CN102430153A - Crosslinking-technology-based bionic bone interface, and construction method and application thereof - Google Patents

Abstract

The invention relates to a crosslinking-technology-based bionic bone interface, and a construction method and application thereof. In the invention, biology signal-recombinant fibronectin/cadherin and a calcium phosphate ceramic micropore interface are subjected to covalent coupling. The invention aims to promote the high-efficiency adhesion and targeted differentiation of cells on the interface and belongs to the field of interface tissue engineering. The signal protein is recombinant protein of fibronectin type III (FNIII)7-10 carrying Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) motifs and cadherin functional domain EC1-2. crosslinking molecules are same bifunctional crosslinking agent ditertiary butyl peroxide (DTBP), and protein and materials can be bridged through amino acetylation. Plantation density and mineralization capacity of seed cells on the interface are improved through isophil and heterophilic adhesion of the signal protein and integrin subtype mediated osteoblasts induction. The interface characteristics are identified in the aspects of appearance topology, surface chemistry, cytocompatibility and the like during implementation. The crosslinking-technology-based bionic bone interface is suitable for preparation of alternative materials of bioactivity tissue engineering of compound cells.

Description

A kind of bionical bone interface, construction method and application thereof based on crosslinking technological

Technical field

The present invention relates to a kind of through connect the bionics interface of albumen/calcium attachment proteins and biphase calcium phosphor pottery with two official's crosslinking technological coupling bio signal recombinant fibers.Belong to Interface Microstructure engineering field.

Background technology

For making bone renovating material have good biological activity and histocompatibility; Can modify according to the bionics principle; Promptly under the prerequisite that does not change substrate performance; Bioactive molecule is fixed on synthetic material or natural material surface, and design has the organizational project formed material of particular organisms, physicochemical property, with the 'inertia' interface change into be beneficial to seed cell provide stick, the bioelectric interface of propagation and differentiation signal.

Cell sticks mainly by the protein molecular layer decision of adsorbing at the specificity of substrate surface.After base material implants; Can from ambient body fluid, select and absorb some protein component (like fibronectin; Laminin; And multiple protein has then determined the biological behaviour of material surface character and seed cell in the competitive adsorption of material interface and the end-state of desorption---Vroman effect vitronectin).Set out thus; The biomimetic modification strategy be intended to through physics, chemistry, biological means host material-cytosis interface " reconstruction " have certain three dimensional structure and molecular signal microenvironment; Improve planting density, adhesive efficiency and the signal intensity of seed cell at material surface; Induce specific cell signal cascade reaction, cell guiding is to particular organization's target spot Growth and Differentiation.It is advantageous that its be fully recognized that extracellular matrix (Exracellular Matrix, ECM) at bone development, keep, heal and rebuild in effect, i.e. the importance of the dialogue of ECM microenvironment and osteoblast, osteoprogenitor cells and communication.

Material is carried out finishing can adopt albumen or polypeptide with adhesion characteristics.The much more early stage native proteins that adopt, like FN, collagen, laminin, but cost is high, and controllability is low, its use that has been prone to cause effects limit such as immune and cross reaction.Proteic cell adhesion site is only determined by the motif that several aminoacid are formed more than the later stage discovery, as is positioned at ¹⁰The RGD motif of FNIII.Further investigation for many years is comparatively ideal to have realized the interface modification of FN to tissue engineering material through bionical means.Wherein select at first, most popular is to contain RGD polypeptide of sequence (simulating peptide), like tetrapeptide RGDS, and six peptide GRGDSY, seven peptide GRGDSPC etc.In fact, RGD exists only in FN, and it is one type of consensus motif that is present in the multiple attachment proteins structure, like bone sialoprotein and osteopontin, therefore it is modified material surface and can improve adhesive efficiency through number of ways.Thus, first generation bionic surface decorative material arises at the historic moment, and soon the RGD peptide is fixed to material surface through the method for passive absorption.Experiment shows that this surface has the positive regulation effect to osteoblastic adhesion and Osteoblast Differentiation.Like Dee, Bearinger etc. draw a circle to approve the RGD peptide at material surfaces such as Pyrex, quartz, interpenetrating polymer networks respectively in experiment in vitro, find that it can be enhanced to osteocyte and adhere to, breeds and differentiation.In addition, the implant that engages through the RGD peptide can more effective promotion implant periphery area of new bone form.Ferris, indexs such as area of new bone amount area, thickness all are superior to blank on this interface of discoveries such as Kantlenheler.Domestic scholars Zheng Qi newly is covalently cross-linking to the PLGA surface with RGD seven peptides, finds the adhesion that it can more effective promotion mescenchymal stem cell, and cytoskeleton is assembled with early stage skeletonization sign alkaline phosphatase activities again and strengthened.

Although compare with native protein, RGD peptide controllability is strong, yet owing to lack attached or the existence of modulability domain, makes its higher structure and space conformation of being difficult to reach native protein, so its biological activity is limited.In addition, surface parameter such as form, conformation, use density etc. can have a strong impact on the function of RGD sequence.If any discovering that cyclic peptide (cyclo-RGDFX) has longer biological half-life and lower basic effect density than linear peptides (GRGDSPC) when rebuilding articular cartilage.Still used density and conformation specific to limit in addition, and exist under the certain force field action and take off absorption problem.

For improving bioavailability; Take into account the specificity (like α 5 β 1) of the plain hypotype of integration and the effect of the plain hypotype of the bonded integration in non-RGD site (like α 2 β 1) simultaneously; The improvement strategy on second filial generation biomimetic modification surface is around the RGD sequence, to draw a circle to approve enough functional sites, to play a role through integrating the plain receptor pathway of element and nonconformity.Wherein one of method is that multiple small peptide mixes and modifies, and can increase like utilization RGD such as Rezania and FHRRIKA (heparin binding motif) mixing modification of surfaces that osteoblast sticks and mineralising.Two of method is that the complementarity of core motif allow RGD and auxiliary sequencel PHSRN is modified.Like Dillow and Kao etc. with as above sequence coupling or modify separately in Polyethylene Glycol polymer surface, find itself and α 5 β 1 integrate plain stiffness of coupling, with all significantly enhancings of adhesion of macrophage.

Modification characteristics to bionical interface of first and second generation; Garcia etc. are on a large amount of bases to FN molecular structure and function assessment research; Develop a kind of HMW aglucon of simulating native protein one-level, secondary and tertiary structure, like FNIII7-10, FNIII9-10.This molecule can be in protokaryon or eukaryote system rational modification, efficiently express, simulated the conformational effect of native protein ideally, have the biological effect identical with plasma F N.Short glue, facilitate have the unrivaled superiority of other peptide molecules aspect the bone differentiation.

Cultler is crosslinked in polystyrene surface with FNIII7-10, finds that through centrifugal adhesion experiment and immunofluorescence this surface significantly improves the adhesiving effect of osteoblast MC3T3-E1 and the packaging efficiency of adhesion plaque.Petrie has analyzed action effect and the mechanism thereof of FNIII7-10 through the experiment in vivo and vitro of system.In the experiment in vitro FNIII7-10 is bonded on alkanethiol self assembly molecule laminar surface; Utilize surface plasma resonance technology and ELISA adjustment to optimize the material surface ligand density; Find its than RGD seven peptides and RGD-PHSRN coupling peptide have stronger promotion cell proliferation, the effect of sticking and focal adhesion kinase phosphorylation level, mainly integrate plain with illustrating its action pathway at 0 o'clock through α 5 β 1.In testing in the body; FNIII7-10 is modified at the glycol molecule layer and is the medical titanium plate surface of background and implants rabbit tibia cortex defect; After finding for 4 weeks; Simple titanium plate in this surface and RGD modification of surfaces can more effectively arrive and promote Osteoblast Differentiation, the substrate mineralising of bone marrow stem cell at material surface, and sclerous tissues's section and dyeing show that host material is substituted by more area of new bone.As above research shows: FNIII7-10 biomimetic modification interface can promote biomechanics to integrate in vivo and in vitro more efficiently, has tremendous potential at bone tissue engineer and regenerative medicine field.

Summary of the invention

The present invention is for solving the deficiency of prior art, and the present invention provides a kind of bio signal albumen (CDH-11-EC based on the crosslinked pattern of DTBP _1-2/ FN III _7-10) the pBCP bioelectric interface modified; Its purpose has two: one of which; " same preferendum " effect enhance bone CFU-GM that is intended to " different preferendum " and CDH 11 (CDHherin 11) through FNIII7-10 acts on intensity between the plantation density of material interface and osteocyte, the inducing action of integrating plain hypotype mediation through specificity simultaneously improves osteogenic ability and mineralization degree.They are two years old; Solve the deficiency of taking off absorption under the low and particular case of absorption efficiency that the used coating technology of tradition exists; The present invention utilizes covalent reaction to realize firm connection the between signal protein and timbering material; Improve the distribution intensity of biological aglucon at the interface, structure has the surface biomimetic microenvironment of good organization's compatibility.

Technical solution of the present invention is following:

The present invention relates to a kind of through connecting albumen/calcium attachment proteins and biphase calcium phosphor ceramic capillary interface with two official's crosslinking technological coupling bio signal recombinant fibers; Said bio signal albumen is to take the FN III7-10 fragment of PHSRN and RGD motif and the recombiant protein of the outer functional domain 1-2 (CDH 11EC1-2) of calcium attachment proteins born of the same parents, this albumen called after CDH-11-EC _1-2/ FN III _7-10, timbering material is a micropore biphase calcium phosphor pottery, called after pBCP, corsslinking molecular are DTBP, and same pair of official's cross-linking agent of a kind imino-ester class, the spacerarm two ends of said corsslinking molecular form CDH-11-EC through the acetylization reaction with amino _1-2/ FN III _7-10And the bridge joint (NH between pBCP ₂... NH ₂).Said bionical interface called after CDH-11-EC _1-2/ FN III _7-10-pBCP; Seed cell is mesenchymal stem cells MSCs, osteoblast or chondroblast, according to the organizational project type selecting at constructed interface.

The main construction step at this bionical interface is following:

1. bio signal PROTEIN C DH-11-EC _1-2/ FN III _7-10Design, preparation and purification, referenced patent document CN101463090 " fusion rotein of fibronectin and calcium attachment proteins-11, method for preparing and application ".

2. the preparation of timbering material pBCP is provided by biomaterial key lab of country of Sichuan University.Its preparation method is wet-chemical deposition and foaming.Sample is tested through X-ray diffraction, and wherein HA and TCP content ratio are 60/40; Surface porosity factor is at 60%-70%, the about 50-500 μ of surface holes diameter m.

3.CDH-11-EC _1-2/ FN III _7-10The assembling at the bionical interface of-pBCP: successively through BSA sealing, PBS washing, DTBP precrosslink, PBS washing, CDH-11-EC _1-2/ FN III _7-10Fusion rotein is hatched, PBS washs, the glycine solution cessation reaction, accomplishes interfacial preparation.

4.CDH-11-EC _1-2/ FN III _7-10The sign evaluation at the bionical interface of-pBCP: utilize methods such as scanning electron microscope (SEM), x-ray photoelectron spectroscopy (XPS), protein adsorption and area density mensuration to accomplish.

5. external function assessment research: mtt assay detects CDH-11-EC _1-2/ FN III _7-10The bionical interface of-pBCP hMSCs cell proliferation situation, SEM observes the hMSCs growthform and changes.Centrifugal short adhesion experiment detects the adhesive capacity to the hMSCs cell.Skeletonization mark Bone Gla protein (OCN) gene, protein expression and calcium tuberosity staining analysis CDH-11-EC _1-2/ FN III _7-10The bionical interface of-pBCP is to the influence of hMSCs Osteoblast Differentiation and substrate calcification.

The present invention adopts the crosslinked strategy based on DTBP, and its reaction condition is gentle, and few with the non-specific responding of nucleophilic histone, the imino-ester product does not influence proteic overall load, has kept proteic native conformation and biological activity.And (S-S-), be convenient to that cross-linking efficiency is carried out XPS detects contained distinctive disulfide bond.

The CDH-11-EC of gained of the present invention _1-2/ FN III _7-10The bionical bone interface of-pBCP has the short dual bio signal albumen that adheres to, facilitates bone in application facet, and composes micropore space structure with timbering material to increase finishing area and advantages such as cell adhesion, substrate mineralising volume.

The present invention can use in the structure of tissue-engineered bone, cartilage, periosteum, for the foundation at biological activity timbering material interface provides a kind of effective technical.

Description of drawings

Fig. 1 shows CDH-11-EC _1-2/ FN III _7-10The structure pattern at the bionical interface of-pBCP: a.CDH-11- _EC1-2/ FN III _7-103-D solid structure shows the lysine group (lysine) that is arranged in fusion rotein; The molecular structural formula of b.DTBP; C.CDH-11-EC _1-2/ FNIII _7-10The structure flow process and the dimension scale at the bionical interface of-pBCP;

Fig. 2 shows CDH-11-EC _1-2/ FN III _7-10Biomimetic modification is to the influence of BCP interfacial microstructure.A-d, the unmodified interface is at 1k, 5k, 10k and the 20k SEM image under doubly; E-h modifies rear interface at 1k, 5k, 10k and the 20k SEM image under doubly; Fig. 3 shows that each surface-element of full analysis of spectrum biomimetic modification front and back distributes under the XPS technology: a, mainly there are four kinds of elements in the simple BCP of negative control group surface: carbon (C _C-C=248.8eV, C _C=O=289.4eV), calcium (Ca 2p=346.6eV, Ca 2s=437.8eV), phosphorus (P 2p=133.0eV, P 2s=190.0eV) and oxygen (531.8eV).D: sample group CDH-11-EC _1-2/ FN III _7-10A tangible nitrogen peak appears in-pBCP surface near 401.0eV; B, c are matched group (CDH 11-pBCP, FN-pBCP).Fig. 4 shows CDH-11-EC _1-2/ FN III _7-10Protein adsorption and area density on the pBCP surface are measured the result: the a.SDS-PAGE standard measure is adhesion protein not; B. surperficial aglucon Statistics of Density figure;

Fig. 5 shows that the hMSCs cell is at CDH-11-EC _1-2/ FN III _7-10The growth curve of-pBCP;

Fig. 6 shows CDH-11-EC _1-2/ FN III _7-10-pBCP interface is to the influence of hMSCs growthform: A-B, C-D, E-F.G-H, low power (250 *) and high power (1800 *) down hMSCs respectively at BCP, CDH-pBCP, FN-pBCP and CDH-11-EC _1-2/ FNIII _7-10The growthform at-pBCP interface;

Fig. 7 shows that hMSCs is adherent quantitatively in different B CP treatment surface, qualitative analysis: (a) relation of BCP area density and cell adhesion quantity under the different condition.(b)-(e) under the fluorescence microscope cell in the adhesion situation on different B CP surface.(scale: 200 μ m)

Fig. 8 shows CDH-11-EC _1-2/ FN III _7-10OCN gene, the protein expression level of-pBCP surface hMSCs;

Fig. 9 shows PCR reaction condition figure;

Figure 10 shows CDH-11-EC _1-2/ FN III _7-10-pBCP surface calcium tuberosity forms situation: before (a) being unstained under light microscopic observed calcium tuberosity; (b) different B CP surface hMSCs calcium tuberosity differential expression after alizarin red dyeing: 1-4 is respectively: blank control group (BCP), matched group 1 (CDH 11-pBCP), matched group 2 (FN-pBCP), sample group (CDH-11-EC _1-2/ FNIII _7-10-pBCP).

The specific embodiment

Specify building process of the present invention below in conjunction with embodiment:

1.CDH-11-EC _1-2/ FN III _7-10The assembling at the bionical interface of-pBCP, referring to Fig. 1:

Adopt with two crosslinked methods of official, with fusion rotein CDH-11-EC _1-2/ FN III _7-10Be fixed to material interface through covalently bound mode.The same pair of official's cross-linking agent that is adopted is DTBP (C ₈H ₁₈N ₂O ₂S ₂Cl ₂), this material belongs to the imidic acid esters, has water solublity, permeable membrane, its spacerarm (spacer arm) two ends can through and the specific reaction of amino form the bridge spline structure between two kinds of molecules, be the common agents that is used for the albumen coupled reaction at present.The concise and to the point step that this bionical interface makes up is described below,

(1). sealing: BCP places the BSA solution soaked overnight (4 ℃) of 85 ℃ of thermal denaturations 1%, and the nonspecific binding site on closed material surface provides competent amino group for material surface simultaneously.

(2). washing: the above-mentioned BCP:5min of PBS solution washing * 2 times.

(3). crosslinked: the DTBP solution of proper volume (20mM, pH 8.5, are as the criterion to flood material surface), incubated at room 1-2hr.

(4) washing: the above-mentioned BCP:5min of PBS solution washing * 2 times.

(5). connect: excessive fusion rotein solution, 4 ℃ of incubated overnight.

(6). washing: the above-mentioned BCP:5min of PBS solution washing * 2 times.

(7). stop: the glycine solution of proper volume, making final concentration is 50mM, cessation reaction.

2.CDH-11-EC _1-2/ FN III _7-10The sign evaluation at the bionical interface of-pBCP,

For investigating CDH-11-EC _1-2/ FN III _7-10The biology at the bionical interface of-pBCP, physicochemical property in the hope of understanding this interface in characteristics such as surface chemistry, microscopic appearance, roughness, hydrophilic and hydrophobic and surperficial aglucon density, and are judged the suitable degree and the histocompatibility of cell growth on this interface.

(1). scanning electron microscope (SEM), referring to Fig. 2:

The electron beam irradiation specimen surface that utilize to focus on is through secondary electron or be scattered electronic imaging and carry out morphology observation.Specimen preparation: after the interface room temperature was dried 48hr, vacuum was drained 24hr, promptly can be used for SEM behind the injection instrument face gold,platinized film and detected.

Operating procedure is following:

1. sample is put into the tem sample chamber: venting → reduction podium level → fixed sample cup → evacuation 10 ^-3Handkerchief.

2. Image Acquisition: high power obtains original image after selecting accelerating potential 30kV → translation, inclination sample stage → first low power according to sample character.

3. graphical analysis: regulate object lens electric current → adjustment focal length → acquisition, analysis image.

(2) .X photoelectron spectroscopy (XPS), referring to Fig. 3:

1.. specimen preparation: after the interface room temperature was dried 48hr, vacuum was drained 24hr and promptly can be used for the XPS detection.

2.. detection method: the sample bin vacuum pressure is 2 * 10 ^-7Torr, the sample stage inclination angle is 20 °, adopts AlK α target source to excite as ion gun, running voltage and electric current are set to 12keV and 15mA.At first through sample is carried out full scan (full spectrum) in overall optical electron energy scope (0-1100eV), with the element of confirming to exist in the sample; And then selected peak-to-peak carried out narrow scan, to confirm chemical state (half spectrum).Calibration standard is the binding energy (284.8eV) of the C1s of organic contamination carbon.

3.. analytical method:

Qualitative analysis: contrast with measured light electronics spectrogram and standard spectrogram; Confirm to exist in the solid sample surface which element (and which kind of chemical compound these elements are present in) according to elemental characteristic peak position (and chemical shift), reference standard is the x-ray photoelectron spectroscopy handbook of Perkin-Elmer company.

Quantitative analysis: sensitivity factor method.Utilize SPSS10.0 software at first each group sample data to be carried out the average check, obtain each class mean and standard deviation, adopting each group of independent sample T check analysis that no difference of science of statistics and effectiveness thereof, p＜0.05 are arranged on this basis is remarkable significant difference.

(3). protein adsorption and area density are measured: referring to Fig. 4:

1. crosslinked (room temperature is 2hr) with gradient concentration CDH-11-EC through 1%BSA sealing (4 ℃ are spent the night), 20mM DTBP successively for BCP _1-2/ FN III _7-10(0,0.1,1.0,2.5,5.0,10.0,15.0 μ g/ml) connects back (4 ℃ are spent the night).

2. BCP collects residual liquid, to wherein add equivalent 2 * SDS sample buffer (0.1mol/L TrisCl, 20% glycerol, 4%SDS, 0.1% bromophenol blue, 10% β-ME), boiling water heating 5min processes sample liquid.

3. SDS-PAGE electrophoresis: as above BIAO and BEN 50ul adds on the gel in the appearance hole with micropipettor respectively, energized, the about 20min of 70V electrophoresis; When treating sample electrophoresis to separation gel interface; Voltage is upgraded to 100V, continues electrophoresis to bromophenol blue indicator and reach the gel lower edge, stop electrophoresis.

4. coomassie brilliant blue R250 dyeing, glacial acetic acid decolouring.

5. Doc 2000 gel scanning system document images are analyzed the band OD value.

6. analyze:, set up protein band optical density standard curve the capable SDS-PAGE electrophoresis of 0,0.1,1.0,2.5,5.0,10.0,15.0 μ g/ml albumen of preset standard concentration.Destination protein band OD value and standard curve contrast, thus measured value obtained.Surface aglucon density adopts The above results, calculates according to the below formula to get:

3. the separation and Culture of human marrow mesenchymal stem cell (hMSCs)

1. after the normal outpatient's informed consent of health check-up, adopt the method for anterior superior iliac spine puncture to obtain bone marrow, add aseptic heparin anticoagulant tube cryopreservation (specifically accomplishing) by hematology of new bridge hospital.

2. adopt the adherent screening method of density gradient centrifugation, the concrete operations step is following:

(1) collects bone marrow 3ml, add the aseptic PBS liquid of equivalent and softly wash the centrifugal 5min collecting cell of 1000rpm/min.

(2) abandon supernatant and fat deposit, collecting precipitation adds aseptic PBS liquid 3ml re-suspended cell, fully mixing.

(3) cell suspension is slowly added on isopyknic percoll cell separation liquid (Pharmacia, the U.S.) 2000rpm/min low-temperature centrifugation 2min, the mononuclearcell of collection tunica albuginea layer.

(4) aseptic PBS liquid washs, centrifugal (1000rpm/min 5min) 2 times, abandons supernatant.

(5) add DMEM/F12 culture medium (Hyclone, the U.S.) re-suspended cell that contains 10%FBS, by 10 ⁵-10 ⁶Density be inoculated in the aseptic 50ml culture bottle.In 37 ℃, 50%CO2, saturated humidity constant temperature cell culture incubator, cultivate 24hr.

(6) row changed liquid first after cell changeed kind of 24hr, discarded not attached cell, added the DMEM/F12 culture medium that contains 10%FBS, continuous culture in 37 ℃, 50%CO2, saturated humidity constant temperature cell culture incubator.

(7) liquid was changed once in every 2-3 days being inverted aberration microscopically observation of cell growing state every day in the back, the band cell grow to 3-6 generation, can be when fusion rate reaches 70%-80% in order to the osteogenic induction Analytical Chemical Experiment.

4.CDH-11-EC _1-2/ FN III _7-10-pBCP is to the influence of the hMSCs rate of increase and vigor, referring to Fig. 5:

Adopt mtt assay to detect hMSCs cell proliferation situation, draw cell growth curve, whether different disposal mode BCP interface influences its propagation and survival.Concrete grammar is following:

(1) interface processing: the BCP interface for preparing is placed 96 porocyte culture plates, and protein powder up.

(2) cell inoculation: conventional method prepares hMSCs single cell suspension (3-6 generation), is inoculated in 96 well culture plates with the first density in 2000/ hole, every hole culture medium total amount 200 μ l.Cultivated 1-9 days in the saturated incubator of 37 ℃, 5%CO2, appropriateness, the next day change liquid once.

(3) MTT liquid (Sigma, the U.S.) the 100 μ l of adding 5mg/ml continue to hatch 3-4hr in the cell culture incubator.

(4) culture fluid in the sucking-off hole adds DMSO liquid (150 μ l/ hole), places microwell plate to pull culture plate and swings the 10-20min that vibrates on the device, makes the crystal dissolving.

(5) each hole solution with variable color changes in another 96 well culture plate, and ELIASA detects and write down each hole OD value (detecting wavelength 570nm).

5.CDH-11-EC _1-2/ FN III _7-10-pBCP is to the influence of hMSCs growthform, referring to Fig. 6:

(1) conventional aseptic technique digestion, centrifugal collection hMSCs are seeded in CDH-11-EC with 300 μ l cell suspension with 1000/ first density _1-2/ FN III _7-10-pBCP interface, continuous culture 2-4hr in 37 ℃, 50%CO2, saturated humidity constant temperature cell culture incubator, fully adherent until cell.Add FBS-DMEM/F12 culture medium to 600 μ l, continue to cultivate 48hr.

(2) take out the CDH-11-EC that is loaded with hMSCs _1-2/ FN III _7-10-pBCP, the rinsing of aseptic PBS liquid once.3% glutaraldehyde is fixing more than 2 hours.

(3) gradient concentration dehydration of alcohol (50%, 70%, 80%, 90%, 95% each 15 minutes, room temperature).

(4) commutation was with gradient concentration tert-butyl alcohol continuous dehydration 15 minutes.

(5) vacuum drying 24hr.Promptly can be used for SEM behind the vacuum coater metal spraying plated film detects.

(6) sample is put into the tem sample chamber: venting → reduction podium level → fixed sample cup → evacuation 10-3 handkerchief.

(7) Image Acquisition: obtain original image according to high power after the 30kV → translation of sample character selection accelerating potential, the inclination sample stage → first low power.

(8) graphical analysis: regulate object lens electric current → adjustment focal length → acquisition, analyze.

6.hMSCs cell is at CDH-11-EC _1-2/ FN III _7-10The adhesiving effect on-pBCP surface: centrifugal short adhesion experiment, referring to Fig. 7:

(1) interface processing: the BCP interface for preparing is placed 96 porocyte culture plates, and protein powder up.

(2) cell is handled: will be cultured to 3-6 generation, growth merge hMSCs to 70%-80% according to the digestion of standard cell lines operating technology, collect, process the single cell suspension that density is 105/ml.

(3) nucleus labelling: Hoechst 33342 (Sigma, the U.S.) mother solution of 0.5mg/ml is added single cell suspension, and making its final concentration is 5 μ g/ml.Lucifuge ice bath 20min fully dyes nucleus.

(4) cell seeding: with cell behind the labelling with the volume in 100 μ l/ holes (promptly 10 ⁴Individual cells/well) adds each hole BCP surface of 96 well culture plates, the operation of omnidistance darkroom lucifuge.

(5) just centrifugal and counting: above-mentioned 96 well culture plates are inserted dull and stereotyped centrifuge draw-in groove, and 4 ℃, the centrifugal 5min of 100rpm/min quickens cell attachment.Under the emission wavelength of the excitation wavelength of 360nm and 465nm, read each hole initial fluorescent intensity 1 and record with ELIASA.

(6) upset is centrifugal: each hole of 96 well culture plates is sealed with preservative film, and dull and stereotyped centrifuge draw-in groove is inserted in upset, and 4 ℃, the centrifugal 5min of 100rpm/min.Abandon film, abandon culture medium, and go unnecessary liquid to make its drying with the sterilized filter paper suction.Under above-mentioned wavelength, read, write down each hole fluorescence intensity 2.

(7) add the adherent hMSCs of pancreatin 100 μ l/BCP digestion, blow and beat repeatedly with pipettor, until abundant eluting.Read, write down each hole fluorescence intensity 3 with method.

(8) place the adherent 2-4hr of cell culture incubator, the fluorescence inverted microscope is observed each porocyte down and is adhered to form, and takes record with the NikonTE-300 camera.

(9) statistics and analysis: read fluorescent value in this testing process altogether 3 times, preceding 2 reflections 10 ⁴The total fluorescence intensity of individual cell has reflected actual adherent cell number for the fluorescence intensity of upset after centrifugal the 3rd time.Be calculated as follows adherent cell quantity:

7.hMSCs cell is at CDH-11-EC _1-2/ FN III _7-10The osteogenic ability and the mineralising on-pBCP surface

(1) cell is handled: the induced osteogenesis differentiation.

1. hMSCs inoculation: will grow to the conventional digestion of hMSCs that 3-6 generation, fusion rate reach 70%-80% and collect, processing density is 10 ⁴The single cell suspension of/ml.Add each hole BCP surface of 24 well culture plates with the volume (i.e. 2000 cells/well) in 200 μ l/ holes, add contain 5%FBS the DMEM/F12 culture medium to 0.5ml, continuous culture 24hr in 37 ℃, 50%CO2, saturated humidity constant temperature cell culture incubator.

2. osteogenic induction: the hMSCs of adhere-wall culture 24hr is abandoned culture medium; Add each 0.5ml of osteogenic induction culture medium (DXM0.01 μ M; β-GP 0.01M; Vit-C 0.05mg/ml is (Roche, the U.S.) product in 37 ℃, 50%CO2, saturated humidity constant temperature cell culture incubator continuous culture 10-14 days.2-3 changes the skeletonization inducing culture once therebetween.

(2) OCN gene expression analysis, referring to Fig. 8:

Method through real time fluorescent quantitative-reverse transcription PCR (Realtime RT-PCR) is identified the gene expression dose of skeletonization mark in late period OCN.

1. the extraction of the total RNA of hMSCs: get 14 days hMSCs of osteogenic induction, aseptic PBS liquid washing BCP surface 2 times.Add RNA extracting solution Trizol (Invitrogen, China) 1ml/ hole, acutely blow and beat cell repeatedly and make its cracking, inverted microscope is observed down and is cracked into fragment fully until cell.Divide to be filled in the aseptic EP pipe of no enzyme, 4 ℃, the centrifugal 5min of 12000 * g is transferred to supernatant in the new 1.5ml EP pipe.In chloroform: Trizol=1: 5 ratio adds freezing chloroform 200 μ l/ pipe, acutely puts upside down 15s up and down, and room temperature leaves standstill 10min, and 4 ℃, 12000 * g, centrifugal 15min.Draw upper strata water sample layer, move to the aseptic EP pipe of new no enzyme, add the isopropyl alcohol (500 μ l/1mlTrizol) of pre-cooling, precipitation at room temperature 20min behind the mixing, 4 ℃, the centrifugal 10min of 12000 * g.Abandon supernatant, add 1ml 75% ethanol (with DEPC treated water fresh), washing precipitation, 4 ℃, the centrifugal 5min of 7500 * g.Abandon supernatant, drying at room temperature 5-10min is when treating the RNA substantially transparent; Add 30 μ l RNase-free water; To dissolving fully, row purity detecting: get 1 μ l RNA solution and add 99 μ l DEPC water, on ultraviolet spectrophotometer, measure the OD value of A260 and A280; Obtain the ratio of A260/A280, require the ratio can be between 1.8～2.0 in order to follow-up reverse transcription.

2. OCN and GAPDH design of primers:

The retrieval people OCN that originates in NCBI gene bank Genebank, it is internal reference that GAPDH is set.According to the requirement of Realtime PCR, the primer of design expanding fragment length 100-200bp is right.Primer information sees following table for details:

3. reverse transcription reaction (mRNA → cDNA)

The universal primer Oligo dT Primer and the Random 6mers that utilize reverse transcription test kit (TaRaKa, Dalian is precious biological) to provide are cDNA with the mRNA reverse transcription of purifying, in order to follow-up quantitative analysis.

By following set of dispense system inverse transcription reaction liquid (operation on ice)

?Component

?Volume?for?20μl?of?reaction?mix

5×PrimeScript TM?Buffer	4μl
		PrimeScript TM?RT?Enzyme?Mix?I	1μl
Oligo?dT?Primer	1μl
		Random?6mers	1μl
Total?RNA	1μg
		RNase?Free?dH2O	11μl

Carry out reverse transcription reaction by following condition

42℃	30min	1cycle
			99℃	5min	1cycle
5℃	5hr	1cycle

4. Real-time PCR detects

With the above-mentioned cDNA that obtains is template, amplified target genetic fragment under the guiding of upstream and downstream primer, " relative quantification " method (Relative Quantitation) of employing ABI7500 system.

Put PCR reactant liquor (reactant liquor is formulated on ice and carries out) by following set of dispense

Condition by shown in the figure is carried out PCR reaction, referring to Fig. 9:

Interpretation: reaction finishes the back and confirms amplification curve and solubility curve, with 2 ^{-Δ Δ Ct}Analytic process is carried out relative quantitative assay.Wherein: Δ Ct=genes of interest Ct value-GAPDH Ct value ,-Δ Δ Ct=normal control group Δ Ct meansigma methods-each sample Δ Ct, 2 ^{-Δ Δ Ct}React relative expression's level of the relative normal control group of each sample sample genes of interest.

(3) the .OCN protein expression is analyzed: Western-blot

1. total protein of cell extracts: conventional aseptic technique trypsinization hMSCs, centrifugal collection (1000rpm, 5min, 4 ℃).

Abandon culture medium, the soft washing of aseptic PBS liquid 2 times.Add RIPA lysate 100 μ l/EP pipe, place cracking 20min on the ice face.Discontinuity piping and druming cell suspension makes it fully broken.Centrifugal collection supernatant is total protein of cell (12000g, 10min, 4 ℃).Determination of protein concentration: micro-BCA method.

②SDS-PAGE+Weatern-blot：

A. sample preparation: in quantitative each histone sample of back (20 μ g), add the equal-volume sample-loading buffer, fully mixing with degeneration 10min in the boiling water, places and treats appearance on the ice face.

B. electrophoresis: as above BIAO and BEN adds on the gel in the appearance hole with micropipettor respectively, energized, and the about 20min of 70V electrophoresis when treating sample electrophoresis to separation gel interface, is upgraded to 100V with voltage, and continuation electrophoresis to bromophenol blue indicator reaches the gel lower edge, stops electrophoresis.

C. change film: after pvdf membrane is soaked into methanol, with gel balance 20min in TBST.Sequentially built according to filter paper-sponge-gel-pvdf membrane-sponge-filter paper changes film " sandwich ", places in the electric turn trough (the pvdf membrane face is towards positive source) energized, 100V constant voltage transfer printing 90min in the ice bath groove.

D. sealing: taking-up, pruning and labelling pvdf membrane, put into confining liquid, room temperature sealing 6hr (or 4 ℃ spend the night).

E. an anti-hybridization: with mouse anti human OCN polyclonal antibody/goat anti GAPDH monoclonal antibody (Santacurz, the U.S.) as first antibody, and preparation working solution (dilution factor 1: 500/1: 2000).Pvdf membrane is placed in one 4 ℃ of incubated overnight.

F. two anti-hybridization: wash film under the TBST room temperature 3 times, each 10min.The anti-mouse monoclonal antibody working solution of the anti-sheep/rabbit of rabbit (middle shirt, Beijing of adding the HRP labelling.Dilution factor 1: 5000), incubated at room 2hr.

G. substrate is luminous: wash film under the TBST room temperature 3 times, each 10min.Preparation ECL chemiluminescence working solution (Sigma, the U.S.) softly drips on pvdf membrane with pipettor, behind the incubated at room 30sec with Chemidoc system scan form images (time of exposure 300sec, every 5sec run-down) and record.

3. add up and analyze: carry software Quantity One with the Chemidoc system corresponding OCN and the GAPDH protein band of each group carried out gray analysis.With the evaluation index of ODOCN/ODGAPDH as the OCN expression.

(4). bionical interface surface calcium tuberosity forms and detects: alizarin red method (GENMED, the U.S.), referring to Figure 10:

1. cell is handled: get osteogenic induction and cultivate 14 days hMSCs conventional method digestion, centrifugal collection, process the single cell suspension of 1 * 105/ml.Transfer into aseptic 96 porocyte culture plates with the density in 1 * 105/ hole.Add 10%FBS+DMEM/F12 culture medium to 200 μ l, continue conventional the cultivation 7 days.

2. the sample set is handled: culture fluid → every hole of carefully taking out in the 24 porocyte culture plates adds 100 μ l GENMED cleaning liquid, cleans the cell surface in the growth; → every hole adds 100 μ l GENMED fixatives, covers the whole growth surface, at room temperature hatches 10 minutes; → remove the GENMED fixative, every hole adds 100 μ l GENMED cleaning liquid, cleans cell surface.

3. sample dyeing is handled: every hole adds 20 μ l GENMED dyeing liquors, covers the whole growth surface; → incubated at room 2 minutes, or until visible salmon pink; → carefully take out the GENMED dyeing liquor, dry in the air.

4. sample clarifying treatment: every hole adds 100 μ l GENMED dehydration liquid, covers the whole growth surface; → remove GENMED dehydration liquid, add 100 μ l GENMED clear liquor, cover the whole growth surface; → remove the GENMED clear liquor, repeat twice of above-mentioned steps; → every hole adds the bright liquid of 100 μ l GENMED, covers the whole growth surface; → promptly being engraved under the optical microscope and observing, calcium deposition positive cell presents salmon pink.

Claims (2)

1. one kind connects albumen/calcium attachment proteins and the ceramic bionical interface of biphase calcium phosphor based on crosslinking technological coupling bio signal recombinant fiber; It is characterized in that; Said bio signal albumen is the fusion rotein of fibronectin (FN) and calcium attachment proteins-11 (CDH 11); Functional domain is FN III7-10 fragment and the outer functional domain 1-2 (CDH 11 EC1-2) of calcium attachment proteins born of the same parents that takes PHSRN and RGD motif, called after CDH-11-EC _1-2/ FN III _7-10

Timbering material is a micropore biphase calcium phosphor pottery, and called after pBCP, corsslinking molecular are DTBP, same pair of official's cross-linking agent of a kind imino-ester class, said bionical interface called after CDH-11-EC _1-2/ FN III _7-10-pBCP;

Seed cell is mesenchymal stem cells MSCs, osteoblast or chondroblast, according to the organizational project type selecting at constructed interface.

2. according to the bionical interface of claim 1, it is characterized in that the spacerarm two ends of said corsslinking molecular form CDH-11-EC through the acetylization reaction with amino _1-2/ FN III _7-10And the bridge joint (NH between pBCP ₂NH ₂), belong to covalent bond and connect, be different from coating/spraying technology based on passive absorption principle.

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