KR101896594B1 - Dual crossslinked biodegradable polymer hydrogel-calcium phosphate complex and a preparation method therof - Google Patents

Abstract

The present invention relates to a bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate composite in which anionic functional groups are crosslinked through divalent metal ions added to a composite in which calcium phosphate particles are inserted into a biodegradable polymer hydrogel, and a method for producing the same .

Description

Crosslinked Biodegradable Polymer Hydrogel-Calcium Phosphate Composite and Dual-Cross-Linked Biodegradable Polymer Hydrogel-

The present invention relates to a bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate composite in which anionic functional groups are crosslinked through divalent metal ions added to a composite in which calcium phosphate particles are inserted into a biodegradable polymer hydrogel, and a method for producing the same .

As tissue engineering studies have been carried out to cultivate cells into a transplantable tissue using a support based on a biomaterial such as a hydrogel having excellent biocompatibility, a biomaterial such as a hydrogel Research is also under way. The hydrogel is largely composed of natural hydrogels such as hyaluronic acid, alginate, collagen, alginic acid, heparin and heparan, and poly (ethylene glycol) ) Can be classified into synthetic hydrogels based on PEG or poly (lactic- co- glycolic acid) (PLGA), and the natural hydrogel is a substance existing in the body, It is a biodegradable polymer that has excellent compatibility and can be naturally absorbed by decomposition by a specific enzyme in the body.

However, in order for the biodegradable polymer to be used as a support for regenerating living tissue, it is necessary to have a mechanical strength capable of withstanding a load of a certain level or higher. However, the polymer itself has a disadvantage of low mechanical strength and is used in a limited manner.

Various studies have been conducted to improve the mechanical strength of these biodegradable polymers. For example, it is a typical method to increase the crosslinking system or add a material capable of increasing the mechanical strength. First, as a method of increasing the crosslinking system, a photocrosslinking method, a chemical crosslinking method using a crosslinking agent, or the like, or a method of increasing the crosslinking degree by using a plurality of crosslinking agents. In addition, there is a method of covalently bonding a polymeric structure reactor using a chemical crosslinking method and further inducing a hydrogen bond or an ionic bond to increase the crosslinking system. A method of increasing the crosslinking system by combining various crosslinking methods, or increasing the crosslinking degree by increasing the concentration and content of the crosslinking agent. However, when the amount of the cross-linking agent is excessively large, the toxicity of the remaining cross-linking agent has a disadvantage in that the biocompatibility is remarkably lowered. Therefore, it is preferable to use a crosslinking agent in an appropriate concentration and amount.

As a method of adding a material capable of enhancing mechanical strength, a hydrogel mixed with two or more kinds of natural polymers having excellent biocompatibility, or a synthetic polymer such as PCL, PLGA and PGA, or a mixture of silica and hydroxyapatite And a method of mixing with a bioceramic material. Specifically, a composite in which calcium phosphate is homogeneously distributed on a biodegradable polymer, for example, a photo-crosslinked hyaluronic acid hydrogel, prepared by coprecipitation of bioceramics particles, particularly calcium phosphate, within the gel has been developed.

However, the composite in which the calcium phosphate particles were embedded in the hyaluronic acid hydrogel could provide enhanced strength as compared to the hyaluronic acid hydrogel itself not containing calcium phosphate, but was still insufficient for use as a tissue engineering support.

The inventors of the present invention have made extensive efforts to find a method for imparting additional strength to the biodegradable polymer hydrogel-calcium phosphate complex. As a result, they have found that a biodegradable polymer hydrogel-calcium phosphate complex containing a free anionic functional group such as a carboxyl group or a hydroxy group It is possible to provide a bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate complex by forming an additional crosslinking without additional crosslinking agent or the like by simply immersing in a solution containing a divalent metal ion, and the double crosslinked biodegradable polymer hydrogel - calcium phosphate complex has a significantly improved strength as compared with the biodegradable polymer hydrogel-calcium phosphate complex which does not form additional crosslinking, and thus completed the present invention.

In order to accomplish the object of the present invention, the first aspect of the present invention provides a biodegradable polymer hydrogel, wherein the biocompatible calcium phosphate particles are inserted into the composite, And a biodegradable polymer hydrogel-calcium phosphate complex.

A second aspect of the present invention is a method for preparing a biodegradable polymer hydrogel, comprising the steps of: preparing a biodegradable polymer hydrogel; A second step of immersing the hydrogel in a solution containing calcium ions and phosphate ions and immersing the hydrogel in an ammonium solution to precipitate calcium phosphate to form a biodegradable polymer hydrogel-calcium phosphate complex; And a third step of immersing the biodegradable polymer hydrogel-calcium phosphate complex in a solution containing a divalent metal ion and crosslinking the biodegradable polymer hydrogel-calcium phosphate complex .

Hereinafter, the present invention will be described in detail.

The present invention relates to a biodegradable polymer hydrogel-calcium phosphate composite which is obtained by treating a biodegradable polymer hydrogel-calcium phosphate complex with a polyvalent metal ion, in particular, a divalent metal ion and forming an additional crosslinking without adding a crosslinking agent or the like to produce a double crosslinked biodegradable polymer hydrogel- And that the double-crosslinked biodegradable polymer hydrogel-calcium phosphate composite thus prepared has a remarkably improved strength.

The present invention can provide a bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate complex in which anionic functional groups are crosslinked through divalent metal ions added to a composite in which calcium phosphate particles are inserted into a biodegradable polymer hydrogel .

The biodegradable polymer may be one prepared from one or more monomers selected from the group consisting of glucosamine, N-acetylglucosamine, glucuronic acid, galactosamine, N-acetylgalactosamine sulfate and iduronic acid. Preferably, the biodegradable polymer may be hyaluronic acid, chitosan, alginic acid, chondroitin, heparin or heparan, but is not limited thereto.

At this time, the calcium phosphate particles inserted into the biodegradable polymer hydrogel may have an average diameter of 50 nm to 500 nm. The size of the calcium phosphate particles can be adjusted according to the concentration of the solution containing calcium ions and phosphate ions to form the calcium phosphate particles. When the average diameter of the calcium phosphate particles is less than 50 nm, the calcium phosphate is recognized by the macrophages at the time of injection into the body and is rapidly decomposed. Therefore, the strength enhancement effect of the complex may be insufficient. If it exceeds 500 nm, It is possible to precipitate calcium phosphate, but the generation uniformity of the formed particles is lowered, and the precipitated particles may be generated non-uniformly on the outer surface of the gel, not in the gel.

The calcium phosphate particles may be contained in an amount of 10 to 50% by weight based on the total weight of the hydrogel composite. When the content of calcium phosphate is less than 10%, the calcium phosphate particles become very small, and as described above, there is a fear that the calcium phosphate particles are decomposed rapidly upon injection into the living body. When the content exceeds 50% by weight, There is a possibility.

The bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate complex according to the present invention may have a shear rate of 500 Pa or more. As an example of the application field, most of the general filler products exhibit a shear rate of 500 Pa or less, which is advantageous in that the calcium phosphate content can be adjusted to various values of shear rate of 500 Pa or more by the present invention. However, it is preferable to have a shear rate of 500 Pa when the support is applied to support the load to some extent, and it is desirable to increase the shear rate to have a higher shear rate in order to withstand the load of the living tissue.

The bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate complex according to the present invention comprises a first step of preparing a biodegradable polymer hydrogel; A second step of immersing the hydrogel in a solution containing calcium ions and phosphate ions and immersing the hydrogel in an ammonium solution to precipitate calcium phosphate to form a biodegradable polymer hydrogel-calcium phosphate complex; And a third step of immersing the biodegradable polymer hydrogel-calcium phosphate complex in a solution containing a divalent metal ion and crosslinking the solution.

The biodegradable polymer hydrogel may be prepared by modifying a polymer with a methacrylate functional group and photo-crosslinking the polymer. However, the biodegradable polymer hydrogel may be prepared by appropriately modifying a method known in the art.

The biodegradable polymer may be a polymer including an anionic functional group capable of binding, such as a free carboxyl group or a hydroxy group. The free carboxyl group or a hydroxyl group, depending on the pH of the anionic functional group ^-COO-There is switched to the addition of 2 can participate in ionic bonding with the metal ^ion-or -O.

The divalent metal ion may be a ^{2 +} Ca, Be ^{+ 2,} Mg ^{+ 2,} Sr ^{+ 2,} Ba ^{+ 2,} Ra ^{2 +,} or a combination thereof. The divalent metal ion can ion-couple with two free anionic functional groups of the biodegradable polymer, so that it is possible to provide additional crosslinking through the plurality of ionic bonds.

The concentration of the solution containing the divalent metal ions may be 0.001 to 1.5 M. If the concentration of the metal ion is less than 0.001 M, it is difficult to expect a remarkable strength enhancement effect. If the concentration is more than 1.5 M, unnecessary crystals may be formed and the biological characteristics may be deteriorated.

Preferably, the third step of forming an additional crosslinking with the divalent metal ion can be carried out in a solution having a pH of 6 or more and 13 or less. The crosslinking reaction occurs through a free carboxyl group or a hydroxyl group, which is an anionic functional group, and the functional groups are preferably present in the form of an anion. Accordingly, when these functional groups are weakly acidic functional groups, they are preferably weakly acidic or basic in order to exist in an anion form. Therefore, prior to carrying out the crosslinking reaction of the third step, a step of adjusting the pH by treating with a basic solution so that the pH of the solution satisfies the above conditions may be further performed.

The third step may be performed for 3 to 12 hours, but is not limited thereto. If the third step is carried out for less than 3 hours, crosslinking may not be sufficiently performed and the strength improving effect may not be exhibited as much as desired. If the step is performed for more than 12 hours, Can delay the reverse reaction. In addition, when the reaction is carried out for a long time in a solution having a low pH, the hydrogel may be decomposed to lower the stability.

The bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate complex according to the present invention is obtained by treating a biodegradable polymer hydrogel-calcium phosphate complex with a polyvalent metal ion, in particular, a divalent metal ion to form additional crosslinking without adding a crosslinking agent or the like The bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate composite thus prepared can provide a significantly improved strength. Therefore, the bi-crosslinkable biodegradable polymer hydrogel-calcium phosphate composite is useful as a tissue engineering support. Can be used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a method for producing a hyaluronic acid hydrogel-calcium phosphate composite produced using double crosslinking.
FIG. 2 is a diagram showing the result of visual inspection of a hyaluronic acid hydrogel-calcium phosphate complex according to an embodiment of the present invention.
FIG. 3 is a graph showing the shear coefficient according to the frequency of a hyaluronic acid hydrogel-calcium phosphate composite prepared using calcium ions according to an embodiment of the present invention.
FIG. 4 is a graph showing the comparison of shear numbers of hyaluronic acid hydrogel-calcium phosphate complexes prepared using various cations (Ca ^{2 +} , Na ⁺ , Al ^{3 +} , Ba ^{2 +} , Sr ^{2 +} and Fe ^{3 +} to be.
FIG. 5 is a graph showing the degree of swelling of a hyaluronic acid hydrogel-calcium phosphate complex prepared using various cations (Ca ^{2 +} , Na ⁺ , Al ^{3 +} , Ba ^{2 +} , Sr ^{2 +} and Fe ^{3 +} ).
FIG. 6 is a graph showing a change in shear rate according to pH of a hyaluronic acid hydrogel-calcium phosphate complex according to an embodiment of the present invention. FIG.
FIG. 7 is a view schematically showing the principle of improving the physical properties of the hyaluronic acid hydrogel-calcium phosphate composite according to an embodiment of the present invention.
8 is a diagram showing the results of TEM observation of changes in calcium phosphate precipitated by co-precipitation.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Comparative Example  And Example : Preparation of sample

Step 1: Photocrosslinking  Hyaluronic acid through Hydrogel  Produce

A methacrylate functional group was introduced into hyaluronic acid and photo-crosslinked to produce a single crosslinking hyaluronic acid hydrogel. Specifically, glycidyl methacrylate hyaluronic acid (GMHA) was synthesized by treating glycidyl methacrylate with a 1% solution of hyaluronic acid, and the glycidyl methacrylate hyaluronic acid (GMHA) . Iragacure 2959 was added to the reaction solution as a photoinitiator and ultraviolet light of 425 nm wavelength was irradiated for 15 minutes to prepare a photocrosslinked hydrogel.

Step 2: Hydrogel  Within the calcium phosphate Precipitation

A complex of hyaluronic acid hydrogel and calcium phosphate prepared in Comparative Example 1 was prepared by coprecipitation. Specifically, CaCl ₂ and H ₃ PO ₄ were prepared at concentrations of 0.153 M and 0.092 M, respectively, to prepare a mixed solution, and the hydrogel was immersed therein. , Re-dipping the Ca ²⁺ and PO ₄ ³ A hydrogel containing a ⁺ ions generated therefrom to the 7.5 wt% solution of ammonium to precipitate a calcium phosphate gel inside. At this time, the precipitated calcium phosphate particles had an average size of 170 nm, and the precipitation amount was 30% by weight relative to hyaluronic acid.

Step 3: Additional by cation addition Ion bridge

The hyaluronic acid hydrogel containing no internally precipitated calcium phosphate produced by the above step 1 or steps 1 and 2 was dissolved in a solution in which various polyvalent metal cations were dissolved at a concentration of 1 M (control; Na ⁺ , bivalent metal ion ; Ca ^{2 +} , Ba ^{2 +} and Sr ^{2 +} , trivalent metal cations; Al ^{3 +} and Fe ^{3 +} ) for 6 hours. In the above process, the non-crosslinked functional group (-COOH or -OH) of hyaluronic acid was additionally cross-linked through polyvalent metal cations, which was called dual crosslinking. As described above, it is possible to use various metal ions The effect of the number of charges and / or kinds of ions was confirmed by proceeding double crosslinking in the solution. NaCl, CaCl ₂ , BaCl ₂ , SrCl ₂ , Al (NO ₃ ) ₃ and FeCl ₃ were used to provide the metal ions. At this time, NaCl, CaCl ₂ , BaCl ₂ and SrCl ₂ solutions were neutral, and Al (NO ₃ ) ₃ and FeCl ₃ solutions showed strong acidity.

Four groups of samples were prepared by performing at least one of the above steps 1 to 3 in combination. First, the samples prepared by the step of w / o precipitation or w / precipitation of the calcium phosphate precipitation step of step 2 are mixed with hyaluronic acid (HAc) pure substance and calcium phosphate ; And CaP (composite) (HAc / CaP). In addition, according to whether or not additional ion crosslinking is performed in Step 3, it is classified into a single crosslinking (denoted as Single-CL in the drawing) and a dual crosslinking (denoted as dual-CL in the drawing). Thus, finally, a negative control (Single-CL HAc) which is single-crosslinked hyaluronic acid prepared according to Step 1; A first comparison group (Single-CL HAc / CaP) which is a mono-crosslinked hyaluronic acid calcium phosphate composite prepared according to Step 1 and Step 2; A second comparative group (Dual-CL HAc) which is a double-crosslinked hyaluronic acid prepared according to steps 1 and 3; And a double-crosslinked hyaluronic acid calcium phosphate composite (Dual-CL HAc / CaP) prepared according to steps 1 to 3. The cross-linked samples were cations used therein.

Experimental Example  1: calcium phosphate Precipitation  And / or Double Bridging  Hyaluronic acid by Hydrogel  Morphological change

In order to confirm the size and morphological changes of the four groups prepared by the above Comparative Examples and Examples, they were arranged side by side, observed with the naked eye, and photographed and directly compared. The results are shown in FIG. As shown in Fig. 2, the size decreased in the double-crosslinked hydrogel as compared to the single crosslinked hydrogel. At this time, a significant size reduction case where monovalent cations ateuna The addition of Na ⁺ reduction rate is rather low, and polyvalent metal cations, particularly bivalent metal ions, e.g., Ca ^{+ 2,} was added to double the cross-Ba ²⁺ and Sr ^{2 +} appear. On the other hand, a solution containing a trivalent metal cation, such as Al (NO ₃ ) ₃ and FeCl _3, has a strong acidity, and thus the hydrogel and / or the precipitated calcium phosphate particles therein tend to decompose. As a result, when immersed in an Al (NO ₃ ) ₃ solution, the hydrogel itself was decomposed and particles could not be recovered. Also, when immersed in a FeCl ₃ solution, a considerable amount of the calcium phosphate particles formed in the hydrogel was decomposed and the double-cross-linked hydrogel was retained in the spherical shape (the uppermost right side) without precipitation process. On the other hand, The final recovered particles exhibited an irregular shape (right bottom right).

Experimental Example  2: Calcium phosphate Precipitation  And / or Double Bridging  Hyaluronic acid by Hydrogel   Property change

(G ') and loss elastic modulus (G' ') of the four groups of samples prepared through the above Comparative Examples and Examples were measured in order to confirm the effect of improving the physical properties by double crosslinking, 3 and Fig.

As shown in FIG. 3, regardless of precipitation of calcium phosphate, the number of shear phases was increased in the double-crosslinked hydrogel as compared with single-crosslinked hydrogel. At this time, in the case of the hydrogel containing calcium phosphate formed therein by further performing the precipitation process before the double crosslinking, a remarkable increase in the number of shear phases was shown by double crosslinking. This can be interpreted as a synergistic improvement effect due to the additional crosslinking of the internal calcium phosphate and the hydrogel.

Further, as shown in Fig. 4, the effect was maximized when a double-crosslinked metal ion was treated by treating with a divalent metal ion as compared with a trivalent metal cation. This is attributed to the fact that the hydrogel and / or the calcium phosphate present therein is decomposed due to the low pH of the solution containing the trivalent metal cations and the physical property improving effect disappears. On the other hand, among the divalent metal ions, in particular, when a solution containing a calcium ion was used to double crosslink, it showed a remarkable property improvement effect because the calcium ions added due to the common ion effect could not remain in the hydrogel And calcium phosphate precipitation effect.

Experimental Example  3: Calcium phosphate Precipitation  And / or Double Bridging  Hyaluronic acid by Hydrogel   Swelling degree change

In order to confirm the degree of swelling of the samples prepared according to the above Comparative Examples and Examples, each sample of 4 groups was dried, weighed and immersed in distilled water (10 ml) to absorb the water. The sample was allowed to stand at 37 DEG C for 24 hours, and the weight of the sample was measured. The ratio is shown in Fig. As shown in FIG. 5, in the case of the double crosslinked sample, particularly, the double crosslinked sample with the divalent metal ion, the swelling of the hydrogel due to the water absorption hardly appeared regardless of the calcium phosphate precipitation, In the case of the samples crosslinked with mono-cations or trivalent metal cations Fe ^{3 +} , the number of crosslinked samples was significantly decreased compared with that of the single crosslinked samples, but the water was still absorbed and swelled. In particular, Respectively.

Experimental Example  4: Calcium phosphate Precipitation  And / or Double Bridging  Hyaluronic acid by Hydrogel   Change of physical properties according to pH

In order to confirm the cause of improving the physical properties in the samples prepared according to the comparative examples and the examples, before proceeding to further crosslinking by polyvalent metal cations, the polyvalent metal cations were immersed in a basic solution of pH 11, And the ion cross-linking was carried out. The shear rate for each was measured, and the results are shown in FIG. As shown in FIG. 6, the shear rate of the sample immersed in the solution of pH 11 was increased, especially in the hydrogel containing calcium phosphate precipitated therein. This is the case of processing with a solution of pH 11 additional cross-linking the functional group to a weak acid of the -COOH and / or -OH group is -COO negative band ^- to facilitate additional crosslinking during polyhydric was converted to the metal cation is added ^- and -OH . This principle is schematically shown in Fig.

Experimental Example  5: Double bridging  By metal ion addition Hydrogel  Internal Of the precipitated particles  change

In order to confirm the influence of the addition of metal ions during double crosslinking according to the present invention on calcium phosphate precipitate formed in the hydrogel, TEM observation was conducted and the results are shown in FIG. The results for the hydrogels treated with the divalent metal ions (Ca ²⁺ , Ba ²⁺ and Sr ²⁺ ) having high ion-crosslinking effect and ion-crosslinked are shown in FIGS. 8A to 8C, respectively. As shown in FIG. 8A, when the Ca ^{2 +} -containing solution was treated, the increase in the number of precipitation particles having a size similar to or smaller than that of the existing precipitation particles was attributed to the additional precipitation of calcium ions added for ion crosslinking . On the other hand, as shown in Figs. 8B and 8C, when Ba ^{2 +} and Sr ^{2 +} -containing solutions were treated, the Ba ^{2 +} or Sr ^{2 +} component was not detected in the precipitated particles and the particles retaining relatively uniform size This indicates that the Ba ^{2 +} and Sr ^{2 +} ions do not affect the precipitated particles and are only involved in additional ion bridging. This also supports previous results showing an additional increase in shear rate when ion bridging with a Ca ²⁺ solution, even with the same ^bivalent metal ion.

Claims (12)

Biodegradable polymer hydrogels in which calcium phosphate particles are inserted into biodegradable polymer hydrogels, biodegradable polymer hydrogels in which anionic functional groups are cross-linked through divalent metal ions added to the calcium phosphate complexes - calcium phosphate As a complex,
^Wherein the divalent metal ion is Ca < 2 ^{+ &} gt ;.
The method according to claim 1,
Wherein the biodegradable polymer is prepared from at least one monomer selected from the group consisting of glucosamine, N-acetylglucosamine, glucuronic acid, galactosamine, N-acetylgalactosamine sulfate and iduronic acid. Hydrogel-calcium phosphate complex.
3. The method of claim 2,
Wherein the biodegradable polymer is hyaluronic acid, chitosan, alginic acid, chondroitin, heparin or heparan. The biodegradable polymer hydrogel-calcium phosphate complex according to claim 1, wherein the biodegradable polymer is hyaluronic acid, chitosan, alginic acid, chondroitin, heparin or heparan.
The method according to claim 1,
Wherein said calcium phosphate particles have a mean diameter of 50 nm to 500 nm. ≪ RTI ID = 0.0 > 20. < / RTI >
The method according to claim 1,
Wherein the calcium phosphate particles are contained in an amount of 10 to 50% by weight based on the total weight of the hydrogel composite.
The method according to claim 1,
Wherein the biodegradable polymer hydrogel-calcium phosphate complex has a shear rate of at least 500 Pa.
A first step of preparing a biodegradable polymer hydrogel;
A second step of immersing the hydrogel in a solution containing calcium ions and phosphate ions and immersing the hydrogel in an ammonium solution to precipitate calcium phosphate to form a biodegradable polymer hydrogel-calcium phosphate complex; And
And a third step of immersing the biodegradable polymer hydrogel-calcium phosphate complex in a solution containing a divalent metal ion and crosslinking the biodegradable polymer hydrogel-calcium phosphate complex, wherein the biodegradable polymer hydrogel-
^Wherein the divalent metal ion is Ca < 2 ^{+ &} gt ;.
8. The method of claim 7,
Wherein the biodegradable polymer is a polymer comprising a free carboxyl group or a hydroxy group capable of bonding.
delete 8. The method of claim 7,
And the concentration of the solution containing the divalent metal ions is 0.001 to 1.5 M. The method for producing a biorthodermal polymer hydrogel-calcium phosphate composite according to claim 1,
8. The method of claim 7,
Wherein the third step is carried out in a solution having a pH of 6 or more and 13 or less.
8. The method of claim 7,
And the third step is carried out for 3 to 12 hours. ≪ RTI ID = 0.0 > 11. < / RTI >

Images