CN105112493A - Method for detecting and evaluating in-vitro cell morphology and osteogenic function of surface of bone substitute implant - Google Patents
Method for detecting and evaluating in-vitro cell morphology and osteogenic function of surface of bone substitute implant Download PDFInfo
- Publication number
- CN105112493A CN105112493A CN201510604370.0A CN201510604370A CN105112493A CN 105112493 A CN105112493 A CN 105112493A CN 201510604370 A CN201510604370 A CN 201510604370A CN 105112493 A CN105112493 A CN 105112493A
- Authority
- CN
- China
- Prior art keywords
- cell
- implant material
- bone implant
- evaluating
- days
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting and evaluating in-vitro cell morphology and osteogenic function of the surface of a bone substitute implant, and belongs to the technical field of biomaterial detection. The method is characterized by comprising the following steps: taking the bone substitute implant to be detected and evaluated, firstly, observing the cell morphology, cytoskeleton morphology and skeleton protein distribution condition, then detecting cell activity, cell adhesion on the surface of the material and alkaline phosphatase activity in cells, then detecting collagen secretion and the external parts of the cells, and remembering the mineralization degree, and finally, determining the cell morphology as well as the expression condition of framework related genes and osteogenesis related genes. According to the method, biological reference is better provided for optimal design of the surface of the bone substitute implant, and on the basis of the prior art, lots of detecting methods are added, and part detecting methods are improved, so that the method is comprehensive and systematical, and passes the cytology and molecule biology cross comprehensive evaluation, and important guiding significances are provided for optimal research of the surface of the bone substitute implant.
Description
Technical field
The invention belongs to biomaterial detection technique field, be specifically related to the methods of testing and evaluating of a kind of bone implant material surface cell in vitro form and osteogenesis function.
Background technology
Large quantity research shows that surperficial bone implant material pattern and surface chemical property have decisive influence for embedded material synosteosis ability, and its surperficial physicochemical characteristics impact on Integrated implant is most important.Therefore, more and more process for treating surface concentrates on the surface characteristic changing bone implant material, makes every effort to improve embedded material and synestotic efficiency.Current bone implant material surface activation treatment process comprises chemical process, physical method, physico-chemical process and biochemical method etc., and concrete grammar mainly contains: the methods such as aciding, alkalinisation treatment, electrochemical oxidation process (anodic oxidation), air heating method, calcium phosphate crystal coating (HA coating), titanium slurry coating, plasma spraying method, laser treatment, sandblasting or sandblasting acid etching, bioactive molecules modification, ion implantation modification, micro-modification.
Good synosteosis is carried out in order to make artificial bone embedded material and nature bone, the properties of artificial bone embedded material and nature bone just must be made as far as possible close, but for how to judge bone implant material cell in vitro form and osteogenesis function, become the focus of attention comprehensive system.Existing bone implant material cell in vitro form and osteogenesis function detection method is single, accuracy is poor, evaluate shallow-layer, comprehensive not, limitation is larger.
Summary of the invention
In order to overcome the defect that above-mentioned prior art exists, the object of the present invention is to provide the methods of testing and evaluating of a kind of bone implant material surface cell in vitro form and osteogenesis function, the method is from multi-orientation detection and evaluate the impact of bone implant material on cellular form and osteogenesis function, ensure that higher detection level, result is more accurate.
The present invention is achieved through the following technical solutions:
A methods of testing and evaluating for bone implant material surface cell in vitro form and osteogenesis function, comprises the following steps:
1) bone implant material with evaluating to be detected is got, observation of cell form, cytoskeleton and skelemin distribution situation;
2) cell viability, material surface cell adhesion forces and cell activity change of Alkaline phosphatase is detected;
3) collagen secretion and mineralization of extracellular matrix degree is detected;
4) expression of cellular form and skeleton genes involved, Bone formation-related gene is measured;
5) performance treating the bone implant material of test and repair according to above detected result is evaluated:
First judge whether bone implant material superficial cell form extends, and whether the filopodia that cell terminal extensions goes out increases, and whether cytoskeleton fiber increases, judge that cytoskeletal protein is expressed and whether strengthen;
Secondly, judge whether cell adhesion forces on bone implant material surface and alkaline phosphatase activities strengthen;
Again, judge whether collagen secretion and mineralization of extracellular matrix degree strengthen;
Finally, judge that skeleton genes involved and Bone formation-related gene are expressed whether to strengthen;
Meet conclusions and then prove that this bone implant material meets biomedical security requirement, otherwise, then do not meet biomedical security requirement.
Step 1) middle observation of cell form, cytoskeleton and skelemin distribution situation, concrete operations are as follows:
Detect cellular form: after being sterilized by bone implant material sample to be detected, getting cell is seeded in 24 well culture plates, after cultivating 72h, fix by 2.5% glutaraldehyde, then adopt the ethanol dehydration of gradient concentration, then with field emission scanning electron microscope, cellular form is observed after drying metal spraying;
Detect matrix morphology: fix with 4% paraformaldehyde, after the Phalloidine incubated at room with 50 μ g/mL rhodamine marks, through DAPI dyeing, then with the anti-agent mounting of quenching of fluorescence, adopt laser confocal microscope to observe the distribution of cytoskeleton and form;
Detection skelemin distributes: functional quality mark is that 4% paraformaldehyde is fixed, 15 minutes are placed by 0.1%triton-X100 room temperature, after add lowlenthal serum, room temperature closes 1 hour, remove serum, add primary antibodie, in 4 DEG C of overnight incubation, room temperature rewarming 1 hour, lucifuge adds two of FITC mark and resists, room temperature lucifuge hatches 1 hour, the Phalloidine that lucifuge adds rhodamine mark hatches 40 minutes, lucifuge adds DAPI solution-dyed 20 minutes, again with the anti-agent mounting of quenching of fluorescence, inverted fluorescence microscope is adopted to observe Cdc42, RhoA and Rac1 cytoskeletal protein distribution situation in cell.
Step 2) middle detection cell viability, material surface cell adhesion forces and cell activity change of Alkaline phosphatase, concrete operations are as follows:
Detect cell viability: after cell to be detected is seeded to bone implant material, by cell cultures after 3 days or 7 days, after sample being transferred to 24 new well culture plates, add CCK-8 and nutrient solution, after hatching 3 hours, solution is transferred to 96 orifice plates, blank well returns to zero, and uses microplate reader to measure absorbance at 450nm place;
Test material superficial cell adheres to: after being sterilized by bone implant material sample to be detected, getting cell is seeded in 24 well culture plates, cell cultures is stopped after cultivating 30min, 1h, 2h and 3h, omnidistance lucifuge after fixing by 2.5% glutaraldehyde, dye with DAPI, by the sample left-hand thread after rinsing at 24 new orifice plates, see Stochastic choice four visuals field with inverted fluorescence microscope and to take pictures record, utilize ImageJ analysis software to carry out cell counting;
Alkaline phosphatase assay: after cell inoculates 48 hours, nutrient solution is replaced by osteogenic induction nutrient solution, cultivate 7 days, fix with 4% paraformaldehyde, use NBT/BCIP alkaline phosphatase activities test kit, experimentally flow process application of sample colour developing, observes under Stereo microscope and takes pictures, carry out alkaline phosphatase activities detection.
Step 3) in detect collagen secretion and extracellular and remember mineralization degree, concrete operations are as follows:
Detect collagen secretion: cell is seeded in 24 well culture plates, nutrient solution is replaced by osteogenic induction nutrient solution, and cultivates 14 days, fixes with 4% paraformaldehyde solution, after using 1% Picro-Sirius red/picric acid solution dyeing 18 ~ 24h, seasoning in room temperature environment; Stereo microscope is used to observe and take pictures; Meanwhile, carry out semi-quantitative analysis to the collagen secretion of specimen surface, be transferred to by sample in 24 new orifice plates, every hole adds collagen staining elutriant, then sucking-off dye liquor is transferred to 96 orifice plates, detects absorbance in microplate reader in wavelength 540nm place;
Detect mineralization of extracellular matrix situation: cell is seeded in 24 well culture plates, nutrient solution is replaced by osteogenic induction nutrient solution, cultivate and induce after 14 days, add sodium alizarinsulfonate dye liquor to specimen surface, left at room temperature 15 minutes, observes under putting Stereo microscope and takes pictures; Semi-quantitative analysis is carried out to the mineralization of extracellular matrix of specimen surface: sample is positioned over new 24 and cultivates orifice plate, every hole adds 1mL dyeing elutriant respectively, shaking table shakes up, after on sample, dyestuff fully dissolves, every hole is sucking-off 3 parts of dye liquors respectively, and every part is 150 μ L, then adds in 96 well culture plates, use the simple elutriant of 150 μ L for contrast zeroing, measure absorbance in wavelength 620nm place.
Step 4) in measure the expression of cellular form and skeleton genes involved, Bone formation-related gene, concrete operations are as follows:
Detect cellular form and skeleton related gene expression: cell is seeded in 24 well culture plates, and cultivate and change nutrient solution after 48 hours, culturing cell stops cell cultures after 3 days or 7 days; Use PBS rinsing cell 3 times, extract cell total rna and carry out real-time quantitative PCR and detect Cdc42, Rac1 and RhoA genetic expression;
Be detected as bone related genes to express: cell is seeded in 24 well culture plates, cultivate and change nutrient solution after 48 hours, culturing cell stops cell cultures after 3 days or 7 days, use PBS rinsing cell 3 times, extract cell total rna and carry out real-time quantitative PCR and detect Ocn, Bsp, Col I, Opn, ALP, BMP and OSX genetic expression.
Described cell to be detected is selected from mesenchymal stem cells MSCs, mouse bone-forming cell system cell, OS-732 cells system cell or original cuiture scleroblast.
The described bone implant material with evaluating to be detected comprises titanium, titanium alloy, phosphorus ash stone biological ceramics and matrix material thereof.
Compared with prior art, the present invention has following useful technique effect:
The detection method of bone implant material surface cell in vitro form disclosed by the invention and osteogenesis function, overcome the deficiencies in the prior art, improve the examination criteria of bone implant material surface cell in vitro form and osteogenesis function, add cytoskeleton and morphologic observation, cellular immunofluorescence is observed, the projects such as DAPI dyeing adhesion and related gene expression detection, ensure that the comprehensive and advanced of detection method, from multi-angle assessment bone implant material on the impact of cellular form and osteogenesis function, ensure that higher detection level, make detection method more accurate, detected result is also more reliable.The inventive method provides biological foundation in order to the design of the better surface optimization for bone implant material, on the basis of existing technology, we add large quantity measuring method, improve part detection method, make the method comprehensive system, by cytology and the evaluation of molecules cross-synthesis, the research of this surface optimization to bone implant material has great directive significance.
Further, more existing mtt assay, adopts CCK8 kit detection cell active, and measuring method is more accurate, and workable, error is little.
Further, dyeing on qualitative observation basis in the past, by using alkaline phosphatase (AKP) and BCA test kit, make collagen elutriant and mineralising elutriant by oneself, in conjunction with microplate reader, quantitative or half-quantitative detection are carried out to alkaline phosphatase, collagen secretion, epimatrix mineralising, detection method is simple, and result is also more accurate.
Accompanying drawing explanation
Fig. 1 is scanning electron microscopic observation different-shape (S, R, R5, R20) surperficial rBMMSCs cellular form when cultivating 1 day;
Fig. 2 is different-shape (S, R, R5, R20) surperficial rBMMSCs cytoskeleton stained photographs;
Fig. 3 is different-shape (S, R, R5, R20) surperficial rBMMSCs cytoskeletal protein stained photographs;
Fig. 4 is that CCK8 method detects cytoactive; A () and (b) are respectively rBMMSCs and grow 3 days and the cytoactive (a: P<0.05 compared with S group when 7 days at specimen surface; B: P<0.05 compared with R group);
Fig. 5 is that rBMMSCs sticks the DAPI stained photographs of 2h in surface of test piece;
Fig. 6 is the DAPI dyeing counting result that rBMMSCs sticks in surface of test piece, and (a), (b), (c), (d) are respectively 30min, 1h, 2h and 3h; (a: P<0.05 compared with S group; B: P<0.05 compared with R group; C: P<0.05 compared with R5 group);
Fig. 7 is different-shape (S, R, R5, R20) surperficial rBMMSCs cell osteogenic induction alkaline phosphatase staining result after 7 days;
Fig. 8 is the surperficial rBMMSCs Cellular alkaline phosphatase (ALP) of different-shape (S, R, R5, R20) 7 days active (a: P<0.05 compared with S group; B: P<0.05 compared with R group);
Fig. 9 is different-shape (S, R, R5, R20) surperficial rBMMSCs cell 14d collagen secretion level detection result;
Figure 10 is the sirius red stains collagen secretion sxemiquantitative of different-shape (S, R, R5, R20) surperficial rBMMSCs cell 14d collagen secretion, (a: P<0.05 compared with S group);
Figure 11 is different-shape (S, R, R5, R20) surperficial rBMMSCs14d mineralization of extracellular matrix level detection result;
Figure 12 is the Alizarin red staining mineralization of extracellular matrix sxemiquantitative (a: P<0.05 compared with S group of the surperficial rBMMSCs14d mineralization of extracellular matrix of different-shape (S, R, R5, R20); B: P<0.05 compared with R group; C: P<0.05 compared with R5 group);
Figure 13 is the expression (a: P<0.05 compared with S group that 7 days qPCR detection RhoGTPases genes involveds are cultivated on different-shape (S, R, R5, R20) surface; B: P<0.05 compared with R group; , wherein, (a) be Rac1 gene c: P<0.05 compared with R5 group); B () is RhoA gene; C () is Cdc42 gene;
Figure 14 is the expression (a: P<0.05 compared with S group that different-shape (S, R, R5, R20) surface cultivates that 7 days qPCR are detected as bone related genes; B: P<0.05 compared with R group; C: P<0.05 compared with R5 group); Wherein, (a) is OPN; B () is OSX; C () is BSP; D () is BMP; E () is ALP.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
One, main raw and instrument
Modified form α-MEM substratum (Hyclone, the U.S.); Foetal calf serum (folium ilicis chinensis, Tian Hang bio tech ltd, Zhejiang); Trypsin Sigma, the U.S.); Penicillin/streptomycin (Sigma, the U.S.); L-glutaminate; CCK-8 test kit (Wuhan Boster Biological Technology Co., Ltd., China); DMSO (Sigma, the U.S.); Alkaline phosphatase detecting reagent box (the green skies Bioisystech Co., Ltd in Shanghai, China); 1% Picro-Sirius red/picric acid staining reagent (the green skies Bioisystech Co., Ltd in Shanghai, China); Sodium alizarinsulfonate reagent (Sigma, the U.S.); 2.5% glutaraldehyde; 75% ethanol (Tianjin Fu Yu Fine Chemical Co., Ltd); Rhodamine mark-Phalloidine (Sigma, the U.S.); E.Z.N.A.TMTotalRNAKitI total RNA extraction reagent box (Omega, the U.S.); PrimeScriptTMRTreagentkit Reverse Transcription box (Takara, China); SYBRPremixExTaqIITM fluorescence real-time quantitative test kit (Takara, China) primer synthesis (Beijing AudioCodes biotech company, China); Bechtop (Shanghai is rich proves to be true after interrogation industry, China); Cell culture incubator (Thermo, the U.S.); Microplate reader (BioTek, the U.S.); Inverted phase contrast microscope (LeicaDM6000B); Stereoscopic microscope (LeciaM125, Germany); Spectrophotometer (ThermoScientificNanoDrop2000, the U.S.), laser confocal microscope (OlympusFV1000, Japan); Real-time quantitative RNA detector (ABI7500); PCR instrument (ABIVeriti, the U.S.); Field emission scanning electron microscope (S-4800, Hitachi, Japan); Freeze drier (E-1045, Hitachi, Japan); Ion sputtering film coating instrument (ES-2030, Hitachi, Japan).
Two, common agents preparation and using method
(1) α-MEM nutrient solution preparation
Modified form α-MEM substratum compound method is volume ratio is 10% foetal calf serum, 1% penicillin/streptomycin, 0.001%L-glutamine add 90% α-MEM substratum mixed preparing and form.
(2) osteogenic induction nutrient solution compound method
Comprise 10% foetal calf serum, 10mM sodium β-glycerophosphate and 0.1 μM of dexamethasone and the ascorbic α of 0.2mM-MEM modified form substratum hybrid filtering is osteogenic induction nutrient solution.
(3) collagen elutriant and mineralising elutriant compound method
Being mixed 500 μ L by 0.2M sodium hydroxide solution and 99% methyl alcohol equal-volume, namely form collagen staining elutriant through sterilizing filter; 500 μ L cetylpyridinium chlorides are dissolved in 2000 μ L10mM sodium phosphates, namely form mineralising dyeing elutriant through sterilizing filter.
(4) CCK-8 (CellCountingKit) test kit using method
CellCountingKit is called for short CCK test kit, a kind of be widely used in cell proliferation and Cytotoxic fast high-sensitive degree detection kit based on WST-8 (chemical name: 2-(2-methoxyl group-4-nitre phenyl)-3-(4-nitre phenyl)-5-(2,4-disulfobenzene)-2H-tetrazolium monosodium salt).
WST-8 belongs to the upgrading products of MTT, and principle of work is: deposit in case at electron coupling reagent, can be generated the orange-yellow formazan product (formazan) of high water soluble by Intramitochondrial desaturase reduction.The depth of color and cell be proliferated into direct ratio, be inversely proportional to cytotoxicity.Microplate reader is used to measure OD value at 450mM wavelength place, indirectly reflection viable cell quantity.
Concrete operation step is as follows: inoculating cell suspension (100 μ L/ hole) in 96 orifice plates.Culture plate is placed in incubator preculture for some time (37 DEG C, 5%CO2).2,10 μ LCCK solution (note not generating bubble in hole, they can affect the reading of OD value) are added to every hole.3, culture plate is hatched 1-4 hour in incubator.4, the absorbancy at 450nm place is determined at by microplate reader.If 5 temporarily do not measure OD value, HCL solution or the 1%w/vSDS solution of 10 μ L0.1M can be added in every hole, hide culture plate and keep in Dark Place at ambient temperature.Measure in 24 hours, absorbancy can not change.
(5) real-time quantitative PCR detects cellular form and skeleton and Bone formation-related gene expression method
Extract total serum IgE concrete grammar as follows: sample is placed and cell is inoculated with cell viability test experience.Stop after cell cultures 3d and 7d cultivating, PBS cleans 3 times.Adopt Trizol method to extract the total serum IgE of each group of cell respectively, key step is as follows:
1) be transferred to new 24 well culture plates by often organizing 6 samples, every hole adds 200ulTrizol, fully blows and beats mixing, make the refrigerant not thickness of its cell homogenates, room temperature leaves standstill 5min-10min, collects and often organizes cell pyrolysis liquid, goes to 0.1%DEPC process rear without in the 1.5mLEP pipe of RNA enzyme;
2) in the ratio of 200ul chloroform/mlTrizol, add chloroform, put upside down mixing 15 seconds, room temperature places 5min, under 4 DEG C of conditions, and the centrifugal 15min of 12000rpm, then draw upper strata aqueous phase (about 400-500ul), be transferred to another in the pretreated EP pipe of DEPC;
3) in the ratio of 0.5ml Virahol/mlTrizol, add Virahol, after putting upside down mixing, leave standstill 2h under-20 DEG C of conditions, then under 4 DEG C of conditions, the centrifugal 15min of 12000rpm, inhales and abandons supernatant;
4) prepare 75% ethanol with the DEPC water of precooling, in the ratio of 1ml75% ethanol/mlTrizol, add ethanol, under 4 DEG C of conditions, the centrifugal 5min of 12000rpm, inhales and abandons supernatant;
5), under room temperature, uncap and dry RNA5-10min;
6) the deionized water dissolving RNA of 20ul through DEPC process autoclave sterilization process is added;
7) RNA purity and concentration is measured with ultraviolet spectrophotometer.
8) be put in-80 DEG C of refrigerators and save backup.
Reverse transcription reaction concrete steps are as follows:
Use PrimeScriptTMRTreagentKit Reverse Transcriptase kit synthesis cDNA first chain.Reverse transcription is totally 20l, prepares reverse transcription reaction liquid (table 2) to specifications.
Table 1RT reaction solution formulation components
Reverse transcription reaction condition is: react 15 minutes at 37 DEG C in PCR instrument, reacted for 5 seconds at 85 DEG C.Using this cDNA synthesized as the template of pcr amplification, be placed in-20 DEG C for subsequent use.
Real-time quantitative PCR reaction concrete steps are as follows:
Use TakaraExTaq test kit, prepare PCR reaction solution (table 3) to specifications.
Table 2PCR reaction solution formulation components
Real-time quantitative PCR reaction conditions is: be placed in by the reaction solution prepared in CFX96 (Bio-rad) real-time PCR and carry out PCR reaction.Response procedures is: predeformation: (1 circulation) 95 DEG C, 30 seconds; Pcr amplification: (40 circulations) 95 DEG C of sex change, 5 seconds, 20 DEG C/sec; 55-60 DEG C of annealing, 20 seconds, 20 DEG C/sec; 72 DEG C of extensions, 30 seconds.
Three, the methods of testing and evaluating of bone implant material surface cell in vitro form of the present invention and osteogenesis function
1, scanning electric mirror observing cell form
RBMMSCs inoculates 24 orifice plates, 5 × 103, every hole cell density, 37 DEG C of 5%CO2 are after 4 groups of specimen surfaces cultivate 1d, sample is transferred to 24 new orifice plates, PBS cleans 3 times, each 5 minutes, 2.5% glutaraldehyde is fixedly spent the night, 50%, 70%, 80%, 90%, 100% ethanol dewaters each 15 minutes successively, metal spraying after seasoning, uses field emission scanning electron microscope to see cellular form as shown in Figure 1.Cell is good at smooth (S) surface spreading, and in the Polygons slightly extended, under high-amplification-factor, visible cell peripheral extension goes out less tabular and filopodia.On micron pattern (R) surface, cell obviously extends, and the elongate, threadlike pseudopodium at the two poles of the earth contacts with flanking cell or base material; At two kinds of micro-nano topographical surface, cells intact form diminishes elongation, and two ends are in sharper fusiformis.Under high-amplification-factor, visible cell also for extend Polygons, comparatively micron pattern stretch, between cell pseudopodium comparatively micron pattern increase and widen, cell terminal extensions goes out a large amount of filopodias.。And these phenomenons are more obvious on R20 surface.
2, the distribution situation of micro-nano topographical surface rBMMSCs cytoskeleton
RBMMSCs inoculation is containing 24 orifice plates of sample, every porocyte density is 5 × 103 cell densities, 37 DEG C of 5%CO2 are after 4 groups of specimen surfaces cultivate 3d, sample is transferred to 24 new orifice plates, PBS cleans 3 times, each 5 minutes, 4% paraformaldehyde fixes 20 minutes on ice, PBS liquid rinsing 3 times, each 5 minutes at every turn, with 200ul/ every hole rhodamine mark Phalloidine (150ug/ml) transfect cell skeleton, lucifuge incubated at room 40 minutes, PBS liquid rinsing 3 times, each 5 minutes at every turn, with DAPI lucifuge transfect cell core 10 minutes, PBS liquid rinsing 3 times, each 5 minutes at every turn, dye as Fig. 2 with confocal microscopy cytoskeleton afterwards.Smooth pattern specimen surface visible cell in slightly circle, red filiform cell's skeleton in endochylema, a small amount of elongated fibers and comparatively assembling.On micron pattern (R) surface, cell is the Polygons extended, and fibres visible is reticulated structure comparatively clearly; At two kinds of micro-nano topographical surface, cells intact form two ends in sharper fusiformis, obviously red fiber reticulated structure clearly in visible endochylema, and red fiber netted in micro-nano pattern (R20) superficial cell slurry is the most obvious.
3, inverted fluorescence microscope observation of cell skelemin distribution situation in cell
The bone implant material sample prepared is after the sterilization of Co60 radiation irradiation, and be placed in 24 well culture plates, cell is with 2 × 10
3the density inoculation in individual/ml/ hole, cultivate and change nutrient solution after 48 hours, cell cultures is stopped after 72 hours, use PBS liquid rinsing cell 3 times, each 5 minutes, 4% paraformaldehyde stationary liquid is used to fix 20 minutes on ice, PBS liquid rinsing 3 times, each 5 minutes, rear use 0.1%triton-X100 adds 500ul/ hole, room temperature places 15 minutes, PBS liquid rinsing 3 times, each 5 minutes, after add 500ul/ hole lowlenthal serum, room temperature closes 1 hour, remove serum, add the primary antibodie solution (PBS dilutes 100 times) that 200ul/ hole prepares, 4 DEG C of overnight incubation, room temperature rewarming 1 hour, PBS liquid rinsing 3 times, each 5 minutes, lucifuge adds the two anti-solution (PBS dilutes 100 times) of the FITC mark that 200ul/ hole prepares, room temperature lucifuge hatches 1 hour, PBS liquid rinsing 3 times, each 5 minutes, the Phalloidine that lucifuge adds 200ul/ hole rhodamine mark hatches 40 minutes, PBS liquid rinsing 3 times, each 5 minutes, lucifuge adds the DAPI solution-dyed 20 minutes that 200ul/ hole prepares, PBS liquid rinsing 3 times, each 5 minutes, use the anti-agent mounting of quenching of fluorescence.Inverted fluorescence microscope is adopted to observe Cdc42 cytoskeletal protein distribution situation in cell.From Fig. 3 Fluorescent Staining Observation, visible Cdc42 has extensive distribution and expression in cell, expresses lower, around nucleus, present high expression level at cytolemma and cytoplasm, especially obvious around micro-nano pattern (R20) nucleus.
4, cell viability detects
As shown in Figure 4, (a) is 3 days to the vigor result of 3 days and 7 days, and (b) is 7 days; Compared with smooth titanium (S), the vigor of the surperficial rBMMSCs cell of micron pattern (R) of two time points all obviously declines, and has significant difference compared with other three groups.Micro-nano pattern (R5, R20) is compared with smooth titanium (S), and when 3 days, cell viability is without considerable change, then obviously declines when 7 days, but fall is less than a micron pattern (R).
5, cell adhesion counting
RBMMSCs inoculates 24 orifice plates according to every hole 2 × 104 cell density, polishing clean after smooth titanium (S) as a control group, micron pattern (R), micro-nano pattern (R5, R20) are experimental group.Cell cultures is stopped after 37 DEG C of 5%CO2 cultivate 30min, 1h, 2h and 3h, with the rinsing 3 times repeatedly of PBS liquid, each 15 minutes, to remove non-attached cell, cell 2.5% glutaraldehyde is fixed, thereafter omnidistance lucifuge, DAPI is used to carry out dyeing 15 minutes, PBS liquid rinsing 3 times, each 5 minutes, by the sample after rinsing, left-hand thread was with inverted fluorescence microscope sight after 24 new orifice plates gently, and excitation wavelength is 400nm, taking pictures record in Stochastic choice four visuals field, utilizes ImageJ analysis software to carry out cell counting.Fig. 5 is that rBMMSCs sticks the DAPI stained photographs of 2h in surface of test piece.RBMMSCs sticks result as shown in Figure 6 in surface of test piece.A (), (b), (c), (d) are 30min, 1h, 2h and 3h respectively, cell adhesion through micro-nano specimen surface counts all higher than control group specimen surface, and along with the increase of nanotube caliber, cell adhesion counting is higher, and the difference between each experimental group has statistical significance (p<0.05).
6, micro-nano topographical surface rBMMSCs alkaline phosphatase synthesis capability
RBMMSCs cell is at the micro-nano pattern of difference (S, R, R5, R20) surface seeding and after osteogenic induction cultivates 7 days, use the expression of test kit to alkaline phosphatase to dye, result as shown in Figure 7.The picture taken under Stereo microscope shows each group of topographical surface and all occurs hepatic alkaline phosphatase crystalline particle, compared with smooth titanium group, micron and micro-nano group of expression amount showed increased, and color is comparatively dark, and increases more obvious in micro-nano pattern (R20) surface expression amount.Along with the change of micron, nanotopography, alkaline phosphatase (ALP) quantum of output entirety is in the trend increased, the quantum of output micron pattern (R) of ALP and micro-nano pattern group (R5/R20) are apparently higher than smooth titanium surface group (S), experimental group and control group significant difference, and also there is between each experimental group significant difference (p<0.05).As shown in Figure 8, the result of ALP Activity determination also presents the identical trend of dyeing.
7, micro-nano topographical surface BMMSCs collagen secretion level
Adopt the method for sirius red stains and semi-quantitative analysis to detect rBMMSCs cell to cultivate after 14d in micro-nano topographical surface collagen secretion level through osteogenic induction, as shown in Figure 9: visible under stereoscopic microscope, all there is the cotton-shaped secretory product of pink silk in four groups of topographical surface, and more obvious at micron pattern (R) and micro-nano pattern group (R5/R20) collagen secretion.See Figure 10, semi-quantitative analysis result shows, compared to smooth titanium surface group (S), micron pattern (R) and micro-nano pattern group (R5/R20) significance have raised cell collagen secretion capacity, but they do not possess significant difference between three groups.
8, micro-nano topographical surface BMMSCs mineralization of extracellular matrix ability
The method of alizarin Soviet Union's red colouring and semi-quantitative analysis is adopted to detect: rBMMSCs cell is after osteogenic induction cultivates 14 days, in the level of four groups of topographical surface mineralization of extracellular matrix, as shown in figure 11: the photo taken from Stereo microscope, all there is red Mineral nodules in four groups of topographical surface, and dyes more obvious at micron and micro-nano group of mineralising.R20 group surperficial Mineral nodules madder Soviet Union red colouring amount is more than R5 and R group.Use spectrophotometer semi-quantitative analysis after using elution dyeing, as shown in figure 12, compared with smooth titanium pattern, micron and micro-nano pattern significantly improve the ability of mineralization of extracellular matrix to result, have statistical significance.And it is the highest at micro-nano pattern (R20) surface extracellular matrix mineralization ability (OD value).
9, the expression of different micro-nano topographical surface cellular form and skeleton genes involved
RBMMSCs cell, after S, R, R5, R20 surface cultivates 7 days, adopts the method for real-time quantitative PCR to detect the expression of RhoGTPases genes involved rac1, rhoA, cdc42.As shown in figure 13, all in all, compare with smooth titanium group (S), micron pattern and micro-nano pattern have all raised the expression level of each RhoGTPases gene, and the promoter action of R20 is more obvious.Wherein, the cdc42 gene expression dose of micron and micro-nano topographical surface apparently higher than smooth surface, and is expressed also apparently higher than R5 group in R20 group, there is significant difference (P<0.05) between two groups.
Table 3 cellular form and skeleton target gene-specific primers
10, the expression of different micro-nano topographical surface Bone formation-related gene
RBMMSCs cell, after S, R, R5, R20 surface cultivates 7 days, adopts the method for real-time quantitative PCR to be detected as the expression of bone related genes Col I, OCN, OPN, OSX, BSP, BMP, ALP.As shown in figure 14: all in all, compare with smooth titanium group (S), micro-nano pattern (R5, R20) has raised each Bone formation-related gene expression level, and especially the rise effect of R20 is the most obvious.Wherein, relative to smooth group, OCN, OPN, ALP gene expression dose of micron topographical surface (R5, R20) obviously raises, and slightly lowers at Col I, OSX, BSP, BMP gene expression dose; Micro-nano topographical surface (R5) only obviously raises at Col I, OCN, OPN, ALP gene expression dose, and micro-nano topographical surface (R20) Col I, OCN, OPN, OSX, BSP, BMP, ALP gene expression dose all obviously raise, between each group, all there is significant difference (P<0.05).
Table 4 skeletonization related objective gene-specific primer
In sum, in order to the surface optimization design of better bone implant material provides biological foundation, on basis in the past, we add large quantity measuring method, improve part detection method, make the method comprehensive system, by cytology and the evaluation of molecules cross-synthesis, the research of this surface optimization to bone implant material has great directive significance.The present invention improves the examination criteria of bone implant material surface cell in vitro form and osteogenesis function, add Laser Scanning Confocal Microscope cytoskeleton and morphologic observation, cellular immunofluorescence are observed, DAPI dyes adheres to and the project such as related gene expression detection, ensure that the comprehensive and advanced of detection method, can effectively from multi-angle assessment bone implant material on the impact of cellular form and osteogenesis function.Relatively mtt assay, adopts CCK8 kit detection cell active, and measuring method is more accurate, and workable, error is little.Dyeing on qualitative observation basis in the past, by using alkaline phosphatase (AKP) and BCA test kit, make collagen elutriant and mineralising elutriant by oneself, in conjunction with microplate reader, quantitative or half-quantitative detection are carried out to alkaline phosphatase, collagen secretion, epimatrix mineralising, detection method is more accurate, and result is also more reliable.
Claims (7)
1. a methods of testing and evaluating for bone implant material surface cell in vitro form and osteogenesis function, is characterized in that, comprise the following steps:
1) bone implant material with evaluating to be detected is got, observation of cell form, cytoskeleton and skelemin distribution situation;
2) cell viability, material surface cell adhesion forces and cell activity change of Alkaline phosphatase is detected;
3) collagen secretion and mineralization of extracellular matrix degree is detected;
4) expression of cellular form and skeleton genes involved, Bone formation-related gene is measured;
5) performance treating the bone implant material of test and repair according to above detected result is evaluated:
First judge whether bone implant material superficial cell form extends, and whether the filopodia that cell terminal extensions goes out increases, and whether cytoskeleton fiber increases, judge that cytoskeletal protein is expressed and whether strengthen;
Secondly, judge whether cell adhesion forces on bone implant material surface and alkaline phosphatase activities strengthen;
Again, judge whether collagen secretion and mineralization of extracellular matrix degree strengthen;
Finally, judge that skeleton genes involved and Bone formation-related gene are expressed whether to strengthen;
Meet conclusions and then prove that this bone implant material meets biomedical security requirement, otherwise, then do not meet biomedical security requirement.
2. the methods of testing and evaluating of bone implant material surface cell in vitro form according to claim 1 and osteogenesis function, is characterized in that, step 1) middle observation of cell form, cytoskeleton and skelemin distribution situation, concrete operations are as follows:
Detect cellular form: after being sterilized by bone implant material sample to be detected, getting cell is seeded in 24 well culture plates, after cultivating 72h, fix by 2.5% glutaraldehyde, then adopt the ethanol dehydration of gradient concentration, then with field emission scanning electron microscope, cellular form is observed after drying metal spraying;
Detect matrix morphology: fix with 4% paraformaldehyde, after the Phalloidine incubated at room with 50 μ g/mL rhodamine marks, through DAPI dyeing, then with the anti-agent mounting of quenching of fluorescence, adopt laser confocal microscope to observe the distribution of cytoskeleton and form;
Detection skelemin distributes: functional quality mark is that 4% paraformaldehyde is fixed, 15 minutes are placed by 0.1%triton-X100 room temperature, after add lowlenthal serum, room temperature closes 1 hour, remove serum, add primary antibodie, in 4 DEG C of overnight incubation, room temperature rewarming 1 hour, lucifuge adds two of FITC mark and resists, room temperature lucifuge hatches 1 hour, the Phalloidine that lucifuge adds rhodamine mark hatches 40 minutes, lucifuge adds DAPI solution-dyed 20 minutes, again with the anti-agent mounting of quenching of fluorescence, inverted fluorescence microscope is adopted to observe Cdc42, RhoA and Rac1 cytoskeletal protein distribution situation in cell.
3. the methods of testing and evaluating of bone implant material surface cell in vitro form according to claim 1 and osteogenesis function, it is characterized in that, step 2) middle detection cell viability, material surface cell adhesion forces and cell activity change of Alkaline phosphatase, concrete operations are as follows:
Detect cell viability: after cell to be detected is seeded to bone implant material, by cell cultures after 3 days or 7 days, after sample being transferred to 24 new well culture plates, add CCK-8 and nutrient solution, after hatching 3 hours, solution is transferred to 96 orifice plates, blank well returns to zero, and uses microplate reader to measure absorbance at 450nm place;
Test material superficial cell adheres to: after being sterilized by bone implant material sample to be detected, getting cell is seeded in 24 well culture plates, cell cultures is stopped after cultivating 30min, 1h, 2h and 3h, omnidistance lucifuge after fixing by 2.5% glutaraldehyde, dye with DAPI, by the sample left-hand thread after rinsing at 24 new orifice plates, see Stochastic choice four visuals field with inverted fluorescence microscope and to take pictures record, utilize ImageJ analysis software to carry out cell counting;
Alkaline phosphatase assay: after cell inoculates 48 hours, nutrient solution is replaced by osteogenic induction nutrient solution, cultivate 7 days, fix with 4% paraformaldehyde, use NBT/BCIP alkaline phosphatase activities test kit, experimentally flow process application of sample colour developing, observes under Stereo microscope and takes pictures, carry out alkaline phosphatase activities detection.
4. bone implant material according to claim 1 surface cell in vitro form and the methods of testing and evaluating of osteogenesis function, is characterized in that, step 3) in detection collagen secretion and extracellular remember mineralization degree, concrete operations are as follows:
Detect collagen secretion: cell is seeded in 24 well culture plates, nutrient solution is replaced by osteogenic induction nutrient solution, and cultivates 14 days, fixes with 4% paraformaldehyde solution, after using 1% Picro-Sirius red/picric acid solution dyeing 18 ~ 24h, seasoning in room temperature environment; Stereo microscope is used to observe and take pictures; Meanwhile, carry out semi-quantitative analysis to the collagen secretion of specimen surface, be transferred to by sample in 24 new orifice plates, every hole adds collagen staining elutriant, then sucking-off dye liquor is transferred to 96 orifice plates, detects absorbance in microplate reader in wavelength 540nm place;
Detect mineralization of extracellular matrix situation: cell is seeded in 24 well culture plates, nutrient solution is replaced by osteogenic induction nutrient solution, cultivate and induce after 14 days, add sodium alizarinsulfonate dye liquor to specimen surface, left at room temperature 15 minutes, observes under putting Stereo microscope and takes pictures; Semi-quantitative analysis is carried out to the mineralization of extracellular matrix of specimen surface: sample is positioned over new 24 and cultivates orifice plate, every hole adds 1mL dyeing elutriant respectively, shaking table shakes up, after on sample, dyestuff fully dissolves, every hole is sucking-off 3 parts of dye liquors respectively, and every part is 150 μ L, then adds in 96 well culture plates, use the simple elutriant of 150 μ L for contrast zeroing, measure absorbance in wavelength 620nm place.
5. the methods of testing and evaluating of bone implant material surface cell in vitro form according to claim 1 and osteogenesis function, it is characterized in that, step 4) in measure the expression of cellular form and skeleton genes involved, Bone formation-related gene, concrete operations are as follows:
Detect cellular form and skeleton related gene expression: cell is seeded in 24 well culture plates, and cultivate and change nutrient solution after 48 hours, culturing cell stops cell cultures after 3 days or 7 days; Use PBS rinsing cell 3 times, extract cell total rna and carry out real-time quantitative PCR and detect Cdc42, Rac1 and RhoA genetic expression;
Be detected as bone related genes to express: cell is seeded in 24 well culture plates, cultivate and change nutrient solution after 48 hours, culturing cell stops cell cultures after 3 days or 7 days, use PBS rinsing cell 3 times, extract cell total rna and carry out real-time quantitative PCR and detect Ocn, Bsp, Col I, Opn, ALP, BMP and OSX genetic expression.
6. the methods of testing and evaluating of bone implant material surface cell in vitro form according to claim 1 and osteogenesis function, it is characterized in that, described cell to be detected is selected from mesenchymal stem cells MSCs, mouse bone-forming cell system cell, OS-732 cells system cell or original cuiture scleroblast.
7. the methods of testing and evaluating of bone implant material surface cell in vitro form according to claim 1 and osteogenesis function, it is characterized in that, the described bone implant material with evaluating to be detected comprises titanium, titanium alloy, phosphorus ash stone biological ceramics and matrix material thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510604370.0A CN105112493A (en) | 2015-09-21 | 2015-09-21 | Method for detecting and evaluating in-vitro cell morphology and osteogenic function of surface of bone substitute implant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510604370.0A CN105112493A (en) | 2015-09-21 | 2015-09-21 | Method for detecting and evaluating in-vitro cell morphology and osteogenic function of surface of bone substitute implant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105112493A true CN105112493A (en) | 2015-12-02 |
Family
ID=54660599
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510604370.0A Pending CN105112493A (en) | 2015-09-21 | 2015-09-21 | Method for detecting and evaluating in-vitro cell morphology and osteogenic function of surface of bone substitute implant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105112493A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018192152A1 (en) * | 2017-04-19 | 2018-10-25 | 中国食品药品检定研究院 | Preparation and application of model for use in detecting in vitro endothelialization of vascular stent material |
| CN110938669A (en) * | 2019-12-20 | 2020-03-31 | 南昌大学第二附属医院 | Method for verifying stimulation of osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| CN112899215A (en) * | 2017-11-30 | 2021-06-04 | 山西加乐医疗科技有限责任公司 | Cell scaffold and preparation method thereof |
| WO2023010660A1 (en) * | 2021-08-03 | 2023-02-09 | 北京大学口腔医学院 | Method for predicting and evaluating function of biomaterial |
-
2015
- 2015-09-21 CN CN201510604370.0A patent/CN105112493A/en active Pending
Non-Patent Citations (7)
| Title |
|---|
| LEI LIU等: "Rapamycin Inhibits Cytoskeleton Reorganization and Cell Motility by Suppressing RhoA Expression and Activity", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| MARC SYMONS: "Rho family GTPases: the cytoskeleton and beyond", 《TRENDS BIOCHEM SCI》 * |
| 王薇: "种植体微纳米形貌对成骨细胞行为影响的分子机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 罗玉敏 等: "《脑血管病实验方法学》", 30 April 2014, 中国医药科技出版社 * |
| 赵领洲: "TiO2纳米管抗菌与生物活性双功能种植体涂层的构建与评价", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 闫钧: "antimiR-138修饰的rBMMSCs膜片的构建及其与种植体复合后的体内外生物学研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 马千里: "纯钛种植体表面纳米形貌通过诱导巨噬细胞M1/M2极化影响宿主成骨功能的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018192152A1 (en) * | 2017-04-19 | 2018-10-25 | 中国食品药品检定研究院 | Preparation and application of model for use in detecting in vitro endothelialization of vascular stent material |
| CN112899215A (en) * | 2017-11-30 | 2021-06-04 | 山西加乐医疗科技有限责任公司 | Cell scaffold and preparation method thereof |
| CN110938669A (en) * | 2019-12-20 | 2020-03-31 | 南昌大学第二附属医院 | Method for verifying stimulation of osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| CN110938669B (en) * | 2019-12-20 | 2023-03-28 | 南昌大学第二附属医院 | Verification method for stimulating osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| WO2023010660A1 (en) * | 2021-08-03 | 2023-02-09 | 北京大学口腔医学院 | Method for predicting and evaluating function of biomaterial |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yu et al. | Effects of 3-dimensional bioprinting alginate/gelatin hydrogel scaffold extract on proliferation and differentiation of human dental pulp stem cells | |
| Baldión et al. | Odontoblast‐Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation | |
| Kim et al. | Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell | |
| Jablonská et al. | A review of in vitro cell culture testing methods for bioactive glasses and other biomaterials for hard tissue regeneration | |
| Vuornos et al. | Bioactive glass ions induce efficient osteogenic differentiation of human adipose stem cells encapsulated in gellan gum and collagen type I hydrogels | |
| CN105112493A (en) | Method for detecting and evaluating in-vitro cell morphology and osteogenic function of surface of bone substitute implant | |
| Zhao et al. | Evaluation of long-term biocompatibility and osteogenic differentiation of graphene nanosheet doped calcium phosphate-chitosan AZ91D composites | |
| KR101721514B1 (en) | Cell scaffold for three dimensional cell culture comprising agarose, decelluarized extracellular matrix and collagen | |
| CN107446885B (en) | Mesenchymal stem cell in-vitro osteogenic induced differentiation scaffold material and application thereof | |
| Altmann et al. | Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions | |
| CN106987555A (en) | Efficiently induce the micromolecular compound composition of human pluripotent stem cells myocardiac differentiation | |
| Zhu et al. | Evaluation of canine bone marrow-derived mesenchymal stem cells after long-term cryopreservation | |
| Nasakina et al. | Biocompatibility of nanostructured nitinol with titanium or tantalum surface composite layers formed by magnetron sputtering | |
| Birru et al. | Improved osteogenic differentiation of umbilical cord blood MSCs using custom made perfusion bioreactor | |
| Ding et al. | Si-doped porous TiO2 coatings enhanced in vitro angiogenic behavior of human umbilical vein endothelial cells | |
| CN108578771A (en) | Preparation method and products thereof with the FGF1 sericin gels for promoting cell-proliferation activity | |
| CN104046589A (en) | Method for inducing in vitro directional differentiation of stem cells by cell co-culture | |
| CN107299079A (en) | A kind of three-dimensional active complex of neural tissue engineering dental pulp stem cell and its three-dimensional construction method | |
| Jiang et al. | Laminin‐521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light‐Induced Cell Sheet Technology | |
| Sun et al. | Bone cell responses to a low elastic modulus titanium alloy surface immobilized with the natural cross-linker genipin | |
| Abay et al. | Bone formation from porcine dental germ stem cells on surface modified polybutylene succinate scaffolds | |
| Li et al. | In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers | |
| CN1884494B (en) | Method for inducing human embryo stem cell differentiation to liver cell and the special-purpose medium | |
| Torres-Sanchez et al. | Physico-chemical characterisation of Ti-Nb-Sn alloys surfaces and their osteogenic properties | |
| CN102350009A (en) | Preparation method of composite bone matrix gelatin polylactic acid porous bioactive material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151202 |