KR101351119B1 - Mussel adhesive protein-added brushited bone graft substitute - Google Patents

Abstract

The present invention relates to a β-TCP / MCPM based brushite bone graft substitute using mussel adhesive protein. In another aspect, the present invention comprises the steps of preparing the mussel adhesive protein fp-151; Preparing a cured liquid using the mussel adhesive protein fp-151; Mixing the curing solution and β-TCP / MCPM (β-tricalcium phosphate / monocalcium phosphate monohydrate); And it relates to a bone graft substitute manufacturing method comprising the step of molding (molding) after mixing.

Description

Mussel adhesive protein-added brushited bone graft substitute}

The present invention relates to a brucite-based bone graft substitute comprising mussel adhesive protein and a method for preparing the same.

Bone graft substitutes have been used to replace local bone defects. The transplantation of autologous bone is known to be the most effective bone graft substitute because it contains cells with bone formation ability as well as bone conduction and bone induction ability. However, the operation time is long, the amount is limited, and the problems such as mortality due to bleeding and chronic pain in donor limit the use thereof. Allogeneic bones and xenografts used as alternatives have bone conduction ability and some osteoinductive ability, but include risks of immune rejection and infection.

Therefore, various synthetic bone graft substitutes for filling bone defect sites have been studied. The bone graft substitute market is growing nearly 10% per year, and synthetic materials account for only about 15% in the bone graft substitute market, but the growth rate is highest at about 15% per year. Various materials are used as bone substitutes. Metals such as iron alloys, magnesium alloys and titanium and polymers such as polylactide, polyurethane and polycaprolactone, silicate-based glass, calcium sulfate hydrate and calcium phosphate Proposed. Since about 60% of bone is actually calcium phosphate, calcium phosphate-based materials are the most popular based on their compositional similarity, and many studies have reported that calcium phosphate is very biocompatible and bone conductive. It has been studied in various ways.

Many bone graft substitutes have been used in granules (typically 0.1-5 mm in diameter), porous blocks, or sponges. Later, a method of stiffening after transplantation or infusion was proposed, and the possibility of injecting a bone graft substitute expanded the field of application to treat bone breakdown with minimal invasive techniques. In the case of calcium phosphate cement, it is a hydraulic cement, and curing begins by mixing a reactive calcium phosphate powder with an aqueous solution. After mixing, the calcium phosphate melts and precipitates in the form of low solubility calcium phosphate. Calcium phosphate crystals grow and entangle during the precipitation, thereby imparting the mechanical rigidity of the cement.

Calcium phosphate-based cement can be divided into apatite and brushite based on the final reaction phase. In the case of apatite cements, much has been studied because of the same advantages as the calcium phosphate phase of bone, mechanically more favorable and curing at neutral pH. In spite of these advantages, since brusite has a metastable calcium phosphate phase in the physiological state unlike apatite, interest in brucite calcium phosphate cement is growing. Brucite calcium phosphate cements are known to have faster absorbency than apatite and are 10-100 times more soluble than apatite at physiological pH. Ideally, high absorbent brucite calcium phosphate cement can be replaced with new bone more easily than apatite because bone substitutes should be replaced with mature bone without loss of mechanical support.

Calcium phosphate cements, on the other hand, are brittle and have low mechanical stability compared to other bone substitute materials. Tensile strength, an important property as a bone graft substitute, is 5-20 times smaller than compressive strength. Most calcium phosphate cements have a compressive strength in the range of 1-100 MPa, while tensile strengths are 1-10 MPa. Thus, current calcium phosphate cements can only be applied in combination with metal implants, where they do not have to withstand or withstand low loads. Also, since brucite calcium phosphate cement is somewhat weaker than apatite calcium phosphate cement, its use is further limited. Therefore, various methods have been proposed to improve mechanical strength, and many studies have been conducted to introduce biocompatible and biodegradable materials into calcium phosphate cement.

On the other hand, mussels, which are marine life, live in a dynamic sea environment by producing and secreting functionally differentiated adhesives and attaching them firmly to the base of the water so that they can adhere underwater in the marine environment. Mussel adhesives are strong natural adhesives that are much more powerful, flexible and flexible than other polymer-based adhesives such as epoxy and phenolic resins. Mussel adhesive proteins can adhere to many types of surfaces, such as plastics, glass, metals, Teflon, and biomaterials, and maintain adhesion even in aquatic environments. Mussel adhesives, which have strong and water-resistant properties, are biodegradable and environmentally friendly, and are harmless to humans and hardly induce immune responses. Therefore, mussel adhesives can be used in various ways as medical adhesives such as cell or tissue adhesives.

Currently commercially available mussel adhesive proteins are used only as cell or tissue culture adhesives due to low extraction yield and high production cost. The team introduced genetic recombination techniques to obtain large amounts of adhesive proteins for practical applications. Mgfp ( Mytilus galloprovincialis foot protein) -1 Designed and constructed a new hybrid mussel adhesive protein fp-151 that genetically connects the structure of 10 repeat amino acids six times to the N- and C-terminus of Mgfp-5, It was expressed as. Hybrid mussel adhesive protein fp-151 has very high productivity and easy separation and purification method, and it has been confirmed that the industrial applicability is very high due to economic mass production (international patent publication WO2006 / 107183 or WO2005 / 092920).

The present inventors confirmed that the mussel adhesive protein obtained through the previous study can be used as a bone substitute additive as a medical material by using it in addition to the hardening solution for improving the mechanical strength of the brothite calcium phosphate cement bone graft substitute.

It is an object of the present invention to provide a brucite-based bone graft substitute comprising mussel adhesive protein. In addition, in order to provide a brucite-based bone graft substitute with improved compressive strength, preparing a mussel adhesive protein fp-151; Preparing a cured liquid using the mussel adhesive protein fp-151; Mixing the curing solution and β-TCP / MCPM (β-tricalcium phosphate / monocalcium phosphate monohydrate); And it is an object of the present invention to provide a method for producing a brushite bone graft substitute comprising the step of molding (molding) after mixing.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a brushite bone graft substitute comprising mussel adhesive protein.

In one embodiment of the invention, the mussel adhesive protein is characterized in that it comprises a fp-151 protein.

In another embodiment of the present invention, the fp-151 protein is characterized in that described by SEQ ID NO: 1.

In another embodiment of the present invention, the brucite is characterized in that β-TCP / MCPM (β-tricalcium phosphate / monocalcium phosphate monohydrate) based.

In another aspect, the present invention comprises the steps of preparing the mussel adhesive protein fp-151; Preparing a cured liquid using the mussel adhesive protein fp-151; Mixing the curing solution and β-TCP / MCPM (tricalcium phosphate / monocalcium phosphate monohydrate); And it provides a method for producing a brushite bone graft substitute comprising the step of molding (molding) after mixing.

The present invention includes a mussel adhesive protein in the β-TCP / MCPM-based Brusite-based bone graft substitutes to prepare a bone graft substitute having improved compressive strength, and thus limited its use to only places with low mechanical properties and low load. It can be manufactured as a bone graft substitute that can support the load by supplementing the bone graft substitute. Therefore, it can be used as a bone graft substitute that is practically applicable to various bone defect sites that need support. In addition, mussel adhesive protein has biocompatibility and biodegradation properties, and since brucite calcium phosphate is metastable calcium phosphate phase, it has high absorbency, so it can be easily replaced with mature bone and can be developed as an ideal bone graft substitute.

1 is a Brucite bone graft substitute prepared by curing by mixing β-TCP and MCPM powder, acidic pyrophosphate.
Figure 2 is a compressive strength of the brucite cement made by mixing acidic pyrophosphate containing protein in β-TCP and MCPM powder. NC is negative control, BSA is 91.7mg / ml bovine serum albumin, 1X MAP is 27.5mg / ml, 2X MAP is 55mg / ml, 3X MAP is 82.5mg / ml, 4X MAP is 110mg / ml, 5X MAP is 137.5 mg / ml of mussel adhesive protein.
3 is a view of the broken surface of the negative control group Brusite observed with a scanning electron microscope. (NCNT is the national center for nanomaterials technology with SEM machines, LEI is the type of detector, 5.0kV is the acceleration voltage of the gun that emits electrons, X500 is enlarged 500 times, and 10㎛ is directly above it. The length of the scale bar located, WD is the working distance, the distance between the lens and the sample through which the beam passes.)
FIG. 4 is a diagram illustrating a broken surface of Brucite cement made of a hardening solution containing 91.7 mg / ml BSA, using a scanning electron microscope.
FIG. 5 is a diagram illustrating a broken surface of Brucite cement made of a curing liquid containing 137.5 mg / ml of fp-151, using a scanning electron microscope.

The present inventors have completed the present invention that can function as a bone graft substitute additive as a medical material by using the mussel adhesive protein in addition to the curing liquid to improve the mechanical strength of the Brucite calcium phosphate cement bone graft substitute.

In one embodiment of the present invention was prepared a Brucite bone graft substitute containing the mussel adhesive protein fp-151, in another embodiment was measured the compressive strength of the Brusite bone graft replacement agent added to the mussel adhesive protein fp-151, In another embodiment, the crystal structure of the Brucite bone graft substitute with mussel adhesive protein fp-151 was observed.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example  One  : Mussel adhesive protein  Added Brusite Bone graft  Production of substitute

1-1. Mussel adhesive protein fp Β- with -151 TCP Of MCPM Based Brusite  making

Mussel adhesion protein fp-151 (SEQ ID NO: 1) synthesizes a fp-1 variant in which a peptide consisting of AKPSYPPTYK is repeated six times in the amino acid sequence of mussel adhesion protein fp-1 (Genbank No. Q27409), which exists in nature, 2 The Mgfp-5 gene (Genbank No. AAS00463) was inserted between the fp-1 variants of dogs, and then produced by E. coli. Preparation of the mussel adhesive protein fp-151 is the same as that shown in International Patent Publication WO2005 / 092920, which is incorporated by reference as a whole.

Using the mussel adhesive protein fp-151 prepared as described above was prepared a cured liquid of the Brusite bone graft substitute.

Specifically, the mussel adhesive protein fp-151 is 30, 60, 90, 120, 150 mg in order to estimate the amount of mussel adhesive protein in response to the color development of the BSA to the degree of color development by the reaction between the mussel adhesive protein and the bradford reagent. Dilution in distilled water at a concentration of / ml, which was confirmed by bradford assay. Mussel adhesive protein solution of each concentration was diluted in a volume ratio of 1:11 in 0.6M sodium pyrophosphate dibasic (Na ₂ H ₂ P ₂ O ₇ ) solution, and 0.05M acid pyrophosphate added with mussel adhesive protein. Sodium solution was used as the curing liquid. As a negative control (NC), a 0.05 M acid pyrophosphate solution containing no protein was used.

Calcium phosphate powder contains 0.3 g of beta-tricalcium phosphate, β-TCP, β-Ca ₃ (PO ₄ ) _2, Sigma-Aldrich, and 0.2 g of calcium phosphate monohydrate (MCPM). , Ca (H ₂ PO _{4 ·} H ₂ O, Sigma-Aldrich) was prepared separately and mixed together when mixing with the curing liquid, where 200ul of the curing liquid per 0.5 g of the total powder was added, and the powder and the curing liquid The ratio of was 2.5 g / ml.

The mixture was kneaded using a spatula and placed in an acrylic mold to cure into a cylindrical shape having a diameter of about 7 mm and a height of about 8 mm. The cured cement obtained a reaction product of calcium diphosphate dihydrate (dicalcium phosphate dihydrate, DCPD, Brushite, CaHPO _{4 ·} H ₂ O) through the following reaction formula.

_{β-Ca 3 (PO 4)} 2 + Ca (H 2 PO 4) · H 2 O + 7H 2 O -> 4CaHPO 4 · H 2 O

The cured Brucite bone graft substitute is shown in FIG. 1.

1-2. Bovine serum albumin  Β- added TCP Of MCPM  Based Brusite  making

In the same manner as in <Example 1-1>, 100 mg / ml bovine serum albumin (BSA) was added to 0.6 M sodium pyrophosphate dibasic (Na ₂ H ₂ P ₂ O ₇ ) solution. A brucite cement was prepared in the same manner by using a 0.05 M sodium pyrophosphate solution added with bovine serum albumin diluted with a volume ratio of 1:11 as a curing solution. Bovine serum albumin (BSA) is the most commonly used protein as a control of the experimental protein, and the result of the experiment is that the effect is due to the inclusion of the mussel adhesive protein, a special protein, not the effect including the general protein Presented for comparison.

Example  2  : Protein added Brusite Bone graft  Compressive strength measurement of substitute

The compressive strength of β-TCP / MCPM-based Brucite bone graft substitute prepared by curing with an acidic pyrophosphate solution containing mussel adhesive protein or bovine serum albumin was measured. The hardened cylindrical brushsite was measured while pressing a 3 mm diameter area of the flat upper portion at a speed of 1 mm / min using a universal testing machine (INSTRON) 2 kN load cell, and the results are shown in FIG. 2.

As shown in Figure 2, the compressive strength of the bone graft substitutes increased as the inclusion rate of the mussel adhesive protein of fp-151 increased compared to the bone graft substitute control group (NC) containing only a hardening solution containing no protein It was confirmed. On the other hand, in the case of a bone graft substitute containing bovine serum albumin (BSA), it was confirmed that the compressive strength is reduced compared to the bone graft substitute control group (NC) that does not contain protein.

Example  3  : Protein added Brusite Bone graft  Observation of Crystal Structure of Alternatives

The crystal structure of the Brucite bone graft substitute prepared by scanning electron microscopy (SEM) was observed. As shown in FIG. 3, the control group (NC) without protein showed a general crystal structure of Brusite, and the BSA had a different crystal structure from that of the control group, and it can be seen from FIG. there was. As shown in FIG. 5, when the high concentration of fp-151 was added (5X MAP), the crystal structure was similar to that of the control group, but showed a higher density.

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Claims (5)

Composition for a brucite bone graft substitute comprising a fp-151 protein represented by SEQ ID NO: 1 as the mussel adhesive protein, and an acidic sodium pyrophosphate solution. delete delete The method of claim 1,
The brucite is characterized in that the β-TCP / MCPM (β-tricalcium phosphate / monocalcium phosphate monohydrate) based, the composition for the bone substitute bone graft substitute. A method for preparing a composition for Bruxite bone graft substitute according to claim 1, comprising the following steps:
(1) preparing a mussel adhesive protein fp-151;
(2) mixing the mussel adhesive protein fp-151 and an acidic sodium pyrophosphate solution to form a cured liquid;
(3) mixing the curing solution and β-TCP / MCPM (β-tricalcium phosphate / monocalcium phosphate monohydrate); And
(4) molding the form after mixing.

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