CN111494219A - Method for biomimetic mineralization of collagen and collagen prepared by using method - Google Patents
Method for biomimetic mineralization of collagen and collagen prepared by using method Download PDFInfo
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- CN111494219A CN111494219A CN201910087347.7A CN201910087347A CN111494219A CN 111494219 A CN111494219 A CN 111494219A CN 201910087347 A CN201910087347 A CN 201910087347A CN 111494219 A CN111494219 A CN 111494219A
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Abstract
According to the method for biomimetically mineralizing collagen and the collagen prepared by the method, the cation polyelectrolyte is grafted on the collagen, a liquid mineralized precursor permeates into collagen fibers through the capillary action and/or the electrostatic action and/or the double balance of charge and osmotic pressure, and then phase transformation is carried out to form a biomineral crystal. The method for biomimetically mineralizing collagen can also be widely applied to other fields of biomimetic mineralization.
Description
Technical Field
The invention relates to a biomimetic mineralization method and collagen prepared by the method, in particular to a biomimetic mineralization method of collagen and collagen prepared by the method.
Background
Under normal physiological conditions, the balance between demineralization and remineralization of dentin and other dental hard tissues influences the health of dental tissues, oral cavity functions and even the whole body. The unbalance between the two will affect the biomechanical performance of the dental tissue, cause the dental tissue to be defective, or even endanger the function of the oral cavity of the patient. With the development of dental materials and related technologies, the direct bonding repair technology of composite resin materials has become a main repair method for defects of hard tissues of teeth and is widely applied. The existing widely used dentin bonding systems have the problem that the resin penetration depth is inconsistent with the collagen demineralization depth, and the exposed collagen in the bonding interface mixing layer is lack of the protection of hydroxyapatite and is easy to be subjected to enzymolysis and hydrolysis, so that the service life of the composite resin restoration body is influenced.
Disclosure of Invention
In order to solve the problems, the invention provides a method for biomimetically mineralizing collagen and the collagen prepared by the method, which can realize in-situ biomimetic mineralization by using the collagen to obtain the collagen mineralized in fibers, thereby protecting the structural integrity of a mixed layer and prolonging the service life of a bonded prosthesis.
In order to realize the technical advantages, the invention adopts the following technical scheme:
a method of biomimetic mineralization of collagen, comprising the steps of:
by grafting the cationic polyelectrolyte to collagen, the liquid mineralized precursor can penetrate into the collagen fibers by capillary action and/or electrostatic action and/or double equilibrium of charge and osmotic pressure, and then phase inversion occurs to form biomineral crystals.
As an improvement of the technical proposal, the cationic polyelectrolyte can be one or a mixture of more than one of polyethyleneimine and ammonium chloride.
As an improvement of the above technical solution, the liquid mineralization precursor comprises calcium phosphate pre-crystal nucleus, amorphous calcium phosphate and polyelectrolyte-induced liquid mineralization precursor, wherein any one of the calcium phosphate pre-crystal nucleus, amorphous calcium phosphate and polyelectrolyte-induced liquid mineralization precursor is formed by calcium phosphate.
Any of the calcium phosphate pre-crystalline nuclei, amorphous calcium phosphate and polyelectrolyte-induced liquid mineralized precursors penetrate into the collagen fibers by capillary action and/or electrostatic action and/or charge and osmotic pressure bi-equilibrium.
As an improvement of the above technical means, wherein the collagen fiber comprises type I collagen, and the collagen fiber is formed by synthesizing the type I collagen from odontoblasts during dentinogenesis, then secreting the type I collagen into extracellular matrix in the form of triple helix procollagen, and further aggregating and assembling the collagen fiber.
As an improvement of the technical proposal, in the process of forming the biomineral crystal, the calcium phosphate pre-crystal nucleus is superposed with a calcium ion to form amorphous calcium phosphate, the amorphous calcium phosphate forms a liquid mineralization precursor induced by polyelectrolytes in the presence of polyelectrolytes, and under certain conditions, the amorphous calcium phosphate is converted into octacalcium phosphate which can be gradually converted into hydroxyapatite crystal.
The invention also provides biomimetic mineralized collagen prepared by the technical method.
The technical effects are as follows: according to the method for biomimetically mineralizing collagen and the collagen prepared by the method, provided by the invention, the cation polyelectrolyte is grafted to the collagen, a liquid mineralized precursor permeates into collagen fibers through the capillary action and/or the electrostatic action and/or the double balance of charge and osmotic pressure, and then phase transformation is carried out to form a biomineral crystal.
Drawings
The following and other advantages and features will be more fully understood from the following detailed description of embodiments thereof, with reference to the accompanying drawings, which must be considered in an illustrative and non-limiting manner, in which:
FIG. 1 is a structural diagram of a mineralized monolayer of collagen with polyelectrolyte-stabilized mineralized precursors formed in solution using a classical method of stabilizing a supersaturated solution of calcium and phosphorus with polyelectrolytes (abbreviated as PI L P method) for 2 days under a transmission electron microscope;
FIG. 2 is a structural diagram under a transmission electron microscope for 2 days of mineralization of a monolayer of collagen with pre-nuclei formed in solution using a saturated calcium phosphorus solution, in accordance with one embodiment of the method of biomimetic mineralization of collagen in accordance with the present invention;
FIG. 3 is a structural diagram of a three-dimensional collagen sponge without modification with polyethyleneimine under a transmission electron microscope, wherein a saturated calcium-phosphorus solution is used and mineralization is carried out for 3 days by using pre-nucleation in the solution;
FIG. 4 is a block diagram of a polyethyleneimine modified three-dimensional collagen sponge under a transmission lens, mineralized with pre-nucleated nuclei in solution using a saturated calcium phosphorus solution for 3 days;
FIG. 5 is a block diagram of the mineralization of rat tail collagen for 14 days under transmission electron microscopy using a classical PI L P method with polyelectrolyte-stabilized mineralizing precursors formed in solution;
fig. 6 is a structural diagram of a collagen biomimetic mineralization method according to an embodiment of the present invention, a rat tail collagen modified with polyethyleneimine is mineralized for 14 days under a transmission electron microscope using a polyelectrolyte stabilized calcium phosphorus supersaturated solution with polyelectrolyte stabilized mineralization precursors formed in the solution.
Detailed Description
As used herein, "and/or" includes any and all combinations of one or more of the associated listed items. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The exemplary invention described herein may suitably lack any one or more of the element limitations, which are not specifically disclosed herein. Thus, terms such as "comprising," "including," "containing," and the like are to be construed broadly and without limitation. Furthermore, the terms used herein are used as terms of description and not of limitation, and there is no intention in the use of such terms to describe only some of their characteristics but, in the light of the claims, various modifications are possible within the scope of the invention. Thus, while the present invention has been particularly disclosed in terms of preferred embodiments and optional features, modification of the invention herein disclosed to embody it may be noted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.
The present invention is broadly described herein. Smaller species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the removal of any substantial amount within the subject invention with a proviso or negative limitation, regardless of whether or not the reduced material is specifically recited herein.
Other embodiments are included in the following claims and non-limiting examples. In addition, the characteristics or related contents of the present invention are subject to the description of Markush group language. Those skilled in the art will recognize that the invention is also described by any independent definite term or subgroup term of markush group.
The conception, the specific contents and the technical effects of the present invention will be clearly and completely described below through specific embodiments to fully understand the objects, the features and the effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The starting materials used in the examples are, unless otherwise specified, commercially available from conventional sources. The preparation methods described in the examples are conventional in the art unless otherwise specified.
The noun explains:
biomineralization and biomineralization: the living organism can induce biomineral formation and affect the site and form of its deposition in tissues. The formation of mineralized substance is beneficial to the hard tissues of the organism to maintain the mechanical property and the biological function.
The invention provides a method for biomimetic mineralization of collagen, which comprises the following steps:
by grafting the cationic polyelectrolyte to collagen, the liquid mineralized precursor permeates into collagen fibers through capillary action and/or electrostatic action and/or double balance of charge and osmotic pressure, and then phase inversion occurs to form biomineral crystals.
In this embodiment, the cationic polyelectrolyte is one or more of polyethyleneimine and poly (ammonium chloride), and the collagen may be a monolayer of two-dimensional collagen or a three-dimensional collagen tissue mass.
In this embodiment, the liquid mineralization precursor includes calcium phosphate pre-crystal nuclei, amorphous calcium phosphate, and a polyelectrolyte-induced liquid mineralization precursor, wherein any one of the calcium phosphate pre-crystal nuclei, amorphous calcium phosphate, and polyelectrolyte-induced liquid mineralization precursor is formed by calcium phosphate, and any one of the calcium phosphate pre-crystal nuclei, amorphous calcium phosphate, and polyelectrolyte-induced liquid mineralization precursor is infiltrated into the collagen fibers by capillary action and/or electrostatic action and/or charge and osmotic pressure double equilibrium.
In this embodiment, the collagen fibrils comprise type I collagen, and the collagen fibrils are formed by synthesis of the type I collagen by odontoblasts, secretion into extracellular matrix in the form of triple helical procollagen, and further aggregation and assembly during histogenesis.
In the present example, wherein the calcium phosphate pre-crystal nuclei form amorphous calcium phosphate upon superposition of a calcium ion during the formation of the biomineral crystals, the amorphous calcium phosphate forms a liquid mineralization precursor induced by polyelectrolytes in the presence of polyelectrolytes, under certain conditions the amorphous calcium phosphate is converted to octacalcium phosphate and finally to hydroxyapatite and can be gradually converted to hydroxyapatite crystals.
In this embodiment, the liquid mineralization precursor includes calcium phosphate pre-crystal nucleus, amorphous calcium phosphate, and polyelectrolyte-induced liquid mineralization precursor, and in a specific environment, the mineralization precursor is formed by aggregation of calcium and phosphate ions.
Example 1
Soaking the prepared monolayer recombinant collagen in 200 μ g/ml branched polyethyleneimine (Mw 800 Kda) solution, reacting for 2 hr under the action of 0.3M 1-ethyl-3- (3-dimethylpropylamino) carbodiimide (EDC), repeatedly washing with deionized water after reaction, and drying.
Soaking the above prepared polyethyleneimine modified monolayer recombinant collagen in supersaturated calcium phosphorus solution (4.2 mM Na)2HPO4，9mM CaCl2 .2H2O), mineralization at 37 degrees for 1 day, 3 days, and 7 days.
Example 2
Soaking the prepared collagen sponge in 200 μ g/ml branched polyethyleneimine (Mw 800 Kda) solution, reacting for 2 hr under the action of 0.3MEDC, repeatedly washing with deionized water, and drying.
Soaking the prepared polyethyleneimine modified collagen sponge in supersaturated calcium-phosphorus solution (4.2 mM Na)2HPO4，9mM CaCl2 .2H2O), mineralization at 37 degrees for 1 day, 3 days, and 7 days.
Example 3
Soaking the prepared dentin collagen in 200 μ g/ml branched polyethyleneimine (Mw 800 Kda) solution, reacting for 2 hr under the action of 0.3M EDC, repeatedly washing with deionized water, and drying.
Soaking the prepared polyethyleneimine modified dentin collagenIn supersaturated calcium-phosphorus solution (4.2 mM Na)2HPO4，9mMCaCl2 .2H2O), mineralization at 37 degrees for 7 days, 14 days and 21 days.
Example 4
Soaking the prepared rat tail collagen in 200 μ g/ml branched polyethyleneimine (Mw 800 Kda) solution, reacting for 2 hr under the action of 0.3MEDC, repeatedly washing with deionized water, and drying.
Soaking the prepared polyethyleneimine modified rat tail collagen in supersaturated calcium phosphorus solution (200 μ g/ml poly- (allylamine hydrochloride) with 4.2mM Na) stabilized by polypropylene ammonium chloride2HPO4，9mM CaCl2 .2H2O), mineralization at 37 degrees for 7 days, 14 days and 21 days.
Example 5
Soaking the prepared monolayer recombinant collagen in 200 μ g/ml solution of ammonium chloride polypropylene (Mw 15 Kda), reacting for 2 hr under the action of 0.3MEDC, repeatedly washing with deionized water, and drying.
Soaking the prepared single-layer recombinant collagen modified by ammonium chloride in supersaturated calcium-phosphorus solution (4.2 mM Na)2HPO4，9mM CaCl2 .2H2O), mineralization at 37 degrees for 1 day, 3 days, and 7 days.
Example 6
Soaking the prepared collagen sponge in 200 μ g/ml polypropylene ammonium chloride (Mw 15 Kda) solution, reacting for 2 hr under the action of 0.3M EDC, repeatedly washing with deionized water, and drying.
Soaking the prepared polypropylene ammonium chloride modified collagen sponge in supersaturated calcium phosphorus solution (4.2 mM Na)2HPO4，9mM CaCl2 .2H2O), mineralization at 37 degrees for 1 day, 3 days, and 7 days.
Example 7
Soaking the prepared dentin collagen in 200 μ g/ml polypropylene ammonium chloride (Mw 15 Kda) solution, reacting for 2 hr under the action of 0.3M EDC, repeatedly washing with deionized water, and drying.
Soaking the above prepared polypropylene ammonium chloride modified dentin collagen in supersaturated calcium phosphorus solution (4.2 mM Na)2HPO4，9mMCaCl2 .2H2O), mineralization at 37 degrees for 7 days, 14 days and 21 days.
Example 8
Soaking the prepared rat tail collagen in 200 μ g/ml polypropylene ammonium chloride (Mw 15 Kda) solution, reacting for 2 hr under the action of 0.3M EDC, repeatedly washing with deionized water, and drying.
Soaking the prepared polypropylene ammonium chloride modified rat tail collagen in a supersaturated calcium phosphorus solution (200. mu.g/ml poly- (allylamine hydrochloride), 4.2mM Na) stabilized with polypropylene ammonium chloride2HPO4，9mMCaCl2 .2H2O), mineralization at 37 degrees for 7 days, 14 days and 21 days.
Referring to fig. 1, fig. 1 is a structural diagram of a conventional multi-polyelectrolyte stabilized calcium-phosphorus supersaturated solution method (abbreviated as PI L P method) under a transmission electron microscope for mineralizing a monolayer of collagen with a multi-polyelectrolyte stabilized mineralized precursor formed in the solution for 2 days, wherein the scale bar is 100 nanometers, in fig. 1, the monolayer of collagen is mostly mineralized in fibers, needle-shaped hydroxyapatite crystals are visible in the partially mineralized collagen and grow parallel to the long axis of the collagen, triangular marks represent the single mineralized collagen, and arrows represent the single partially mineralized collagen.
Referring to fig. 2, fig. 2 is one embodiment of a method of biomimetic mineralization of collagen according to the present invention. The polyethyleneimine modified collagen uses saturated calcium phosphorus solution to mineralize a structural diagram of a single-layer collagen under a transmission electron microscope for 2 days by using a pre-crystal nucleus formed in the solution, wherein the scale bar in the diagram is 100 nanometers, the single-layer collagen is completely mineralized in fibers, and needle-shaped hydroxyapatite crystals are visible in the collagen and grow parallel to the long axis of the collagen. The triangle indicates singly mineralized collagen, and the arrow indicates fully mineralized singly collagen.
Referring to fig. 3, fig. 3 is a structural diagram of a three-dimensional collagen sponge without modification by polyethyleneimine under a transmission electron microscope, which is mineralized for 3 days by using a saturated calcium-phosphorus solution and using pre-crystal nuclei in the solution; there was no evidence of mineralization within the collagen fibrils, and the arrows indicate that no mineralized collagen fibrils were formed, and the periodic striations characteristic of collagen fibrils were clearly visible under transmission electron microscopy, with a scale bar of 0.5 microns.
Referring to fig. 4, fig. 4 is one embodiment of a method of biomimetic mineralization of collagen according to the present invention. The polyethyleneimine modified three-dimensional collagen sponge is mineralized by using saturated calcium-phosphorus solution and utilizing a pre-crystal nucleus in the solution for 3 days, and a structural diagram under a transmission electron microscope is shown, wherein a scale bar in the diagram is 100 nanometers. As shown in fig. 4, the collagen sponge was partially mineralized. Arrows indicate mineralized collagen fibers, and hydroxyapatite crystals can be seen to be orderly arranged in the collagen fibers under a transmission electron microscope to present a periodic striation structure which is characteristic of the collagen fibers.
Referring to fig. 5, fig. 5 is a block diagram of a rat tail collagen mineralized using a polyelectrolyte-stabilized mineralized precursor formed in solution using the classical PI L P method under transmission electron microscopy for 14 days.
Referring to fig. 6, fig. 6 is a structural diagram of a collagen biomimetic mineralization method according to an embodiment of the present invention, a rat tail collagen modified by polyethyleneimine is mineralized for 14 days under a transmission electron microscope by using a polyelectrolyte-stabilized supersaturated solution of calcium and phosphorus, and using polyelectrolyte-stabilized mineralization precursors formed in the solution. Rat tail collagen has been substantially completely mineralized, and arrows indicate that hydroxyapatite crystals are orderly arranged inside collagen fibers and reflect the striation structure of collagen.
The invention also provides biomimetic mineralized collagen prepared by the method.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (6)
1. A method for biomimetic mineralization of collagen, comprising the steps of:
by grafting cationic polyelectrolytes to collagen, the liquid mineralized precursor permeates into collagen fibers through capillary action and/or electrostatic action and/or charge and osmotic pressure double equilibrium, and then phase inversion occurs to form biomineral crystals.
2. The method according to claim 1, wherein the cationic polyelectrolyte is selected from one or more of polyethyleneimine and ammonium chloride polypropylene.
3. The method of claim 1, wherein the liquid mineralization precursor comprises calcium phosphate pre-crystalline nuclei, amorphous calcium phosphate, and a polyelectrolyte-induced liquid mineralization precursor, wherein any of the calcium phosphate pre-crystalline nuclei, amorphous calcium phosphate, and polyelectrolyte-induced liquid mineralization precursor are formed from calcium phosphate,
any of the calcium phosphate pre-crystalline nuclei, amorphous calcium phosphate and polyelectrolyte-induced liquid mineralized precursors penetrate into the collagen fibers by capillary action and/or electrostatic action and/or charge and osmotic pressure bi-equilibrium.
4. The method of claim 1, wherein the collagen fibrils comprise type I collagen, and wherein the collagen fibrils are formed by secretion into the extracellular matrix in the form of triple helical procollagen, and further aggregation and assembly during dentinogenesis, following synthesis of the type I collagen by odontoblasts.
5. The method according to claim 1, characterized in that during the formation of the biomineral crystals, the calcium phosphate pre-crystal nuclei form amorphous calcium phosphate on addition of a calcium ion, which forms a liquid mineralization precursor induced by polyelectrolytes in the presence of polyelectrolytes, and under certain conditions is converted into octacalcium phosphate and can be gradually converted into hydroxyapatite crystals.
6. A biomimetic mineralized collagen prepared by the method according to any one of claims 1 to 5.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112426367A (en) * | 2020-12-11 | 2021-03-02 | 西安邮电大学 | Markable luminous repair material for dentistry and preparation method thereof, adhesive and preparation method thereof, and repair structure |
| CN115970067A (en) * | 2023-01-17 | 2023-04-18 | 中山大学附属口腔医院 | Intelligent guided bone tissue regeneration membrane and preparation method and application thereof |
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| CN107224609A (en) * | 2017-05-03 | 2017-10-03 | 中国人民解放军第四军医大学 | The method and its application of bionical calcification in complex polyelectrolyte particle induction collagenous fibres |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112426367A (en) * | 2020-12-11 | 2021-03-02 | 西安邮电大学 | Markable luminous repair material for dentistry and preparation method thereof, adhesive and preparation method thereof, and repair structure |
| CN115970067A (en) * | 2023-01-17 | 2023-04-18 | 中山大学附属口腔医院 | Intelligent guided bone tissue regeneration membrane and preparation method and application thereof |
| CN115970067B (en) * | 2023-01-17 | 2023-10-17 | 中山大学附属口腔医院 | Intelligent guiding bone tissue regeneration membrane and preparation method and application thereof |
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