CN103893826A - Titanium alloy surface modification method for regulating and controlling stem cell differentiation and promoting in-vivo bone formation - Google Patents
Titanium alloy surface modification method for regulating and controlling stem cell differentiation and promoting in-vivo bone formation Download PDFInfo
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Abstract
本发明公开一种调控干细胞分化及促进体内骨生成的钛合金表面改性方法。首先通过多巴胺偶联将带负电荷的明胶固定在Ti6Al4V（TC4）材料表面，将其作为引发层引发层层自组装程序。其次，利用层层自组装技术在明胶修饰的TC4表面构建壳聚糖－明胶－BMP2多层膜。最后，壳聚糖－明胶－BMP2多层膜修饰的TC4材料表面沉积一层纤粘蛋白分子，从而构建出一种能调控干细胞分化和促进体内骨生成的钛合金界面细胞微环境。
The invention discloses a titanium alloy surface modification method for regulating stem cell differentiation and promoting bone formation in vivo. Firstly, negatively charged gelatin was immobilized on the surface of Ti6Al4V (TC4) material by dopamine coupling, which was used as the initiator layer to initiate the layer-by-layer self-assembly procedure. Secondly, chitosan-gelatin-BMP2 multilayer film was constructed on the surface of gelatin-modified TC4 by layer-by-layer self-assembly technology. Finally, a layer of fibronectin molecules was deposited on the surface of the chitosan-gelatin-BMP2 multilayer modified TC4 material, thereby constructing a titanium alloy interface cell microenvironment that can regulate stem cell differentiation and promote bone formation in vivo.
Description
Technical field
The invention belongs to field of medical materials, relate to the construction method at a kind of functional material interface.
Background technology
The complicated network system that cell micro-environment is made up of the multiple protein such as dissolubility signal (as somatomedin etc.) and non-dissolubility signal (hormone) and glycoprotein.Building required extracellular microenvironment at material surface is the important directions of the research of biomaterial of new generation.In recent years, research worker is all expected the biological response by build extracellular study on microenvironment regulation cell at biomaterial surface.At present, by different technologies means, various active molecule comprises that natural derived extracellular matrix (ECM) albumen, liposome, cell pad, cell adhesion molecule etc. are fixed to biomaterial surface to the outer microenvironment of analog cell.
Desirable bone implant should possess two features: the first, and material surface energy is raised osteoblast or osteoprogenitor cells and is promoted cell adhesion; The second, material surface energy provides suitable bioactive molecule (somatomedin or cytokine) promote cell proliferation and Cell differentiation inducing activity and then promote new osteogenesis.At present, bone implant field of material preparation, still has the space of larger research.
Summary of the invention
The object of the invention is to build a kind of regulation and control differentiation of stem cells and promote osteogenetic titanium alloy interface cell micro-environment in body.
Be such for realizing the technical scheme that the object of the invention adopts, a kind of regulation and control differentiation of stem cells and promote osteogenetic titanium alloy surface method of modifying in body, comprises the following steps:
1) by dopamine coupling, electronegative gelatin is fixed on to the surface of titanium alloy material TC4, obtains the TC4 modifying through gelatin;
2) TC4 surface construction chitosan, gelatin and the bone morphogenic protein BMP-2 2 modified through the gelatin overlapping multilayer film successively obtaining in step 1), obtains the TC4 modifying through multilayer film;
3) adopt the mode of physical absorption, in step 2) TC4 surface deposition one deck FN molecule of modifying through multilayer film of obtaining.
Further, the detailed process of step 1) is: first dopamine is dissolved in Tris buffer solution, is configured to the dopamine solution that concentration is 2mg/mL; Again titanium alloy material TC4 is immersed in described dopamine solution; Finally take out the titanium alloy material TC4 soaking through dopamine, used two to steam after water rinse, be immersed in gelatin solution, obtain the TC4 modifying through gelatin.
Further, step 2) detailed process be:
A) chitosan solution that configuration concentration is 1～10mg/mL, the gelatin solution that concentration is 1～10mg/mL and concentration are the BMP2 solution of 0.5～5 μ g/mL;
B) steam with two the TC4 modifying through gelatin that water rinse step 1) obtains; In chronological sequence order is carried out c～f step;
C) TC4 is placed in to chitosan solution, after immersion 5～15min, takes out, with two steaming water rinses;
D) TC4 is placed in to gelatin solution, after immersion 5～15min, takes out, with two steaming water rinses;
E) TC4 is placed in to BMP2 solution, after immersion 5～15min, takes out, with two steaming water rinses;
F) TC4 is placed in to gelatin solution, after immersion 5～15min, takes out, with two steaming water rinses;
G) repeating step c～f several times, the TC4 surface construction chitosan-gelatin-BMP2-modifying at gelatin ... multilayer film.
Further, the detailed process of step 3) is: configuration concentration is the FN solution of 0.5～5 μ g/mL, by step 2) TC4 modifying through multilayer film that obtains is immersed in FN solution and takes out after 0.5～2h, with two steaming water rinse 2 times, deposit to the TC4 surface of modifying through multilayer film by FN.
What deserves to be explained is, the object of the invention is to improve the bone conformability between titanium or titanium alloy implant and natural bone tissue, simulate n cell epimatrix in the microenvironment of implant surfaces structure expection and can promote the bone of implant to integrate.In previous research, we use layer-by-layer that chitosan and gelatin are assembled into titanium material surface.Studies confirm that chitosan/gelatin multilayer film on titanium material surface can improve the biocompatibility of titanium film.But the passivity response of this simple extracellular matrix to natural fabric, has certain limitation in induction osteogenesis application aspect.Desirable bone implant should possess two features: the first material surface energy is raised osteoblast or osteoprogenitor cells and promoted cell adhesion; The second material surface energy provides suitable bioactive molecule (somatomedin or cytokine) promote cell proliferation and Cell differentiation inducing activity and then promote new osteogenesis.
Based on above-mentioned research, we are assembled into by bone morphogenetic protein (BMP2) and FN (FN) the Ti6Al4V surface that chitosan/gelatin multilayer film is modified in Ti6Al4V surface construction class ECM sample microenvironment.On the one hand, BMP2 is a kind of somatomedin that maintains bone dynamic equilibrium, not only can promote propagation, the migration of mescenchymal stem cell and divide but also the interior osteogenesis of energy inductor.On the other hand, FN (main ECM protein) energy mediated cell is in the initial adherence of biomaterial surface.After implant implants, when cell contacts with material, we expect by adhesion and the differentiation cascade respondent behavior of mesenchymal stem cells MSCs in FN and BMP2 regulation and control pulp cavity.In addition, when bioactie agent (as BMP2) is exposed in external environment, be easy to degeneration or degraded loses activity.Studies confirm that, LbL multilayer film can maintain the biological activity of medicine/albumen in gentle environment.Multilayer film can avoid the BMP2 isoreactivity factor to be directly exposed in external environment.
Therefore, the TC4 that the designed BMP2 hydridization multilayer film of this patent is modified can control the release of BMP2 and regulate and control adhesion and the differentiation of MSCs, thereby promotes the bone of TC4 implant to integrate.
The present invention also discloses a kind of regulation and control differentiation of stem cells obtaining by said method and promotes osteogenetic titanium alloy material in body.
Beneficial effect of the present invention is: the method has simple to operate, with low cost, highly versatile, does not need special installation.The adhesion that can regulate and control mescenchymal stem cell and the differentiation that utilize the method to prepare, and promote the bone of implant to integrate, in osteopathia treatment, bone implantation technique, there is important clinical value.
Accompanying drawing explanation
Fig. 1 contact angle detection, even level is gelatin; 3,7,11,19,27,31 layers is chitosan; 5,9,13,17,21,29 is BMP2; 32 layers is FN; Except the TC4 base material (n=3) of the 1st layer;
The laser confocal microscope picture (artwork master, former figure submits in examination as to substances reference material) of Fig. 2 TC4/LbL/BMP2 surface deposition FN;
The pure TC4 scanning electron microscope (SEM) photograph of Fig. 3;
Fig. 4 TC4/LbL/BMP2/FN scanning electron microscope (SEM) photograph;
Fig. 5 BMP2 and gelatin be the cumulative release curve from TC4/LbL/BMP2/FN and TC4/LbL respectively.
The expression of Fig. 6 mesenchymal stem cells MSCs Bone Gla protein after different substrate surface are cultivated 21 days, n=4, * * p<0.01.
Fig. 7 TC4 and TC4/LbL/BMP2/FN implant 4 weeks and 12 weeks rear implant surrounding bone density, n=3.
The specific embodiment
Below in conjunction with drawings and Examples, the invention will be further described, only limits to following embodiment but should not be construed the above-mentioned subject area of the present invention.Without departing from the idea case in the present invention described above, according to ordinary skill knowledge and customary means, make various replacements and change, all should be included in protection scope of the present invention.
In the present embodiment, first by dopamine coupling, electronegative gelatin is fixed on to Ti6Al4V(TC4) material surface, set it as trigger layer and cause self assembly program layer by layer.Afterwards, the TC4 surface construction chitosan-gelatin-BMP2 that utilizes layer-by-layer to modify at gelatin ... multilayer film.Finally, TC4 material surface deposition one deck FN molecule of modifying at multilayer film, can regulate and control differentiation of stem cells and promote osteogenetic titanium alloy interface cell micro-environment in body thereby construct one.
In preparation process, factors can affect the structure of titanium alloy interface cell micro-environment, deposition concentration and the sedimentation time etc. of for example fixing working concentration, polycation chitosan and the polyanion gelatin of concentration, the active factors of the dopamine solution of trigger layer gelatin, different preparation methoies can obtain having the titanium material interface cell micro-environment of different-shape feature.The process that the present embodiment is modified multilayer film is investigated.Result shows, in the time that the concentration of dopamine is 2mg/mL, can preferably gelatin be fixed on to titanium alloy TC 4 surface.When the concentration of chitosan and gelatin is 5mg/mL, when BMP2 concentration is 2 μ g/mL, in titanium alloy surface deposition 10min, the titanium alloy that can be prepared into multilayer film modification is TC4/LbL/rhBMP2.In the titanium alloy surface deposition 1h modifying at multilayer film in the time that FN concentration is 2 μ g/mL, can be regulated differentiation of stem cells and promote osteogenetic titanium alloy interface cell micro-environment in body.
Embodiment 1: the preparation of osteogenetic titanium alloy interface cell micro-environment in regulation and control differentiation of stem cells and promotion body
A. gelatin is fixing: appropriate dopamine is dissolved in Tris buffer solution (10mM, pH8.5), is configured to the dopamine solution that concentration is 2mg/mL.By Ti6Al4V(TC4) to be immersed in dopamine solution, lucifuge is spent the night, i.e. the static 12h that deposits of lucifuge.With two steaming water rinse TC4 samples.Subsequently, the TC4 sample that dopamine was processed is immersed in gelatin solution (5mg/mL, pH7.4), hold over night, the i.e. static 12h that deposits of lucifuge.
The preparation of the titanium alloy TC 4 that b.BMP2 multilayer film is modified: first, configuration solution.Chitosan solution (5mg/mL) is configured by 0.3% acetic acid.Gelatin (5mg/mL) is configured by PBS (pH7.4).RhBMP2 (2 μ g/mL) solution is by 100mM sodium acetate (pH5.1) solution allocation.With the TC4 sample of two steaming water rinse gelatin modifieds, rinse 3 times.The TC4 sample that gelatin is modified is soaked in chitosan (5mg/mL), gelatin (5mg/mL), rhBMP2 (2 μ g/mL) and gelatin (2mg/mL) successively ... in solution, in every kind of solution, soak 10min, change before soaking solution, with distilled water rinse TC4 twice, each 1min.After above-mentioned steps is carried out, the TC4 surface construction chitosan-gelatin-BMP2 modifying at gelatin ... multilayer film, i.e. desired (Gel/Chi/Gel/rhBMP2)
nmultilayer film is assembled into TC4 surface.In an embodiment, n=3, the TC4 sample of modifying by gelatin is soaked in chitosan (5mg/mL), gelatin (5mg/mL), rhBMP2 (2 μ g/mL), gelatin (2mg/mL), chitosan (5mg/mL), gelatin (5mg/mL), rhBMP2 (2 μ g/mL), gelatin (2mg/mL), chitosan (5mg/mL), gelatin (5mg/mL), rhBMP2 (2 μ g/mL) and gelatin (2mg/mL) solution successively, obtains (Gel/Chi/Gel/rhBMP2)
3the TC4 that multilayer film is modified.
The deposition of c.FN: FN is deposited to (Gel/Chi/Gel/rhBMP2)
nthe TC4 surface that multilayer film is modified.First,, with PBS (pH7.4) configuration FN (Fibronectin, FN) solution, concentration is 2 μ g/mL.By (Gel/Chi/Gel/rhBMP2)
nthe TC4 sample that multilayer film is modified is immersed in FN solution 2 μ g/mL, 37 ℃ of standing 1h.With two steaming water rinse 2 times, can complete the structure that can regulate and control differentiation of stem cells and promote osteogenetic titanium alloy interface cell micro-environment in body, sample is designated as TC4/LbL/rhBMP2/FN.
With the structure of contact angle detection monitoring titanium alloy TC 4 surface multilayer film.As shown in Figure 1, from low 6 layers, contact angle is about 45 degree, 42 degree, 41 degree and 36 degree, the contact angle size when corresponding respectively to gelatin, chitosan, gelatin and BMP2 and being outermost layer.After follow-up multilayer film assembling, contact angle has also produced similar alternate rule.Result shows that gelatin, chitosan and BMP2 multilayer film have deposited to TC4 surface.After above-mentioned c step process, FN deposits to TC4/LbL/BMP2 surface in the mode of physical absorption.Utilizing monoclonal antibody immunity fluorescent method to detect FN distributes.Laser co-focusing is observed and is shown that green fluorescence is distributed in TC4/LbL/BMP2/FN surface (Fig. 2) equably.Show that FN has successfully deposited to TC4 surface.Scanning electron microscope (SEM) photograph without the titanium material surface after titanium material surface and each step process of example a-d of any processing is respectively Fig. 3 and Fig. 4; As can be seen from Figure, pure TC4 surface ratio more coarse (Fig. 3), this is caused by polishing.After multilayer film covers, TC4/LbL/BMP2/FN surface is relatively level and smooth and occur tiny granular texture (Fig. 4).Result shows that multi-layer film structure forms on TC4 surface.
Experimental example 1: the release of the titanium alloy interface cell micro-environment regulation activity factor
The release dynamics of this study tour BMP2 active factors from the cell micro-environment of titanium alloy interface.
Cumulative release behavior with the detection BMP2 of ELISA test kit discontinuity from multilayer film.First, by sample prepared embodiment 1 (TC4/LbL/BMP2/FN) and only gelatin/chitosan multilayer film modify TC4(TC4/LbL) be placed in respectively 0.5mL PBS solution, place 37 ℃.In predetermined point of time, after taking-up 50 μ L culture fluid, add the fresh PBS solution of equivalent volumes.Detect the BMP2 amount of TC4/LbL/BMP2/FN release with BMP2 quantitative ELISA test kit.Detect the gelatin amount discharging in TC4/LbL by BCA detection kit.
As shown in Figure 5, BMP2 and gelatin have similar release profiles.All there is slight violent release phenomenon in BMP2 and gelatin in 24h.In initial 2h, about 30%BMP2 discharges, and reaches 50% after 24h.This is directly released in PBS solution relevant to the BMP2 around being exposed to multilayer film.After 14 days, in the multi-layer film structure on TC4 surface, still there is approximately 16% BMP2.Result shows the degraded along with titanium alloy TC 4 surface multilayer film, sustained release in BMP2 is long-time.
Experimental example 2: the differentiation of titanium alloy interface cell micro-environment regulation and control stem cell
Choose prepared by embodiment 1 and the sample that obtains is investigated the osteocalcin level that material surface mesenchymal stem cells MSCs produces.First by the 4th generation former culture mesenchymal stem cells MSCs be seeded in different titanium alloys and TCPS surface.Cultivate after 21 days, collect culture medium.With the content of Bone Gla protein in Bone Gla protein detection kit culture fluid, be expressed as ng/mL.Bone Gla protein, by expressing in the well differentiated osteoblast of mineralization stage, is the mark in late period of osteoblast differentiation.As shown in Figure 6, compared with TC4 and TC4/LbL, the expressed Bone Gla protein of the MSCs on TC4/LbL/BMP2/FN surface obviously and have a significant difference ((p<0.01)).Result shows that the titanium alloy interface with cell micro-environment is conducive to the mineralising that inducing bone mesenchymal stem cell to osteoblast breaks up and promote cell.
Experimental example 3: osteogenesis in the cell micro-environment inductor of titanium alloy interface
Choose embodiment 1 and prepare and obtain TC4/LbL/rhBMP2/FN pole sample, and itself and implant the femur epiphysis of ripe new zealand rabbit (average weight is 2.5-3Kg) without any processing TC4 pole.With intravenous injection sodium phenobarbital anesthesia rabbit.Rabbit operative site is shaved after hair sterilization, cut skin (about 3cm) and expose femur epiphysis.Be that 3cm length is that the operation electricity of 13mm turns at rabbit femur epiphysis and turns hole with diameter.Different samples is inserted and turned in hole.Last skin suture, and operative site is sterilized.Implant after 4 weeks and 12 weeks, rabbit is put to death with intravenous injection air.Fixedly contain the femur sample 48h of implant with 10% formaldehyde.Scan with Mmiro-CT, and analyze with software mimic10.1, bone density size is expressed as Hounsfield units.
As Fig. 7 has shown the rear implant of the Micro-CT scanning density of new bone around.Micro-CT can observe internal structure and the area of new bone of bone effectively.As shown in Figure 4, implant surrounding bone density increases along with the increase of time.The more important thing is, TC4/LbL/BMP2/FN surrounding bone density is significantly higher than matched group TC4 at 4 weeks and 12 Zhou Shijun.Research shows that the titanium alloy interface (TC4/LbL/BMP2/FN) with cell micro-environment is having more advantage than undressed titanium alloy aspect the new osteogenesis of induction.
Generally speaking, bioactie agent is as shorter in BMP2 is exposed in external environment life cycle.And in this patent, the effect of Chi/Gel multilayer film is the release of regulation and control BMP2 the activity of phase maintenance BMP2.In body, result confirms that the BMP2 discharging has good biological activity, can induce host bone cell to osteoblast differentiation, causes the generation of implant/bone interface area of new bone.This result is consistent with previous report.To sum up, the prepared titanium alloy interface with extracellular microenvironment of this patent can regulate cell function to promote the bone of implant and in-vivo tissue to integrate.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN104353109A (en) * | 2014-09-30 | 2015-02-18 | 广西中医药大学 | Method for modifying titanium with antibacterial property and effect of promoting growth of osteoblasts |
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| CN111939317A (en) * | 2020-07-14 | 2020-11-17 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A method for constructing bone morphogenetic protein sustained-release system |
| CN112546300A (en) * | 2020-11-24 | 2021-03-26 | 温州医科大学附属口腔医院 | Raloxifene modified MOF coating mediated local anti-osteoporosis metal substrate implantation material and preparation method thereof |
| CN116407677A (en) * | 2023-05-30 | 2023-07-11 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | A kind of magnesium alloy layered coating with wear-resistant self-healing function and preparation method thereof |
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| CN104353109A (en) * | 2014-09-30 | 2015-02-18 | 广西中医药大学 | Method for modifying titanium with antibacterial property and effect of promoting growth of osteoblasts |
| CN104353114A (en) * | 2014-09-30 | 2015-02-18 | 广西中医药大学 | Medical titanium with osteoblast growth promotion function and surface modification method of medical titanium |
| CN104258468B (en) * | 2014-09-30 | 2015-12-30 | 广西中医药大学 | A kind of titanium method of modifying possessing antibiotic property and promote Oesteoblast growth |
| CN104258468A (en) * | 2014-09-30 | 2015-01-07 | 广西中医药大学 | Modification method of titanium metal having antibacterial property and capable of promoting growth of osteoblast |
| CN105214140B (en) * | 2015-09-22 | 2018-02-09 | 重庆大学 | The functionalization interface construction method of local bone remoulding and the titanium alloy of healing in coordinated regulation osteoporosis |
| CN105214140A (en) * | 2015-09-22 | 2016-01-06 | 重庆大学 | The functionalization interface construction method of the titanium alloy of local bone reconstruction and healing in coordinated regulation osteoporosis |
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| CN106729964A (en) * | 2016-12-02 | 2017-05-31 | 江苏师范大学 | It is a kind of to have promoting bone growing concurrently and suppress titania nanotube of bone resorption and preparation method thereof |
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| CN107402293B (en) * | 2017-07-20 | 2023-09-08 | 上海交通大学医学院附属第九人民医院 | Experimental device for promote osteogenic differentiation based on titanium alloy elastic deformation |
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| CN111529754A (en) * | 2020-05-08 | 2020-08-14 | 重庆大学 | Titanium-based active bone implant with composite coating and preparation method thereof |
| CN111939317A (en) * | 2020-07-14 | 2020-11-17 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A method for constructing bone morphogenetic protein sustained-release system |
| CN111939317B (en) * | 2020-07-14 | 2021-12-17 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A method for constructing bone morphogenetic protein sustained-release system |
| WO2022012339A1 (en) * | 2020-07-14 | 2022-01-20 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Method for constructing bone morphogenetic protein sustained-release system |
| US12337077B2 (en) | 2020-07-14 | 2025-06-24 | The 2nd Affiliated Hospital AND Yuying Children's Hospital of Wenzhou Medical University | Method for constructing bone morphogenetic protein slow-release system |
| CN112546300A (en) * | 2020-11-24 | 2021-03-26 | 温州医科大学附属口腔医院 | Raloxifene modified MOF coating mediated local anti-osteoporosis metal substrate implantation material and preparation method thereof |
| CN112546300B (en) * | 2020-11-24 | 2024-03-15 | 温州医科大学附属口腔医院 | Raloxifene modified MOF coating-mediated local osteoporosis-resistant metal substrate implantation material and preparation method thereof |
| CN116407677B (en) * | 2023-05-30 | 2023-11-03 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | A magnesium alloy layered coating with wear-resistant self-healing function and preparation method thereof |
| CN116407677A (en) * | 2023-05-30 | 2023-07-11 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | A kind of magnesium alloy layered coating with wear-resistant self-healing function and preparation method thereof |
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