CN101745150B - Gene activation titanium and preparation method thereof - Google Patents
Gene activation titanium and preparation method thereof Download PDFInfo
- Publication number
- CN101745150B CN101745150B CN 200910250890 CN200910250890A CN101745150B CN 101745150 B CN101745150 B CN 101745150B CN 200910250890 CN200910250890 CN 200910250890 CN 200910250890 A CN200910250890 A CN 200910250890A CN 101745150 B CN101745150 B CN 101745150B
- Authority
- CN
- China
- Prior art keywords
- titanium
- layer
- titanium material
- chitosan
- plasmid
- 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.)
- Expired - Fee Related
Links
Images
Abstract
The utility model discloses a gene activation titanium which a titanium body, a pEGFP-hBMP2 plasmid layer and chitosan layer which are sequentially coated on the titanium from interior to exterior with interval to form an activation layer; the gene activation titanium has good biocompatibility and osteoinduction property, can improve the transfection efficiency of host bone cells and effectively regulate and control the gene delivery, and improve the cell vitality, and induce the cell osteogenesis and accordingly enables the titanium body to be well combined with the bone tissues initiatively; the invention also provides the preparation method of the gene activation titanium, which includes that the chitosan layer and the plasmid pEGFP-hBMP2 layer are sequentially precipitated on the surface of the titanium through the static effect between the chitosan molecular and the plasmid DNA; the preparation method not only has simple operation and low cost, but also has simple implementation and ensures the prepared gene activation titanium to effectively induce the conversion from osteogenic cells to osteocyte, and also to be well combined with bone tissues firmly for long time; and the invention is applicable to industrial development.
Description
Technical field
The present invention relates to a kind of titanium material and preparation method thereof.
Background technology
The titanium material is the medical metal material of commonly using, because its good biocompatibility and mechanical performance just are used to medical field as far back as mid-term in 20th century, and begin to obtain extensive use the seventies as embedded material.Along with biotechnology and biomedical development, the titanium material has obtained important breakthrough in the application of the aspects such as human body implant, substituted device and human organ, mainly be as (Mater Sci Forum, 2003 such as dental implant, artificial joint and tooth implant frames; 416:669-674.) (J Mater Chem 2004; 14:2282-2290).
The titanium material belongs to the biologically inert biomaterial, and on the one hand, the biologically inert of material surface is given titanium material good biocompatibility.But on the other hand, this inertia is also so that the application of titanium material has been subject to challenge, because the titanium material of inertia surface is a kind of mechanical locking mode with the combination of bone, tissue reaction shows as non-specific and randomness (Biomed Pharmacother after implanting, 2006,60 (8): 365-369), so that titanium material implant can not form firmly with osseous tissue, the combination of long-term stability, easily cause the problems such as inflammation, displacement, cause graft failure.Therefore, how making titanium material surface have biological activity, improve the binding ability of biocompatibility and the bone of titanium material, is the matter of utmost importance in the titanium material research design.
At present, for improving titanium material surface bioactive, what mainly use is the methods such as face coat, biochemical modification.Studying more coating technology is to carry out hydroxyapatite coating layer on titanium material surface.Form the bioactive hydroxyapatite of one deck on titanium material surface and can improve the surface activity of titanium material implantation body.As far back as 1991, the hydroxyapatite coating layer on the confirmation titanium material surfaces such as L.L.Hench can be strengthened the fixation between titanium implants and the host bone, impelled osteocyte to implant growth (J Am Ceram Soc 1991; 74:1485-1510.).And biochemical modification is another effective way that obtains the activated titanium material of tool surface.The confirmations such as Le Guillou-Buffello D more are conducive to osteoblastic adhesion through the titanium material surface that RGD modifies, sprawl, break up the biological behaviour (Tissue Eng Part A.2008,14 (8): 1445-55) such as survived.
These methods have been improved biocompatibility and the synosteosis ability of titanium material to a certain extent, but these methods still can not make the titanium material have good osteoinductive energy, the titanium material is difficult to being combined with body of active and regulates and control the biological behaviors such as osteocyte adhesion, increment, migration and differentiation, so that the titanium material can't have with osseous tissue good synestotic ability occurs.
Chitosan is a kind of natural cationic polysaccharide (pKa=6.3) that chitin obtains through deacetylation, has degradability, good biocompatibility and certain excellent properties such as antibiotic, is widely used in the fields such as organizational project, drug release.The pEGFP-hBMP2 plasmid is a kind of recombiant plasmid, can be in eukaryotic cell expressing green fluorescent protein and bone morphogenetic protein, green fluorescent protein is used for monitoring transfection efficiency, bone morphogenetic protein belongs to bone inductive factor, can induce bone source sexual cell to bone cell growth, promote osteogenesis.The multilayer film that chitosan and plasmid pEGFP-hBMP2 form can be degraded under physiological condition, so that plasmid DNA or chitosan-plasmid DNA granule continues to produce in long-time, keeps the concentration of plasmid DNA.After the cell (such as bone marrow stroma stem cell) that is grown in the titanium material surface of this gene activation absorbs composite particles, within the long time, make plasmid DNA continuous expression and complex functionality albumen---bone morphogenetic protein, original position induces bone marrow stroma stem cell to osteoblastic generation, gives the titanium material and initiatively with osseous tissue good synosteosis ability occurs.
Summary of the invention
In view of this, one of purpose of the present invention provides a kind of gene activation titanium, have good biocompatibility and osteoinductive energy, can improve the transfection efficiency of host bone cell, controlling gene discharges and the enhancing cell viability effectively, induce the generation of osteocyte, thereby make the titanium material body can be initiatively and osseous tissue generation good combination.
Gene activation titanium of the present invention comprises titanium material body, titanium material body surface from inside to outside successively the interval be covered with pEGFP-hBMP2 plasmid layer and chitosan layer and form active layer.
Further, described titanium material body is made by medical titanium material, and total number of plies of pEGFP-hBMP2 plasmid layer and chitosan layer is the 9-21 layer in the described active layer.
Two of purpose of the present invention provides the method for the described gene activation titanium of preparation, comprises the steps: that (1) is dipped in described titanium material body in the polyethyleneimine: amine aqueous solution after 10-20 minute, soaks 1-3 time 1-2 minute at every turn with deionized water;
(2) step (1) gained titanium material body is immersed in the phosphate buffer contain plasmid pEGFP-hBMP2 after 10-20 minute, soak 1-3 time 1-2 minute at every turn with deionized water;
(3) step (2) gained titanium material body is dipped in the acetic acid solution of chitosan after 10-20 minute, soaks 1-3 time 1-2 minute at every turn with deionized water;
(4) successively repeating step (2) and step (3) are until obtain required gene activation titanium.
Further, contained component and the concentration of respective components in solution are respectively in the described polyethyleneimine: amine aqueous solution: sodium chloride 100-150mM and polymine 2-5mg/ml;
Further, the described phosphate buffer pH value that contains plasmid pEGFP-hBMP2 is 7.4, and plasmid concentration is 0.1-1mg/ml;
Further, in the acetic acid solution of described chitosan, quality of acetic acid percentage ratio is 1-3%, and chitosan concentration is 3-5mg/ml.
The invention has the beneficial effects as follows:
Gene activation titanium of the present invention has good biocompatibility and osteoinductive energy, can improve transfection efficiency and the release of Effective Regulation gene of host bone cell and strengthen cell viability, induce the generation of osteocyte, thereby make the titanium material body can be initiatively and osseous tissue generation good combination, be fit to very much clinical practice.
The method for preparing gene activation titanium of the present invention utilizes the electrostatic interaction between chitosan molecule and the plasmid DNA that chitosan layer and plasmid pEGFP-hBMP2 layer are deposited successively on titanium material surface, not only simple to operate, with low cost, and implement easily, the gene activation titanium that makes can effectively induce the bone source cell to the conversion of osteocyte and with osseous tissue firmly, steady in a long-term combination, the inventive method is suitable for commercial application very much.
Other advantages of the present invention, target and feature will be set forth to a certain extent in the following description, and to a certain extent, based on being apparent to those skilled in the art to investigating hereinafter, perhaps can be instructed from the practice of the present invention.
Description of drawings
Accompanying drawing is that embodiment gained gene activation titanium carries out the spatial induction rat bone marrow mesenchymal stem cells in the osteocyte conversion process, the testing result of alkali phosphatase, among the figure, the 1-orifice plate, the orifice plate of 2-surface-coated liposome 2000, the titanium material of 3-surface-coated liposome 2000,4-embodiment gained gene activation titanium, the titanium material that 5-chitosan/DNA Layer by layer assembly is modified; White box is cell produced alkali phosphatase in the time of the 7th day total amount, and cell produced the total amount of phosphatase when black box was fortnight.
The specific embodiment
Below with reference to specific embodiment the present invention is described in detail.Should be appreciated that selected embodiment only for the present invention is described, rather than in order to limit protection scope of the present invention.
Embodiment
1. titanium material body is dipped in the polyethyleneimine: amine aqueous solution 10 minutes, then soaked 1 minute with deionized water, again gained titanium material was immersed in the phosphate buffer of the pH7.4 that contains plasmid pEGFP-hBMP2 20 minutes, then soaked 1 minute with deionized water; At last the titanium material is dipped in the acetic acid solution of chitosan, after 10 minutes, soaked 1 minute with deionized water;
2. repeating step operation 1. gets required gene activation titanium until to form pEGFP-hBMP2 plasmid layer and the total number of plies of chitosan layer at titanium material outer surface be 15 layers active layer.Described polyethyleneimine: amine aqueous solution is take the 100-150mM sodium chloride solution as solvent, and polymine concentration is 2-5mg/ml in the solution
Contained component and the concentration of respective components in solution are respectively sodium chloride 150mM and polymine 5mg/ml in the described polyethyleneimine: amine aqueous solution of the present embodiment, the pEGFP-hBMP2 plasmid concentration is 1mg/ml in the described phosphate buffer, in the described chitosan acetic acid solution, quality of acetic acid percentage ratio is 1%, and chitosan concentration is 5mg/ml.
In the present embodiment, in the polyethyleneimine: amine aqueous solution, sodium chloride concentration is that 100-150mM, polymine concentration are that 2-5mg/ml all can, the pH value that contains the phosphate buffer of pEGFP-hBMP2 plasmid is 7.4, plasmid concentration is that 0.1-1mg/ml all can, in the chitosan acetic acid solution, quality of acetic acid percentage ratio is that 1-3%, chitosan concentration are that 3-5mg/ml all can.
In the present embodiment, in the active layer pEGFP-hBMP2 plasmid layer and the total number of plies of chitosan layer be not less than five layers all can, reason is total number of plies greater than 5 layers active layer, its structure is just comparatively perfect; The multilayer film that chitosan and plasmid pEGFP-hBMP2 form can be degraded under physiological condition, so that plasmid DNA (both plasmid pEGFP-hBMP2) or chitosan-plasmid DNA granule continue to produce within a certain period of time, and keep finite concentration, titanium material surface is after chitosan and the modification of plasmid DNA layered self-packaging, the cell (such as bone marrow stroma stem cell) that is grown in this gene activation titanium surface can absorb plasmid DNA or composite particles and make in a long time plasmid DNA continuous expression and complex functionality albumen---bone morphogenetic protein, original position induces bone marrow stroma stem cell to osteoblastic generation, give the titanium material and initiatively with osseous tissue good synosteosis occurs, user can limit total number of plies of titanium material body surface active layer as required, to realize the sustained release of plasmid DNA in the special time, the total number of plies of active layer is best with the 9-21 layer, reason is that the plasmid DNA number of plies more than 9 layers is to guarantee the total content of plasmid DNA, reach the purpose of long-time sustained release, but too much the number of plies may cause plasmid DNA excessive or restive in special time period release.
In the described gene activation titanium preparation method, titanium material body is at the polyethyleneimine: amine aqueous solution, soak time in the acetic acid solution of phosphate buffer and chitosan can be respectively 10-20 minute, but do not represent in the inventive method, the time in each solution soaking stage is confined to 10-20 minute, the purpose of soaking is to make plasmid layer and chitosan layer to be attached to the outer surface of titanium material body in the interval successively, because the same sex electrical charge rejection with layer medium (polyanion or polycation), tend in theory form single layer structure, soak time is long, polyanion or polycation layer are formed more abundant, more complete to titanium material surface coverage, the cover layer that forms is more smooth, and soak time can get final product by complete covering titanium material surface formation single layer structure with plasmid DNA layer or chitosan layer.
In the present embodiment, after the immersion end of each solution to the titanium material, all need adopt deionized water to carry out washing by soaking, the inferior number average that each deionized water soaks in stage be advisable for 1-3 time, the deionized water soak time all is advisable with each 1-3min, reason is: the main purpose of immersion is to remove to form bilayer or multilamellar polyanion or polycation in same layer, in order to avoid they cause back laying structural instability, but long soaking time then may cause established multiple structure to dissociate.
Result verification:
The present embodiment gained gene activation titanium is carried out the evaluation that spatial induction rat bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) transforms to osteocyte
Operate as follows: adherent method is cultivated Rat Mesenchymal Stem Cells (MSCs) when adopting density－gradient centrifuga－tion method and difference, MSCs is inoculated into respectively the orifice plate of orifice plate, surface-coated liposome 2000, the titanium material of surface-coated liposome 2000, the gene activation titanium of chitosan/pGB modification and the titanium material surface that chitosan/DNA layered self-packaging is modified, and the cell inoculum density is 40000 cells/cm2; The new-born calf serum that adds 10% (w/v) with the DMEM culture medium is cultivated, and changes liquid once in per 3 days; Cell is after the gene activation titanium surface seeding is cultivated respectively 7 and 14 days, and with 0.25% trypsinization, collecting cell is used the 1%Triton-X100 cell lysis, detects the expression of cell pyrolysis liquid alkaline phosphatase.
Result such as accompanying drawing, from figure as can be known, cell is on the surface of gene activation titanium, and the 7th day and the amount that produced alkali phosphatase in 14 days all are higher than other matched group, the differentiation of filling stem cell to osteoblast between the titanium material that this gene activation is described can more be conducive to promote.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (2)
1. gene activation titanium is characterized in that: comprise titanium material body, titanium material body surface from inside to outside successively the interval be covered with pEGFP-hBMP2 plasmid layer and the chitosan layer forms active layer; Described titanium material body is made by medical titanium material, and total number of plies of pEGFP-hBMP2 plasmid layer and chitosan layer is the 9-21 layer in the described active layer.
2. prepare the method for gene activation titanium claimed in claim 1, it is characterized in that: comprise the steps:
(1) described titanium material body is dipped in the polyethyleneimine: amine aqueous solution after 10-20 minute, soaks 1-3 time with deionized water, 1-2 minute at every turn; Contained component and the concentration of respective components in solution are respectively in the described polyethyleneimine: amine aqueous solution: sodium chloride 100-150mM and polymine 2-5mg/ml;
(2) step (1) gained titanium material body is immersed in the phosphate buffer contain plasmid pEGFP-hBMP2 after 10-20 minute, soak 1-3 time 1-2 minute at every turn with deionized water; The described phosphate buffer pH value that contains plasmid pEGFP-hBMP2 is 7.4, and plasmid concentration is 0.1-1mg/ml;
(3) step (2) gained titanium material body is dipped in the acetic acid solution of chitosan after 10-20 minute, soaks 1-3 time 1-2 minute at every turn with deionized water; In the acetic acid solution of described chitosan, quality of acetic acid percentage ratio is 1-3%, and chitosan concentration is 3-5mg/ml;
(4) successively repeating step (2) and step (3) are until obtain required gene activation titanium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910250890 CN101745150B (en) | 2009-12-31 | 2009-12-31 | Gene activation titanium and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910250890 CN101745150B (en) | 2009-12-31 | 2009-12-31 | Gene activation titanium and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101745150A CN101745150A (en) | 2010-06-23 |
| CN101745150B true CN101745150B (en) | 2013-02-27 |
Family
ID=42473161
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910250890 Expired - Fee Related CN101745150B (en) | 2009-12-31 | 2009-12-31 | Gene activation titanium and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101745150B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112107725A (en) * | 2020-09-05 | 2020-12-22 | 湖南师范大学 | Bone induction material and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6994883B2 (en) * | 2001-01-30 | 2006-02-07 | Isotis S.A. | Method for applying a bioactive coating on a medical device |
| CN101411894A (en) * | 2008-11-20 | 2009-04-22 | 重庆大学 | Self-assembly modified titanium or titanium alloy material from layer to layer as well as preparation method and use thereof |
| CN101411893A (en) * | 2008-11-20 | 2009-04-22 | 重庆大学 | Titanium or titanium alloy material with modified surface as well as preparation method and use thereof |
-
2009
- 2009-12-31 CN CN 200910250890 patent/CN101745150B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6994883B2 (en) * | 2001-01-30 | 2006-02-07 | Isotis S.A. | Method for applying a bioactive coating on a medical device |
| CN101411894A (en) * | 2008-11-20 | 2009-04-22 | 重庆大学 | Self-assembly modified titanium or titanium alloy material from layer to layer as well as preparation method and use thereof |
| CN101411893A (en) * | 2008-11-20 | 2009-04-22 | 重庆大学 | Titanium or titanium alloy material with modified surface as well as preparation method and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101745150A (en) | 2010-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Miranda et al. | Three-dimensional culture of rat BMMSCs in a porous chitosan-gelatin scaffold: A promising association for bone tissue engineering in oral reconstruction | |
| Feng et al. | Application of 3D printing technology in bone tissue engineering: a review | |
| Kashte et al. | Artificial bone via bone tissue engineering: current scenario and challenges | |
| Balla et al. | Tantalum—A bioactive metal for implants | |
| Polo-Corrales et al. | Scaffold design for bone regeneration | |
| EP1385449B1 (en) | Biologically-functionalised, metabolically-inductive implant surfaces | |
| Martins et al. | Responsive and in situ-forming chitosan scaffolds for bone tissue engineering applications: an overview of the last decade | |
| Liu et al. | Advances of adipose-derived mesenchymal stem cells-based biomaterial scaffolds for oral and maxillofacial tissue engineering | |
| Green et al. | Evolving marine biomimetics for regenerative dentistry | |
| Zhao et al. | Enhanced osteogenic activity and antibacterial ability of manganese–titanium dioxide microporous coating on titanium surfaces | |
| CN105903084B (en) | A kind of 3D printing porous support and preparation method thereof with antibacterial functions coating | |
| CN102090982B (en) | Artificial tooth root or joint material and microarc oxidation preparation method thereof | |
| Zhao et al. | Osteogenic activity and antibacterial ability on titanium surfaces modified with magnesium-doped titanium dioxide coating | |
| Triplett et al. | New frontiers in biomaterials | |
| CN107213529B (en) | Preparation method of degradable medical high-molecular three-dimensional material for improving adhesion and osteogenic performance of osteoblasts | |
| De Olyveira et al. | First otoliths/collagen/bacterial cellulose nanocomposites as a potential scaffold for bone tissue regeneration | |
| CN105688274A (en) | Preparation technology of PCL/GE (polycaprolactone/gelatin) electrospinning composite stent | |
| WO2008029612A1 (en) | Bioimplant | |
| CN101411894B (en) | Self-assembly modified titanium or titanium alloy material from layer to layer as well as preparation method and use thereof | |
| CN104645414A (en) | Titanium-based surface antibacterial and bone tissue regeneration induced functional coating as well as preparation method and application thereof | |
| CN102743789A (en) | Artificial tooth root with micro-nano hierarchical topologic surface structure and preparation method of artificial tooth root | |
| CN103598919B (en) | A kind of preparation method of dental implant surface biological gradient coating | |
| CN107761148B (en) | A method of fibroin albumen hydroxyapatite coating layer is prepared in metal surface | |
| Liu et al. | Vascularized bone tissue formation induced by fiber-reinforced scaffolds cultured with osteoblasts and endothelial cells | |
| Nikolova et al. | Advances in multifunctional bioactive coatings for metallic bone implants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130227 Termination date: 20141231 |
|
| EXPY | Termination of patent right or utility model |