrared scanner.Presto Blue Cell ViabilityReagent is for detection of cell viability, and uses microplatereader to detect the absorbance of 570and650nm.Anyway cell dyeing is undertaken by LIVE/DEAD Cell Viability Assays.F-actin detects by fluorescently-labeled phalloidin.Anti-Ki67 antibody is used for detecting the Ki67 expression of BMSC, and DAPI is used for the nucleus of labelling BMSC.Finally use Laser Scanning Confocal Microscope to observe.CFSE labelling experiment is used CFDA-SE(Sigma – Aldrich, St.Louis, MO, USA) hBMSC is carried out to labelling, CFDA-SE is formed CFSE after nonspecific lipase excision acetate after entering cell, can non-specific irreversible stable covalent bonds occur with polypeptide in cell and protein.Plant in being positioned in six orifice plates (Corning, NY, USA) through the hBMSC of labelling kind
sample on, after 24 hours, sample proceeds to new six orifice plates and continues to cultivate.Cultivate after 5 days, use flow cytometer (Franklin Lakes, NJ, USA) to detect the hBMSC of labelling.
The ALP dyeing of 4.2 coating surface BMSC
sample put into six orifice plates (Corning, NY, USA) and add BMSC(5 × 10
4cells/well), α-MEM culture medium culturing proceeds to six new orifice plates and cultivates 24h again after 24 hours, then cultivates with osteogenic induction liquid, within every 3 days, changes liquid once.Use BCIP/NBT ALP Color Development Kit(Beyotime, P.R.China) dye, and with Odyssey near-infrared scanner(LI-COR Biosciences, Millipore, Billerica, MA, USA) and scanner (Cannon, Tokyo, Japan) scan.
4.3In-Cell?Western
4% paraformaldehyde is for fixing, Odyssey Blocking Buffe(LI-COR Biosciences, Millipore, Billerica, MA, USA) for sealing and antibody dilution, ALP antibody(SantaCruz Biotechnology, SantaCruz, CA, USA) for detection of the ALP protein expression of coating surface BMSC.Hatch rear Infra-Red Secondary Antibody IRDye(Rockland Immunochemicals, Gilbertsville, PA, USA) anti-as two, finally pass through Odyssey Infrared Imaging System(LI-COR Biosciences, Millipore, Billerica, MA, USA) detect.
The gene expression of BMSC Osteoblast Differentiation in embodiment 5 porous Ta coatings
This tests required reagent preparation and key instrument equipment
(1) preparation of DEPC water: 1ml DEPC is added in 1 liter of Milli-Q ultra-pure water, and stirring is spent the night; After high temperature sterilize 15min inactivation DEPC, room temperature storage is for subsequent use;
(2) reverse transcription test kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA);
(3) each PCR primer: Invitrogen(Invitrogen, Carlsbad, CA) synthetic, be made into 10mmol/L concentration ,-20 DEG C of preservations with DEPC water;
GAPDH:
Forward primer (SEQ ID NO:1) GCACCACCAACTGCTTA
Reverse primer (SEQ ID NO:2) AGTAGAGGCAGGGATGAT
Runx2:
Forward primer (SEQ ID NO:3) TGTTCTCTGATCGCCTCAGTG
Reverse primer (SEQ ID NO:4) CCTGGGATCTGTAATCTGACTCT
(4) PCR kit for fluorescence quantitative (SYBRPremixExTaq, TaKaRa);
(5) TriPure Isolation Reagent(Roche, Basel, Switzerland), 4 DEG C of preservations;
(6) chloroform (analytical pure);
(7) isopropyl alcohol (analytical pure);
(8) RealTime PCR instrument Lightcycler480(Roche, Basel, Switzerland);
(9) low temperature desk centrifuge (Eppendorf).
Real-time quantitative PCR
Extract total RNA by TriPure Isolation Reagent; Equivalent 1 μ g RNA is by Revert Aid FirstStrand cDNA Synthesis Kit(Fermentas, Thermo Fisher Scientific, Waltham, MA, USA) carry out reverse transcription; The RNA that adds 1 μ g in every 20 μ l reaction systems, water to the 6.5 μ l of 1 μ l oligo (dT) 16 and RNA-free, hatches 5min at 70 DEG C after mixing, and is placed on rapidly on ice; Add 5 × buffer, 4 μ l, 2.5mM dNTP8 μ l, mixes, (0.5 μ is (Fermentas, Thermo Fisher Scientific, Waltham l) to add the Revert Aid M-MuLV reverse transcription of 100 units, MA, USA), mix latter 42 DEG C and hatch 60min; Stopped reaction after 70 DEG C of 10min, sample is put in-20 DEG C of preservations; Negate is transcribed the cDNA of acquisition as the expression of template inspection Bone formation-related gene, uses SYBR Premix ExTaq
tMsystem (Takara, Otsu, Shiga, Japan); 10 μ l
Reaction system is as follows: 5 positive anti-primer+0.5 of μ l SYBR Premix ExTaq supermix+2pmol/l μ lcDNA templates, all the other water polishings.Each sample at least adopts 4 multiple holes; Lightcycler480(Roche, Basel, Switzerland) carry out real-time PCR reactions;
Reaction condition: 95 DEG C, 10s-(95 DEG C, 10s; 60 DEG C, 20s, 40 circulations)-solubility curve-4 DEG C; Adopt Roche Lightcycler480software(Roche, Basel, Switzerland) carry out data analysis;
Embodiment 6 bodies are implanted into experimentation
This tests required reagent preparation and key instrument equipment
(1) at porous Ta coating prosthetic material diameter 2mm, length 10mm, is provided by Shanghai Silicate Inst., Chinese Academy of Sciences;
(2)Technovit7200VLC（Exakt，Norderstedt，Hamburg，Germany）；
(3)EXAKT400CS?MicroGrinding?System?with?AW110controller（Exakt，Norderstedt，Hamburg，Germany）
(4)EXAKT510Dehydrationand?Infiltration?System（Exakt，Norderstedt，Hamburg，Germany）
(5)EXAKT520Light?Polymerization?System（Exakt，Norderstedt，Hamburg，Germany）
(6) pinkish red picric acid dyeing liquor (VanGeison): 1% the acid fuchsine solution 10ml, picric acid saturated aqueous solution 100ml;
(7) 2% pentobarbital sodium liquid injection solutions;
(8) drill bit of diameter 2mm and electricity turn;
(9) a complete set of operating theater instruments: mainly comprise knife blade, handle of a knife, shears, tweezers, mosquito forceps, needle holder, suture needle, suture, drag hook, syringe etc.;
(10) draping surgery and operating coat;
(11) disinfectant solution: povidone iodine, 75% ethanol;
(12) calcein (Sigma – Aldrich, St.Louis, MO, USA);
(13) sclerous tissues's microtome (Leica, SP1600Germany);
(14) fluorescence inverted microscope (LEICADM4000B, German);
(15) image analysis software (BIOQUANT)
6.1 animal models build
The animal surgery apparatus that makes arrangements for surgery the previous day, prosthese is through autoclave sterilization post-drying; Operating theater instruments autoclave sterilization is dried; The sterilizing-dryings such as draping surgery; Anesthetics configuration; Operating room cleans ultraviolet sterilization.
Adopt male new zealand white rabbit (March, 2.8 – 3.0kg) to make femur defect prosthese model, intramuscular injection 2% pentobarbital sodium solution (20mg/Kg) anesthesia; Fully fixing after anesthesia, rabbit is got and faces upward position, lower limb condyle of femur art district preserved skin, routine disinfection, drape; Longitudinally cut skin, subcutaneous tissue along condyle of femur, should avoid muscle group as far as possible, between flesh, expose condyle of femur; Push periosteum open and check condyle of femur surrounding border, determine the position of getting into the cave;
drill bit is vertical with surface of bone gets into the cave, and the degree of depth should be advisable not bore offside cortical bone, fixed position anti-skidding; Put into prosthese, ensure that material surface is not higher than cortical bone; Successively stitch from the inside to the outside skin; The conventional raising of Animal House is put back in animal recovery after recovering to observe.For avoiding wound infection, postoperative 3 day every day penicillin injection once.
The two marks of 6.2 fluorescence and specimen are obtained
Be marked on for the first time sacrifice of animal injection in first 10 days, be marked on for the second time sacrifice of animal injection in first 3 days.The two marks of calcein (8mg/kg).Excessive anesthesia is put to death, and obtains its condyle of femur and key part.
6.3 histological observation
6.3.1 specimen embedding
Specimen is fixed: 10% formalin is fixed 10d, and tap water rinses and spends the night;
Gradient alcohol dehydration: 75%, 85%, 90%, 95%, 100%, 5 gradient is to specimen row processed.
Technovit7200VLC(Exakt, Norderstedt, Hamburg, Germany) specimen is carried out to vacuum infiltration 2d.
By EXAKT520Light Polymerization System(Exakt, Norderstedt, Hamburg, Germany) make Technovit7200VLC photopolymerization 10h.
6.3.2 sclerous tissues's section
Adopt Saw Microtome LeicaSP1600(Leica, Wetzlar, Germany) sclerous tissues's microtome section, slice thickness is 100 μ m, EXAKT400CS Micro Grinding System with AW110controller(Exakt, Norderstedt, Hamburg, Germany) polishing is to 50 μ m, polishing.
6.3.3 picric acid-product red colouring
The ultrasonic 2min that develops a film; 0.1% formic acid 3min; Flowing water rinses 2min; 20% methanol 2h; Flowing water rinses 2min, dries; The 60 DEG C of blue dyeing of Stevenol 5-15min; 60 DEG C of distillation washings; VG dye liquor dyeing 3-8min; 100% ethanol cleans; Distillation washing, dries.
Universal method
Tissue morphology metering
Before and after dyeing, tissue slice, in the lower shooting of microscope (LEICA DM4000B, German), uses semi-automated analysis software (BIOQUANT) to carry out quantitative analysis.
Statistical analysis
Statistical analysis adopts SAS JMP(Cary, NC, USA) carry out.Two sample averages are Aspin-Welch-Satterthwaite-Student's t-test, and data represent with mean ± standard deviation, and P<0.05 is significant difference, with * labelling.
Experimental result
1.BMSC qualification
In order to assess biocompatibility and the osteoinductive of porous tantalum coating, we adopt described method in this research material and method part to extract purification BMSC.Detect by flow cytometer, the surface marker CD29 of the cellular expression MSC that separation obtains, CD105, CD44 and CD90, but do not express hemopoietic and show to indicate CD34 and leukocyte mark CD45(Fig. 1), show that the cell characteristics of the BMSC that we extract and known BMSC cell are similar.In addition we also by ALP with detected with oil red O BMSC to skeletonization and the differentiation capability (Fig. 2) that becomes fat direction.These experimental datas show that BMSC has skeletonization and the potential that becomes fat differentiation, can be for follow-up experiment.
2. coating surface pattern
Fig. 3 has shown XRD figure, and experimental result shows, porous Ti coating surface main component is Ti, and porous Ta coating surface is mainly Ta and a small amount of Ta oxide.Oxide layer (the Ta of porous Ta coating surface
2o
5) all very stable under multiple pH value.
As shown in Figure 4 and Figure 5, the contact angle detection of porous Ta coating surface and porous Ti coating surface has no obvious difference (n=3; Ticoating, 131.917 ± 5.10882; Tacoating, 130.455 ± 5.55294; P=0.7125).
As shown in Figure 6, we by SEM observe porous Ta and Ti coating configuration of surface (300 ×, 500 ×, 1000 ×, and 3000 ×), the surface roughness of visible porous Ta and Ti coating is close.
3. porous Ta coating surface hBMSC cellular morphology and cytoskeleton tension force
BMSC shows relative Ti coating at porous Ta coating surface and better sprawls state (Fig. 7, Fig. 8).The experimental result of SEM shows to show the flat state of sprawling at the BMSC of porous Ta coating surface.F-actin cytoskeleton dyeing (Fig. 8) also shows to show and the different cytoskeleton tension force of porous Ti coating surface BMSC and the flat state of sprawling at the BMSC of porous Ta coating surface.
4. adhesion and the propagation of porous Ta coating surface BMSC
We apply DRAQ5 and detect the growing state of the BMSC of coating surface.Fig. 9 shows that BMSC has than porous Ti coating surface growth rate (48h, n=3 faster at porous Ta coating surface; Ti coating, 65.377 ± 18.2972; Ta coating, 164.80 ± 46.1212; P<0.0498*).
As shown in figure 10, the result of PrestoBlue shows the 4th and have than at porous Ti coating surface growth rate (n=3 faster at porous Ta coating surface BMSC after 6 days; The 2nd day: Ti coating, 0.45268 ± 0.097954; Ta coating, 1.0767 ± 0.495686, P=0.992; The 3rd day: Ticoating, 0.56950 ± 0.089554, Ta coating, 1.26228 ± 0.458399, P=0.0620; The 4th day: Ti coating, 0.90275 ± 0.082739, Ta coating, 2.13239 ± 0.699593, P<0.0390*; The 6th day: Ti coating, 0.86752 ± 0.089092, Ta coating, 1.68688 ± 0.386935, P<0.0233*).
What anyway cell viability experiment (Figure 11) and anti-Ki67 experiment (Figure 12) showed not only to breed at the BMSC of porous Ta coating surface is faster, and apoptosis still less.
CFSE experiment (Figure 13) shows BMSC, and at porous Ta coating surface, growth rate/cell viability is just because BMSC can better stick to porous Ta coating surface relatively faster, and its faster speed of growth at porous Ta coating surface also plays a role.
5. the ALP of the BMSC of porous Ta coating surface and RUNX2 expression
To carrying out osteogenic induction at the BMSC of porous Ta coating surface by osteogenic induction liquid, can detect that the mrna expression of RUNX2 of BMSC is at the relative rise (n=3 of porous Ta coating surface; Ti coating, 1 ± 0.1404; Ta coating, 1.9678 ± 0.2370; P<0.01*) (Figure 14).
Show to have at porous Ta coating surface BMSC the expression of higher ALP by immunofluorescence (Figure 15) and ALP dyeing (Figure 16).The above results porous surface Ta coating is more conducive to the osteogenetic process of hBMSC with respect to porous Ti coating.
6. the body internal skeleton of porous Ta coating prosthese is integrated
We use sclerous tissues's section of Van Gieson ' s Picric – Fuchsin dyeing to be used for observing porous Ta, the bone integration of Ti coating prosthese.New bone formation rate detects by the two marks of calcein.Prosthese implanted after 3 months, and tectology metering prompting porous Ta coating area of new bone is around obviously more than porous Ti coating prosthese (Figure 17, Figure 18, n=3; Ti coating, 14.0741 ± 6.46293; Ta coating, 32.6501 ± 0.90721; P<0.0358*).Calcein biplot 17 shows that the relative porous Ti coating of porous Ta coating Periprosthetic Periprosthetic has higher new bone formation rate.
Discuss
Over 20 years of past, many different types of coatings and material are all used to improve the biological firmness of prosthese.Because Ti alloy has good reliable mechanical attributes, it is a kind of biomaterial of most suitable load-bearing.But the same with other most metals, the osteoinductive of Ti is not strong.In biomedical metal material, Ta is a kind of more and more by novel biomaterial that everybody noted.In vivo, Ta shows very strong anticorrosive and good biological activity, but relatively high manufacturing cost and the more difficult processability of himself have hindered the extensive use of Ta.In order to have given play at load prosthese the good bio-compatible performance of Ta, porous Ta coating has been prepared in this research on the base material of Ti by Vacuum Plasma Spray Technology.Then we have assessed biocompatibility and the osteoinductive of the Ta coating of preparing by vacuum plasma spray coating.
As testing at SEM morphocytology, seen in F-actin cytoskeleton Coloration experiment and the experiment of PrestoBlue cell viability, external BMSC and material interaction result clearly point out porous Ta coating surface to significantly improve sticking of BMSC and cell proliferation.Prosthetic surface properties of materials and biocompatibility depend on the interaction of material and cell very much, and biomaterial can affect by the interaction of this cell material reaction and the behavior of cell.Our experimental result shows that BMSC has much better cell adhesion and cell proliferation at the relative porous Ti coating surface of porous Ta coating surface.These results suggest Ta surface has biocompatibility, and biological behaviour that can appreciable impact BMSC.In general, initial adhesion has affected follow-up differentiation of stem cells, and the different cytoskeleton signals that come from adhesion are delivered to nucleus, and they can change the expression of gene signal and further change cell differentiation procedure there.The proof cellular morphologies such as McBeath can regulate the skeletonization of BMSC to become fat differentiation destiny.Such as, can adhere to the BMSC stretching that flattens and can enter skeletonization process, and those are assembled perfectly round cell and can become lipoblast.In this research, the dyeing of F-actin cytoskeletal protein discloses BMSC and is flat stretch-like at porous Ta coating surface.So at porous Ta, the different BMSC F-actin cytoskeleton of Ti coating surface can partial interpretation BMSC at the higher osteogenic ability of porous Ta coating surface.Further, the relative porous Ti coating of porous Ta coating has not only increased adhesion and the propagation of BMSC, is also more conducive in vitro the Osteoblast Differentiation of stem cell simultaneously.These conclusions are confirmed at the high expressed of the BMSC of porous Ta coating surface by ALP and RUNX2.A kind of possible explanation is that the approximate substrate surface of elasticity and bone may be conducive to the new life of bone and the differentiation of BMSC.Like this, owing to having close elasticity with bone trabecula, therefore porous Ta coating may more be conducive to skeletonization.Experiment in vivo, by the femur defect model of rabbit, because Ta has good biocompatibility and biological activity, so relative porous Ti coating prosthese, porous Ta coating prosthese has better bone conformability.In addition, the determinant attribute of porous Ta coating is its surface oxide layer that can form self-passivation.This layer of surface oxide layer generation water and effect generate Ta-OH in vivo, the Ca in itself and body
2+ion forms certain calcification tantalum, then with phosphate anion and follow-up a large amount of Ca
2+ion and phosphate anion form apatite layer, and this layer of surface caused the formation of bone sample apatite in vivo, thereby bone and fibre structure can easierly be grown into, and adhere to the tune condition that provides for rear symplectic bone-soft tissue rapid, high volume.Experiment in our body also shows in porous Ta coating with respect to have higher new bone formation rate in porous Ti coating.This has higher cell adhesion propagation with BMSC in our external porous Ta coating and also conforms to Osteoblast Differentiation.
These results show porous Ta coating have in vitro support differentiation of stem cells be osteoblastic ability, have in vivo support mature osteocytes ability.Porous Ta is a kind of biomaterial in vivo with excellent biocompatibility and safety in utilization, is that a kind of attractive clinical practice is selected.Although still need long-term experiment and clinical research to prove its advantage and curative effect, the porous Ta coating of application VPS promotes the application prospect of osteanagenesis to be opened as orthopaedics graft coating.
Conclusion
We have prepared porous Ta coating by VPS on Ti alloy base material surface.Porous Ta coating has effectively reduced the mechanical incompatibility of metal implants and osseous tissue.We determine by detecting biocompatibility osteoinductive and the bone conformability of porous Ta coating prosthese whether porous Ta coating prosthese is applicable to clinical practice.In vitro, result shows the cell adhesion at porous Ta coating surface BMSC, and propagation and Osteoblast Differentiation are all compared with increasing at porous Ti coating surface.Further, in body, femoral defects in rabbits prosthese experimental model shows that porous Ta coating prosthese has good bone and integrates and new bone formation rate.Our result shows that porous Ta coating prepared by VPS is a kind of bone repair mode likely.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a composite, is characterized in that, comprising:
(a) matrix, wherein said matrix is metallic matrix; With
(b) be coated on the coating on matrix, described coating is Ta coating.
2. composite as claimed in claim 1, is characterized in that, all or part of coating of described matrix surface.
3. composite as claimed in claim 1, is characterized in that, described coating has the one or more features that are selected from lower group:
Described coating is loose structure; And/or
Described coating layer thickness is 30～500 microns; And/or
Described coating forms by vacuum plasma spray coating.
4. composite as claimed in claim 1, is characterized in that, described metallic matrix is selected from lower group: Ti metallic matrix, Ti alloy substrate, stainless steel base, Co alloy substrate, or its combination.
5. the purposes of medical composite material as claimed in claim 1, is characterized in that, for the preparation of medical implant.
6. a medical implant, is characterized in that, described graft comprises:
(a) matrix, wherein said matrix is metallic matrix; With
(b) be coated on the coating on matrix, described coating is Ta coating.
7. graft as claimed in claim 6, is characterized in that, all or part of coating of described matrix surface; And/or;
Described coating is positioned at the position that described graft contacts with bone.
8. graft as claimed in claim 6, is characterized in that, described coating has the one or more features that are selected from lower group:
Described coating is loose structure; And/or
Described coating layer thickness is 30～500 microns; And/or
Described coating is standby by vacuum plasma spray coating legal system.
9. the method for making of composite as claimed in claim 1, is characterized in that, described composite is standby by vacuum plasma spray coating legal system.
10. goods, is characterized in that, described goods have composite claimed in claim 1 or made by composite claimed in claim 1.
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| WO2017214614A1 (en) * | 2016-06-10 | 2017-12-14 | Bioventus, Llc | Protein delivery with porous metallic structure |
| US10130678B2 (en) | 2014-12-29 | 2018-11-20 | Bioventus, LLC. | Systems and methods for improved delivery of osteoinductive molecules in bone repair |
| CN110075354A (en) * | 2018-01-26 | 2019-08-02 | 中国科学院上海硅酸盐研究所 | A kind of tantalum base coating and its preparation method and application with significant rush cell Osteoblast Differentiation performance |
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