KR101317515B1 - Bone regeneration scaffold coated with fibronectin-osteocalcin fusion protein - Google Patents

Abstract

PURPOSE: A bone regeneration scaffold is provided to promote the attachment and maintenance of osteoblast and to improve the specialization of the osteoblast. CONSTITUTION: A bone regeneration scaffold comprises: a step of manufacturing a polymer scaffold; a step of coating the scaffold with hydroxyapatite polymers; a step of synthesizing fibronectin-osteocalcin fusion protein; and a step of coating the hydroxyapatite-coated polymer scaffold with fibronectin-osteocalcin fusion protein. The step of coating the scaffold with hydroxyapatite polymers comprises a step of dipping the polymer scaffold in a CaCl2 solution and a Na2HPO4 solution by turns and treating the polymer scaffold with a mineralization solution at 35 deg. C for 1-10 days.

Description

Bone regeneration scaffold coated with fibronectin-osteocalcin fusion protein}

The present invention relates to a scaffold for bone regeneration comprising a hydroxyapatite coated with a polymer scaffold surface and a fibronectin-osteocalcin fusion protein bonded to hydroxyapatite and a method for producing the same.

Treatment of massive bone defects is an important task in modern medicine. Research on regenerative medicine and tissue engineering continues, and stem cell-based therapies are being considered for bone regeneration in the clinic. This overcomes the problem that early osteoblast acquisition is difficult in patients with partial bone defects that have already advanced or have aged tissue. Mesenchymal stem cells (MSCs) differentiate directly into the phenotype of osteoblasts when supplied with appropriate nutrients, such as bone forming proteins.

Scaffolds for application in bone tissue engineering must meet several key conditions for optimized tissue formation of host tissues. These conditions include cell affinity, adequate porosity for nutrient and oxygen permeation, interfacial activity to promote cell adhesion and differentiation, and the like. In addition, ideally the scaffold should be continuously degraded and replaced by host cells. Extracellular matrix (ECM) molecules such as collagen are suitable scaffold materials. However, synthetic degradable plastics such as polylactic acid (PLA), polycaprolactone (PCL) and copolymers thereof have the advantage of being capable of accurate processing and being more economical and clinically widely available than naturally occurring materials.

However, since synthetic biopolymers have low bone bioactivity, high hydrophobicity, low affinity for bioproteins and cells, synthetic biopolymer scaffolds need to be optimized for bone tissue engineering. Thus, several attempts have been made, such as surface modification with hydrophilic materials to increase bioactivity. The adhesion protein covalently bonds to the surface of the synthetic polymer, increasing initial cell adhesion. Such adhesive proteins include collagen or fibronectin. However, linking the adhesion protein to the scaffold does not provide adequate surface conditions for stem cells to differentiate into osteoblasts to form functional bone tissue. Composite scaffolds consisting of PCL and bioactive ceramics such as hydroxyapatite (HA) or tricalcium phosphate (TCP) have increased biological capacity in bone regeneration, such as improved cell adhesion, differentiation of osteoblasts, and bone formation. Shows. Thus, although complex scaffolds of bioactive ceramics and PCL are preferred for bone tissue engineering, this may not be preferred for early cell responses and undifferentiated stem cells. Ideally, the biomimetic environment is suitable for both early stem cell adhesion and growth, and for continuous differentiation to mature into the phenotype of osteoblasts.

The present inventors have studied scaffolds that are suitable for both the attachment and continuous bone formation of early mesenchymal stem cells, and when the binding protein of fibronectin-osteocalcin is linked to a porous scaffold mineralized with hydroxyapatite, The present invention was completed by confirming that adhesion is increased and bone formation is promoted.

The present invention provides a scaffold for bone regeneration comprising hydroxyapatite coated with a polymer scaffold surface and fibronectin-osteocalcin fusion protein bonded to hydroxyapatite.

The present invention also provides a process for preparing a polymer scaffold, b) coating a polymer scaffold with hydroxyapatite, c) synthesizing a fibronectin-osteocalcin fusion protein, and d) fibronectin-osteocalcin. It provides a method for producing a scaffold comprising coating with a fusion protein.

In order to solve the above problems, the present invention provides a scaffold for bone regeneration comprising a polymer scaffold, a hydroxyapatite coated on the surface of the polymer scaffold, and a fibronectin-osteocalcin fusion protein combined with hydroxyapatite.

The material of the polymeric scaffold should have biodegradability that can be maintained until the scaffold fully functions and serves and then completely degraded in vivo. Accordingly, preferred biodegradable polymers include polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), and polycaprolactone (PCL). desirable.

The fibronectin-osteocalcin fusion protein can bind to hydroxyapatite and promote integrin mediated cell adhesion. In the present invention, "fibronectin" is a protein having a molecular weight of about 220 kDa, has an amino acid sequence of SEQ ID NO: 4, includes several motifs including an RGD motif, and works with various integrins to regulate cell function. Means. Among various motifs of fibronectin that interact with integrin, a cell surface adhesion receptor, RGD is present in the ninth to tenth type III repeat units as cell adhesion motifs. Fibronectin domains of the present invention are the ninth to tenth type III repeat units of fibronectin and comprise the RGD sequence. Preferably, the fibronectin domain may have an amino acid sequence of SEQ ID NO: 1.

In the present invention, "osteocalcin" includes 2-3 gamma-carboxylglutamate (Gla) residues, and is a vitamin K-dependent calcium-binding non-collagenic protein having a molecular weight of about 5,900 Da. It means a protein having an amino acid sequence. Osteocalcin in the bone plays an important physiological role in the metabolism of bone mineralization, such as the local adjustment or control the movement of the bones and Ca ^{2 +} between fluids. Osteocalcin is synthesized in osteoblasts and then glazed by vitamin K dependent carboxylase in the cells. Gladed Osteocalcin (Gla-OC) binds to hydroxyapatite (HA) in bone and accumulates in bone matrix and is involved in bone formation. Unglazed Osteocalcin (Glu-OC) has affinity with bone matrix. It is weak and released into the blood, which is an indicator of bone resorption. Osteocalcin has a high affinity for hydroxyapatite, and the osteocalcin domain of the present invention may comprise 18-95 amino acid sites that bind hydroxyapatite (HA). Preferably, the osteocalcin domain may have an amino acid sequence of SEQ ID NO: 2.

The fusion protein binds to hydroxyapatite (HA) to promote integrin mediated cell adhesion, more specifically to promote osteoblast adhesion and maintenance on tooth and bone mineral components, and to promote osteoblast differentiation. . The fusion protein of the present invention may be a structure in which the osteocalcin domain is connected to the C terminus of the fibronectin domain or a structure in which the osteocalcin domain is connected to the N terminus of the fibronectin domain. Preferably, it has the amino acid sequence of SEQ ID NO. Representative examples of the fibronectin-osteocalcin fusion protein are described in Korean Patent Publication No. 10-2012-0057429, which is incorporated by reference of the present invention.

In addition, the present invention to solve the above problem is a) preparing a polymer scaffold, b) coating the polymer scaffold with hydroxyapatite, c) synthesizing the fibronectin- osteocalcin fusion protein, d) a polymer Provided are methods of making a scaffold comprising coating the scaffold with a fibronectin-osteocalcin fusion protein.

Step a) is a step of preparing a polymer scaffold. In this case, the polymer scaffold used may be made of polyglyrrolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), and the like. Most preferably, the resulting polymer scaffold has a three dimensional structure with pores of controlled size and arrangement.

Step b) is a step of coating the polymer scaffold with hydroxyapatite. The coating increases the adhesion between the polymer scaffold and the fibronectin-osteocalcin fusion protein.

Step b) comprises a) alternately immersing the scaffold in CaCl ₂ and Na ₂ HPO ₄ solution, and b) treating the mineralization solution at 35 ° C. for 1 to 10 days.

Step a) is a step of alternately immersing the scaffold in CaCl ₂ and Na ₂ HPO ₄ solution to induce CaP nucleation. At this time, prior to immersion in CaCl ₂ and Na ₂ HPO ₄ solution it is preferable to activate by immersion in NaOH gently stirred. Wash thoroughly with distilled water to remove the remaining residue and alternately immerse the scaffold in CaCl ₂ and Na ₂ HPO ₄ solution. At this time, the concentration of the CaCl ₂ and NaHPO ₄ solution is preferably 10 mM to 1000 mM.

Step b) is a step of forming hydroxyapatite on the polymer scaffold by treating the polymer scaffold in the mineralization solution.

Mineralization solution to be used this time is ^{Na +, K +, Mg 2} +, Ca 2 +, Cl - is similar body fluid containing _{^{-, HCO 3 -, HPO 4}} 2 -, SO 4 2. Preferably, the mineralization solution ^{Na + 213.0 mM, K + 7.5} mM, Mg 2 + 2.25 mM, Ca 2 + 3.75 mM, Cl - 221.7 mM, HCO 3 - 6.3 mM, HPO 4 2- 1.5 mM and SO ₄ is similar to the body fluid of 1.5mM, including ^2- 0.75 mM.

The temperature of the mineralization solution of step b) is preferably 35 ° C., and the immersion time is 1 day to 10 days. If the temperature of the mineralization solution is lower or higher than 35 ° C., the temperature difference from the body temperature is large and thus does not provide a suitable environment for the reaction to occur.

Step c) is a step of synthesizing the fibronectin-osteocalcin fusion protein. In this case, the fusion protein including the fibronectin domain and the osteocalcin domain may be prepared by a chemical peptide synthesis method known in the art, or the genes encoding the respective domains may be amplified by PCR (polymerase chain reaction) or synthesized by a known method. By linking in frame (in frame) it can be expressed by operably linked and cloned in the expression vector.

The expression vector is a recombinant vector capable of expressing a protein of interest in a suitable host cell, and refers to a gene construct containing essential regulatory elements operatively linked to express a gene insert. Expression vectors of the present invention include expression control elements such as promoters, operators, initiation codons, polyadenylation signals, and enhancers as elements generally possessed by suitable expression vectors. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

As the expression vector, all conventional expression vectors can be used. Depending on the host cell, the expression level and expression of the protein are different, so select the most suitable host cell for the purpose. In an embodiment of the present invention, pBAC / His-FNIII9-10 / OC was prepared as an expression vector containing a gene encoding the fusion protein of the present invention.

The vector of the present invention may further comprise a protein tag, which can be optionally removed using an endopeptidase, to facilitate detection of the fusion protein. As used herein, the term "tag" refers to a molecule exhibiting quantifiable activity or properties, and refers to a polypeptide fluorescent substance such as a chemical fluorescent substance (fluoracer), a fluorescent protein (GFP) or a related protein such as fluorescein. It may be a fluorescent molecule included, or may be an epitope tag such as a Myc tag, a flag tag, a histidine tag, a leucine tag, an IgG tag, and a straptavidin tag. Suitable tag polypeptides generally have 6 or more amino acid residues and typically have about 8 to 50 amino acid residues, wherein 6 histidine tags were used in the present invention.

Step d) is a step of connecting the fibronectin-osteocalcin fusion polymer to the polymer scaffold by immersing the scaffold in a fibronectin-osteocalcin solution in PBS (phosphate buffered saline). At this time, the concentration of fibronectin-osteocalcin in the PBS solution is preferably 100 ng / ml to 100 µg / ml.

In addition, the immersion time in the solution of the scaffold is preferably 8 hours to 24 hours. If the immersion time is shorter than 8 hours, a sufficient amount of fibronectin-osteocalcin fusion polymer will not bind to the scaffold, and if the immersion time is longer than 24 hours, it will already be saturated and no more fibronectin-osteocalcin fusion polymer will bind to the scaffold. It is not efficient.

The present invention provides a scaffold for bone regeneration comprising hydroxyapatite coated with a polymer scaffold surface and fibronectin-osteocalcin fusion protein coupled to hydroxyapatite and a method for preparing the same, which promotes adhesion and maintenance of osteoblasts. In addition, it can be usefully applied to bone tissue engineering and regenerative medicine by improving osteoblast differentiation.

1 is a diagram schematically showing the structure of a PCL scaffold according to the present invention.
FIG. 2 is a graph of signal strength showing the amount of FN-OCN fusion protein bound to the PCL scaffold over immersion time.
3 is a graph showing the degree of attachment of MSCs to PCL scaffolds to which FN-OCN fusion proteins are linked.
Figure 4 shows the mRNA expression of the osteogenic genes of each marker OPN, OCN, BSP in the FN-OCN-bound scaffold.

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 to further understand the present invention, and the present invention is not limited by the examples.

Example  1. the rat bone marrow Intermediate lobe  Stem Cells( MSCs Isolation and culture

MSCs were isolated from the tibia and femur bone marrow of Sprague-Dawley rats. All protocols involving animals were conducted in accordance with the guidelines of the Animal Ethics Committee of Dankook University. The bone marrow of tibia and femoral call was extracted from Hank's Balanced Salt Solution (HBBS) containing 0.1% of Type I collagen and 0.2% of Dispase II. Mononuclear cells were obtained using a centrifuge and enzyme solution at 1500 rpm. Cells were placed in two parallel culture dishes at a concentration of 2 × 10 ³ / cm ² (one for proteum analysis and one for culture). Supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan UT), 2 mM l-glutamine, 100 U / mL penicillin, and 100 mL / mL streptomycin (all purchased from Sigma-Aldrich, St. Louis) In a general growth medium consisting of a-Minimal Essential Medium (a-MEM; WelGene, DaeGu, Korea), incubated for 7 days in a humid air containing a temperature of 37 ℃, 5% CO ₂ . In the experiment, cells passaged 2-3 times were used.

Example  2. PCL Scaffold Robot type  Spraying and HA Mineralization .

PCL (MW = 80,000, Sigma-Aldrich) was dissolved in acetone at 50 ° C. to prepare a PCL solution (PCL / acetone = 25 wt.%). A three-dimensional PCL scaffold was prepared using an Ez-ROBO3 robocasting machine (Iwashita, Japan). Specifically, the solution was added to the syringe and maintained at 50 ° C. using a heating jacket. Thereafter, the solution was injected into a metal nozzle using a force-controlled plunger to control the mass flow rate. Partially controlled units operate to induce a two-dimensional fiber dispersion path with constant velocity. 2D fibers were laminated in multiple layers to create 3D porous scaffolds with controlled pore size and geometry. The fibers were then immersed in cold ethanol to effectively solidify the PCL solution. Thereafter, the scaffold was heat treated at 50 ° C., and the fibers were contact fused. .

For mineralization of the scaffold, the gently stirred 1N NaOH scaffold was immersed to activate the surface and washed with distilled water. Thereafter, the scaffolds were alternately replaced with 150 mM CaCl ₂ and 150 mM Na ₂ HPO _4. Soaking in solution induced calcium phosphate nucleation. Between the alternating dipping procedures, the scaffolds were cleaned thoroughly. Mineralization to scaffold the CaP nucleus resulting solution (1.5 x simulated body fluid, 1.5 mM SBF (213.0 mM Na +, 7.5 mM K +, 2.25 mM Mg 2 +, 3.75 mM Ca 2 +, 221.7 mM Cl -, 6.3 was further treated at 37 ° C. for up to 7 days with mM HCO ₃ ⁻ , 1.5 mM HPO ₄ ^2- and 0.75 mM SO ₄ ^2- ).

Example  3. FN - OCN  Construction, Expression and Purification of Fusion Proteins

PCR primers were constructed as follows to recognize the ninth to tenth type III domains of human fibronectin (FNIII9-10) containing the specific cell binding sequence RGD. FNIII9-10 forward primer, 5'-CTCGAGCAACAATCAACAGTTTC-3 '(introduced SacI restriction site before N-terminus of FNIII9-10), FNIII9-10 reverse primer, 5'-CTCGAGTGGTTTGTCAATTTC-3' (COOH-terminus of FNIII9-10 SacⅠ Restricted Sites). PCR was performed with 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 100 μg / ml gelatin, 0.2 mM dNTPs, 1.25 units of taq polymerase (iNtRON) and 50 pmol each of reverse and forward primers. It was performed in 30 μl reaction solution. PCR was performed by annealing at 55 ° C. for 1 minute, extending at 72 ° C. for 1 minute, and denaturing at 94 ° C. for 1 minute. After 30 repetitions, the amplified PCR product was digested with restriction enzyme SacI. After cleavage, the PCR product was linked to the SacI position of the pBAD / His-OC vector to generate the construct pBAD / His-FNIII9-10 / OC.

For expression of recombinant FNIII9-10 / OC, Escherichia 　 coli TOP10 cells were incubated overnight at 37 ° C. in LB-Amp medium. When A600 = 0.6, transcription induction was initiated with 0.02% (w / v) of the transcription inducer L-arabinose. After 3 hours, the bacteria were pelleted by centrifugation, lysed and sonicated. The dissolved extracts were prepared by centrifugation in a refrigerated centrifuge at 14,000 × g for 30 minutes, and the supernatant was transferred to a new tube.

The crude protein separated from the bacterial supernatant sonicated with a nickel-nitrilotriacetic acid (Ni-NTA) resin column (Invitrogen, Carlsbad, Calif.) Was purified by binding a hexahistidine tag (located at the amino terminus of the protein). The degree of purification of the recombinant protein was confirmed by Coomassie bule staining of the dissolved protein by 10% (v / v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions.

Example  4. Mineralized PCL On the scaffold FN - OCN  Fusion protein linkage

The mineral HA phase produced on the surface of the PCL scaffold has a high binding affinity for OCN. Therefore, as shown in FIG. 1, FN-OCN was linked to the mineralized PCL scaffold. To determine the optimal coating time, FN-OCN solution was prepared at a concentration of 1 μg / ml in phosphate buffered saline (PBS). PCL-HA scaffolds were immersed in the solution for 2, 4, 8 and 16 hours at room temperature, respectively. Each hour the samples were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 5 minutes. By immunolabelling, the amount of FN-OCN bound to the surface was measured. Scaffolds were incubated with rabbit anti-OCN antibody for 1 hour at room temperature. After washing several times with PBS, a second antibody, goat anti-rabbit Rhod labeled antibody was applied to generate the fluorescent signal. The labeling intensity of FN-OCN coated PCL-HA scaffolds was measured using an LSM700 confocal microscope (Carl Zeiss, Germany). The relative amount of FN-OCN coated on the PCL-HA surface was calculated as the intensity of the red fluorescence signal. As a result, it was found that the intensity of fluorescence intensity was saturated when coated for 8 hours or more (FIG. 2).

Example  5. Intermediate lobe  Stem Cells( MSCs Evaluation of adhesion

Cultured MSCs were harvested by trypsinization, washed and re-suspended in standard growth media at a concentration of 1 × 10 ⁵ / ml. 100 μl portions of cell containing media were mounted in each scaffold and incubated for various times to adhere the cells. In order to calculate the amount of cells that successfully adhered to the scaffold, all the cells that did not adhere to each time were collected and counted using a hemocytometer. The amount of adhered cells was judged as the inverse of the amount of non-bonded cells.

As a result, it was confirmed that the amount of MSC contact was higher than that of the scaffold to which the FN-OCN-coupled scaffold was not bound, and after 8 hours, about 80 to 90% of the cells were attached to the scaffold (FIG. 3).

Example  6. Gene Expression RT - PCR  analysis

After incubation for 21 days, cells were harvested by trypsinization, washed, pelleted by centrifugation, and stored at -80 ° C until use. Total RNA was isolated from each cell pellet using RNeasy mini kit 74104 RNA isolation kit (Qiagen, Valencia, CA). RNA samples (1 μg) were reversely transcribed into cDNA in 40 μl reaction using Quantitect RT kit (Qiagen) according to the manufacturer's protocol. The reaction proceeded at 95 ° C. for 5 minutes. Similar reaction mixtures containing no transcribed enzyme were prepared and used as samples to show no contaminated genomic DNA. Microliter cDNA was PCR amplified using specific primers of mouse osteopontin and osteocalcin and a pre-mixed PCR kit (Bioneer, Daejon, Korea). The PCR reaction was repeated 40 times, 30 seconds at 95 ° C., 30 seconds at 55 ° C., 30 seconds at 75 ° C., and repeated three times for each cDNA. Representative PCR bands of each sample are shown. (Figure 4)

Attach an electronic file to a sequence list

Claims (16)

a) preparing a polymer scaffold;
b) coating the polymer scaffold with hydroxyapatite;
c) synthesizing the fibronectin-osteocalcin fusion protein; And
d) coating the polymer scaffold coated with hydroxyapatite with fibronectin-osteocalcin fusion protein,
Here, the coating step of step b)
A) alternately immersing the polymer scaffold in CaCl ₂ solution and Na ₂ HPO ₄ solution; And
B) treatment with mineralization solution at 35 ° C. for 1 day to 10 days,
Method of making scaffolds.
The method of claim 1, wherein the concentration of CaCl ₂ solution and Na ₂ HPO ₄ solution of step a) is 10 mM to 1000 mM, respectively.
The method according to claim 1, wherein the mineralization solution of step b) is an analog solution comprising Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ , Cl ⁻ , HCO ₃ ⁻ , HPO ₄ ^2- and SO ₄ ^2- . Manufacturing method.
4. The method of claim 3 wherein the mineralization solution ^{Na + 213.0 mM, K + 7.5} mM, Mg 2+ 2.25 mM, Ca 2+ 3.75 mM, Cl - 221.7 mM, HCO 3 - 6.3 mM, HPO 4 2- 1.5 mM And SO ₄ ^2- 0.75 mM analogue solution of 1.5 mM.
The method of claim 1, wherein step d) comprises coating the scaffold by dipping the fibronectin-osteocalcin solution in phosphate buffered saline (PBS).
The method of claim 5, wherein the concentration of fibronectin-osteocalcin solution is between 100 ng / ml and 100 μg / ml.
The method of claim 5, wherein the immersion time is 8 hours to 24 hours. delete delete delete delete delete delete delete delete delete

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