KR102067211B1 - Porous bone substitutes comprising dialdehyde starch and collagen, and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a porous bone graft and a method for producing the same, provides a porous bone graft comprising a polymer complex of dialdehyde starch, collagen, and heparin, and a method for producing the porous bone graft.

Description

Porous bone substitutes comprising dialdehyde starch and collagen, and method for manufacturing the same

A porous bone graft comprising dialdehyde starch and collagen, and a method of making the same.

Bone tissue consists of a complex of organic and inorganic compounds. The organic compound is composed of collagen, proteoglycan, and the like, and collagen type 1 is the main component of 90% of the organic component. The main component of the inorganic component is hydroxyapatite, an inorganic compound of the calcium phosphate family. Bone tissue is composed of cortical bone, cancellous bone, periosteum, and endosteum. Cortical bones make up the outer surface of bone, protect bone tissue, and make up 85% of bone tissue. The inside of the bone tissue is composed of a highly porous reticular bone, which accounts for about 15% of the bone tissue. Bone tissue composed of an organic-inorganic complex has a hard and flexible mechanical property, and serves to store or quickly provide calcium, which is involved in various metabolisms occurring in the human body. Thus, bone tissue not only performs structural functions of the human body, but also plays an important role in biological function. Nevertheless, the study of bone tissue is less than that of other living tissues.

Meanwhile, various types of bone graft materials for replacing and regenerating damaged bone tissue have been studied. Implants to replace damaged bone tissue should be designed to facilitate the biological functions performed by bone tissue, along with their morphological and mechanical roles. In addition, it should be excellent in biocompatibility, and should not cause toxicity, carcinogenicity, allergy, inflammation, foreign body reaction, etc., and at the same time, it should be excellent in osteointegration and osteoconduction. Materials used as bone grafts can be classified into autograft, allograft, xenograft, and synthetic material. Autologous bones have problems of secondary surgery and limitation of supply, and allogeneic bone and xenograft have risks of inducing immune response or infection. Synthetic bone can be manufactured and mass-produced in various forms and components, and has the advantage of minimizing the impact on the body. In the study of substitute material of bone tissue, metals and metal alloys were mainly used for the purpose of replacing high strength of bone tissue, but recently, in consideration of the biocompatibility and biodegradability of materials, polymers or ceramics In particular, research on composite materials has been actively conducted to simulate the unique mechanical properties of bone tissue (Korean Patent Registration 10-0957546).

Under this background, the present inventors have experimentally confirmed the excellent efficacy of dialdehyde starch and collagen-based bone grafts based on the present invention to develop a bone graft derived from a polymer having excellent biocompatibility and strength. Was completed.

One aspect includes mixing a dialdehyde starch solution, a collagen solution, and a heparin solution to prepare a mixed solution; Neutralizing the mixed solution to form a gel; And it provides a method for producing a porous bone graft, comprising the step of lyophilizing the gel formed.

Another aspect is to provide a porous bone graft produced by the method.

Other objects and advantages of the present application will become apparent from the following detailed description taken in conjunction with the appended claims. Content not described herein is omitted because it can be sufficiently recognized and inferred by those skilled in the art or similar technical field of the present application.

One aspect includes mixing a dialdehyde starch solution, a collagen solution, and a heparin solution; Neutralizing the mixed solution to form a gel; And it provides a method for producing a porous bone graft, comprising the step of lyophilizing the gel formed.

The method for producing the porous bone graft is described in detail for each step, as follows.

First, the method may include mixing a dialdehyde starch solution, a collagen solution, and a heparin solution to prepare a mixed solution.

In one embodiment, the step of mixing may be to mix the dialdehyde starch solution, collagen solution, and heparin solution simultaneously or sequentially, for example, by mixing the dialdehyde starch solution and the collagen solution Preparing a dialdehyde starch-collagen mixed solution; And adding and mixing the heparin solution to the prepared dialdehyde starch-collagen mixed solution.

In one embodiment, the concentration of the dialdehyde starch solution may be 12 to 30% (w / v). If the concentration of the dialdehyde starch in the solution is too low outside the above range, the mechanical strength may be too weak or the manufactured bone graft may shrink over time, on the contrary, if the concentration of the dialdehyde starch is too high, the dialdehyde Starch may not be homogeneously dissolved and may have difficulty molding. For example, the concentration of the dialdehyde starch solution is 12 to 25% (w / v), 12 to 20% (w / v), 12 to 15% (w / v), 14 to 30% (w / v). ), 14 to 25% (w / v), 14 to 20% (w / v), 14 to 15% (w / v), 16 to 30% (w / v), 16 to 25% (w / v) ), 16 to 20% (w / v), 18 to 30% (w / v), 18 to 25% (w / v), or 18 to 20% (w / v), but is not limited thereto. no.

In one embodiment, the concentration of the collagen solution may be 5 to 10% (w / v). If the concentration of collagen in the solution out of the above range is too low, the biocompatibility may be lowered, on the contrary, if the concentration of collagen is too high, it may not dissolve homogeneously or the bone graft produced may shrink over time. . For example, the concentration of the collagen solution may be 5 to 9% (w / v), 5 to 8% (w / v), 5 to 7% (w / v), 5 to 6% (w / v), 6 to 10% (w / v), 6 to 9% (w / v), 6 to 8% (w / v), 6 to 7% (w / v), 7 to 10% (w / v), 7 to Number of 9% (w / v), 7-8% (w / v), 8-10% (w / v), 8-9% (w / v), or 9-10% (w / v) However, it is not limited thereto.

  The dialdehyde starch and collagen will form a polymer complex through a cross-linking reaction to be carried out later, according to the formation of these complexes may serve as a structural support which is a major function of bone tissue. The volume ratio of the mixed dialdehyde starch solution and collagen solution may be 1: 6 to 1:12, for example, 1: 6 to 1:10, 1: 6 to 1: 8, 1: 8 to 1: 12, 1: 8 to 1:10, or 1:10 to 1:12, but is not limited thereto.

In one embodiment, the concentration of the heparin solution may be 0.01 to 1% (w / v), for example, 0.01 to 0.5% (w / v), 0.01 to 0.1% (w / v), 0.05 To 1% (w / v), 0.05 to 0.5% (w / v), 0.05 to 0.1% (w / v), 0.1 to 1% (w / v), or 0.1 to 0.5% (w / v) days May be, but is not limited thereto. The heparin is distributed in the bone graft material, thereby enabling binding or adhesion between the growth factor and the bone graft material provided from the outside or existing in vivo, and thereby may play a role in improving osteoadhesion and bone conduction. According to one embodiment, the addition of heparin salt in solution form, for example, heparin sodium salt, was able to homogeneously distribute heparin in bone grafts compared to the use of heparin in powder form, thereby improving efficacy as a bone graft. You can.

On the other hand, the method may further comprise the step of supporting the porous bone graft in a solution containing a growth factor.

In one embodiment, the step is to induce binding between heparin and growth factors on the surface of the porous bone graft by immersing the porous bone graft in a solution containing growth factors, and then storing or leaving the solution under specific conditions. Can be. For example, by leaving the solution for 6 to 24 hours at a temperature of 2 to 10 ℃, it may be to induce binding between heparin and growth factors on the surface of the porous bone graft, but is not limited thereto.

The growth factors include, for example, bone morphogenic protein (BMP), platelet-derived growth factor (PDGF), transgenic growth factor (TGF-beta), insulin-like growth factor (IGF-I), IGF-II, and FGF. (Fibroblast growth factor), BGDF-II (beta-2-microglobulin), osteocalcin, boneloloprotein (bonesialo protein), and osteogenin (osteogenin), and the like can be used, for example, BMP2, BMP4, BMP7 / PDGF-AA, PDGF-BB, PDGF-AB, FGF-1, FGF-2, FGF-4, FGF-7, FGF-9, FGF-18, FGF-20, FGF-22, FGF- 23, the listing factor is harmless to the human body and can be used without limitation as long as it promotes bone growth.

Thereafter, the method may include neutralizing the mixed solution to form a gel.

In one embodiment, the step of forming the gel may include adding a neutralizing solution to the mixed solution, the neutralizing solution may be a basic solution, ammonia, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide , Magnesium hydroxide, zinc hydroxide, cesium hydroxide, barium hydroxide, rubidium hydroxide, ferrous hydroxide, ferric hydroxide, aluminum hydroxide, methylamine, ethylamine, n-propylamine, n-butylamine, calcium carbonate, potassium bicarbonate , Sodium carbonate and sodium bicarbonate solution may be at least one selected from the group consisting of, for example, may be a solution containing sodium hydroxide. Here, the concentration of sodium hydroxide in the solution may be 0.5 to 1N. For example, the concentration may be 0.5 to 0.9 N, 0.5 to 0.8 N, 0.5 to 0.7 N, 0.5 to 0.6 N, 0.6 to 1 N, 0.6 to 0.9 N, 0.6 to 0.8 N, 0.6 to 0.7 N, but is not limited thereto. It doesn't happen.

In one embodiment, the step of forming the gel may be performed for 1 to 10 minutes at 30 to 37 ℃ conditions. For example, the temperature conditions are 30 to 35 ° C, 30 to 33 ° C, 30 to 31 ° C, 32 to 37 ° C, 32 to 35 ° C, 32 to 33 ° C, 34 to 37 ° C, 34 to 35 ° C, or 36 to It may be carried out at 37 ℃ conditions, crosslinking reaction time is 1 to 9 minutes, 1 to 8 minutes, 1 to 7 minutes, 1 to 6 minutes, 1 to 5 minutes, 1 to 4 minutes, 1 to 3 minutes, or It may be 1 to 2 minutes, but is not limited thereto.

In the step of forming a gel according to an embodiment, the concentration of the neutralization solution and / or the crosslinking reaction time may affect the strength and the porous structure of the bone graft.

In one embodiment, the step of forming the gel induces a cross linking reaction, wherein the collagen may form an imine bond with dialdehyde starch, and the heparin may form an amide bond with collagen. .

Specifically, the collagen is cross-linked by dialdehyde starch to form an imine bond (Schiff's base reaction). Such addition of dialdehyde starch to collagen is converted into C = N group by Schiff's base formation between the -amino group of the lysine or hydroxylysine side chain of collagen and the group of dialdehyde starch to form a crosslink. By the above reaction, the collagen is crosslinked with each other by dialdehyde starch and imine bond, and the crosslinking with collagen and dialdehyde starch may impart excellent mechanical strength and physical properties as described above.

In addition, the heparin is interconnected by collagen and amide bonds to form a COL-DAS-HEP complex, crosslinking with the heparin may contribute to improving the binding to growth factors as described above.

Meanwhile, before or after forming the gel, the method may further include shaping the bone tissue according to the shape of the damaged bone tissue. As an example, the mixed solution or gel may be injected into a syringe to which an extruder is connected, and then a molded body may be manufactured by applying pressure to the extruder based on a three-dimensional rapid molding method, wherein the extruder uses a piston or screw method. To extrude by applying pressure to the material, but is not limited thereto. As another example, the molded body may be manufactured using a three-dimensional printing method known in the art. At this time, the detailed conditions, such as the nozzle, the radial shape of the three-dimensional printer can be changed according to the shape and area of the damaged bone.

Thereafter, the method may comprise freeze drying the formed gel.

In one embodiment, the freeze-drying step may be to freeze-dried after adding glycerol to the formed gel.

According to one embodiment, the addition or use of glycerol in the freeze-drying step may impart physical properties to the bone graft material, such as physical properties, such as suture potential, suitable for bone graft surgery. In addition, the step may be carried out by freezing the gel formed at -70 to -90 ℃ and dried for 2 to 5 days, it is possible to change the enemy.

Another aspect provides a porous bone graft produced by the method.

Among the terms or elements mentioned in the following porous bone grafts, those mentioned in the description of the method of making the same are as described above.

According to one embodiment, the bone graft material containing a dialdehyde starch and collagen in a specific range of concentration, it was possible to mimic the strength and porosity similar to the bone tissue structure, by mixing heparin in the form of a solution with growth factors The adhesion of was significantly improved. In addition, the addition of glycerol in the freeze-drying process has made it possible to provide a bone graft material suitable for bone graft surgery. Therefore, the bone graft can be usefully used as a substitute for damaged bone tissue.

The bone graft includes a porous structure, which provides space for cells to attach and proliferate, facilitates the flow of nutrients and metabolic waste, and has sufficient surface area for cell growth and surface chemistry suitable for cell adhesion. Make sure

In one embodiment, the bone graft comprises 4-8% (w / v) collagen, 0.05-0.15% (w / v) heparin, and 1-2% (w / v) dialdehyde starch. It may be.

In one embodiment, the bone graft may be a homogeneous distribution of heparin. The homogeneous distribution of heparin may promote binding between growth factors and bone grafts, thereby contributing to improving osteoadhesion and bone conduction.

In one embodiment, the bone graft may further comprise the growth factor described above. In addition, various materials may be added to increase the moldability of the bone graft. That is, a linear material, a tubular material, a particulate material, an amorphous material, and the like, which are excellent in biocompatibility, may be further added to improve physical properties of the support for bone regeneration. Materials of this type may be selected from materials such as carboxymethylcellulose (CMC) or animal-derived gelatin, and may be applied without limitation, as long as it does not cause an immune response to the transplant target.

According to the method for producing a porous bone graft according to one aspect, by using a dialdehyde starch solution and a collagen solution having a specific content, it is possible to produce a porous bone graft with excellent biocompatibility and strength, as well as at room temperature The advantage is that mass production is possible in a short time.

According to the porous bone graft material according to one aspect, having mechanical properties as a bone graft material, that is, excellent strength and porosity, and excellent binding to the growth factor, it can effectively replace damaged bone tissue.

1 is a view schematically showing a manufacturing process of a porous bone graft material according to an embodiment.
2 is a view illustrating a process of forming and extruding in the manufacturing process of the porous bone graft material according to an embodiment.
Figure 3 is a porous bone graft according to an embodiment, the strength change of the bone graft material according to the concentration of dialdehyde starch visually confirmed, Figure 3A is 6% Col-10% DAS and 6% Col-20 The results of observing the% DAS bone graft, and Figure 3 B is the result of observing 8% Col-10% DAS and 8% Col-20% DAS bone graft.
4 is a result of confirming the porosity change of the bone graft material according to the concentration of dialdehyde starch in the porous bone graft material according to an embodiment through visual and microscope.
Figure 5 is a porous bone graft according to an embodiment, confirming the change in physical properties of the bone graft material according to the concentration of sodium hydroxide in the reconstitution solution, Figure 5A is a result of comparing the strength of the bone graft material, Figure 5 B is the result of comparing crosslinking reaction time.
Figure 6 is a porous bone graft according to an embodiment, heparin distributed in the bone graft was confirmed through toluene blue staining, Figure 6 A is a result of confirming the distribution of heparin in the bone graft prepared using heparin powder, 6B is a result of confirming the distribution of heparin in the bone graft prepared by using the heparin solution.
7 is a result of confirming the effect of the treatment process on the suture to the bone graft when lyophilized without immersion or pre-treatment in glycerol in the manufacturing process of the porous bone graft according to one embodiment.
8 is a result of confirming the change in proteoglycan production capacity of the cell according to the addition of the growth factor.
Figure 9 is a porous bone graft according to one embodiment, confirming the change in the mobility of the cells according to the addition of growth factors, Figure 9A is a schematic diagram for the present experiment, Figure 9B is DAS- The result of microscopic observation of the area where the bone graft containing COL-HEP and PDGF-BB is located, C of FIG. 9 is the result of microscopic observation of the area where the bone graft containing DAS-COL-HEP is located. .
10 is a result of confirming the improvement of efficacy according to the addition of the growth factor in the porous bone graft according to one embodiment.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Porous Bone Grafts

1-1. Experimental material

Dialdehyde starch (C ₉ H ₁₆ O ₄ , molecular weight: 188.223 g / mol, DAS) was obtained from MONOMER-POLYMER AND DAJAC LABS (USA), type 1 or type 2 collagen (COL), heparin sodium salt ( H3129, HEP), and acetic acid were obtained from Sigma-Aldrich. 10 × DPBS (10 × Dulbecco's Phosphate-Buffered Salines) was obtained from ThermoFisher Scientific, phenol red solution, sodium hydrogencarbonate (NaHCO ₃ ), HEPES sodium salt (4- (2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt), and sodium hydroxide were obtained from Sigma-Aldrich.

1-2. Experiment preparation

The DAS was dissolved in 10 × DPBS to prepare a 20% (w / v) DAS solution, which proceeded with stirring at 500-700 rpm on a heating block at 90 ° C.

8% (w / v) collagen was added to 1 mM acetic acid solution to prepare an 8% (w / v) COL solution, the dissolution process proceeded with stirring at 700 rpm. By way of example, to prepare 3 ml of COL solution, 240 mg of collagen powder was added to 1 mM acetic acid solution (2.7 ml).

HEP solution was prepared by dissolving 0.1% (w / v) heparin sodium salt in 10ХDPBS. As an example, 3 mg of heparin powder was dissolved in 375 ul of 10 × DPBS.

A reconstituted solution for pH neutralization was prepared by dissolving 2.2% (w / v) sodium bicarbonate and 200 mM HEPES in 0.75N sodium hydroxide solution. By way of example, 6.6 g of sodium bicarbonate and 15.9 mg of HEPES were dissolved in 0.75 N sodium hydroxide solution to prepare 300 ml of reconstitution solution.

0.5% phenol red was added to 10 × DPBS to prepare a phenol red solution for measuring the change in pH.

1-3. Preparation of Porous Bone Grafts

A process for preparing a porous bone graft according to one embodiment is shown in FIG. 1. First, 3 ml of DAS-COL mixed solution was prepared by mixing 0.3 ml of 20% (w / v) DAS solution and 2.7 ml of 8% COL solution in a volume ratio of 1: 9. Thereafter, a HEP solution was added to the DAS-COL mixed solution to prepare a DAS-COL-HEP mixed solution. Thereafter, after filling the syringe with the DAS-COL-HEP mixed solution, the empty syringe was connected to the filled syringe using a connector, and the DAS-COL-HEP mixture existing in the syringe using a pistol. The solution was mixed several times. To this was added 12.5% (v / v), i.e. 0.375 ml of reconstituted solution and mixed in the same manner as above to neutralize the DAS-COL-HEP mixed solution. Thereafter, the neutralized mixed solution was injected into the mold, and gelled at about 37 ° C. for about 1 to 30 minutes, and then extruded. After immersing the extruded gel in glycerol, it was frozen at -80 ° C and dried for 3 days to prepare a porous bone graft.

Additionally, in order to compare the efficacy with the bone graft material according to one embodiment, conditions for 1) the concentration of DAS and collagen solution, 2) crosslinking time, 3) mode of addition of heparin, and 4) presence of glycerol in the manufacturing process Was modified to prepare a comparative mixed solution or comparative bone graft. On the other hand, when bone grafts were prepared using 6% (w / v) collagen solution, the porous bone graft was 4.8% (w / v) collagen, 0.08% (w / v) heparin, and 1.6% ( w / v) dialdehyde starch. In addition, when bone grafts were prepared using 8% (w / v) collagen solution, the porous bone graft had 6.4% (w / v) collagen, 0.08% (w / v) heparin, and 1.6% ( w / v) dialdehyde starch.

Experimental Example 1. Changes in physical properties of bone graft material according to the concentration of dialdehyde starch

In this experimental example, the physical properties of the bone graft material were examined according to the concentration of dialdehyde starch. To this end, a bone graft (6% Col-20) according to one embodiment prepared by mixing a 6% (w / v) or 8% (w / v) collagen solution and a 20% (w / v) dialdehyde starch solution Bone graft (6% Col-10) prepared by mixing% DAS and 8% Col-20% DAS) with 6% (w / v) or 8% collagen solution and 10% (w / v) dialdehyde starch solution % DAS, and 8% Col-10% DAS) were visually and microscopically observed to confirm their strength and porosity changes.

As a result, as shown in FIG. 3, the strength of the bone graft when 20% (w / v) dialdehyde starch solution was used as compared to the bone graft prepared using the 10% (w / v) dialdehyde starch solution. It was visually confirmed that is significantly improved. In addition, as shown in Figure 4, the increased concentration of dialdehyde starch was found to further improve the porous structure inside the bone graft. That is, the results of these experiments show that by increasing the concentration of dialdehyde starch, not only can a good mechanical strength be inserted into the damaged site to support the structure, but also the network structure that is involved in the biological function of bone, that is, porous It indicates that the structure can be reproduced.

Experimental Example 2 Changes in Physical Properties of Bone Graft Materials with Concentration of Reconstituted Solution

In this experimental example, the physical properties of the bone graft material were examined according to the concentration of sodium hydroxide in the reconstitution solution. Specifically, DAS-COL-HEP mixed solution prepared in the same manner as in Example 1 using 8% (w / v) collagen solution, each containing 0.25N, 0.5N, 0.75N sodium hydroxide By addition of the reconstitution solution, gelation, ie crosslinking reaction was induced. The strength and crosslinking reaction time of the gelled bone grafts were then compared.

As a result, as shown in Figure 5, when the reconstitution solution containing 0.75N (normality) sodium hydroxide was added, the strength of the bone graft significantly improved, wherein the crosslinking reaction time is about 3-4 minutes It was found that a very fast crosslinking reaction proceeded. That is, these experimental results indicate that as the concentration of sodium hydroxide in the reconstitution solution is increased, the strength of the bone graft can be improved and the crosslinking reaction time can be shortened. In addition, as a condition applicable to the production of bone graft material, it was derived that the sodium hydroxide concentration in the reconstitution solution is about 0.75N, the crosslinking reaction time is 3-4 minutes.

Experimental Example 3 Changes in Physical Properties of Bone Grafts by Mixing Heparin Solution and Addition of Glycerol

3-1. Changes in Adhesion to Growth Factors after Mixing with Heparin Solution

In this experimental example, the physical properties of the bone graft material were examined with crosslinking time. Specifically, toluene blue staining was performed on the bone graft prepared in Example 1 to confirm heparin distributed in the bone graft. In the comparative group, a bone graft prepared by adding heparin powder, not heparin solution, to the DAS-COL mixed solution was used.

As a result, as shown in Figure 6, heparin was heterogeneously distributed in the bone graft prepared using heparin powder, while heparin was homogeneously distributed in the bone graft prepared using heparin solution. In other words, these results indicate that the bone graft material according to an embodiment may be more readily distributed with endogenous growth factors because heparin is evenly distributed.

3-2. Changes in Physical Properties of Glycerol Addition in Freeze-Drying Process

In this experimental example, the effect of the addition of glycerol in the freeze-drying process on the physical properties of the bone graft. Specifically, the gelled and neutralized mixed solution of Example 1 was immersed in glycerol or lyophilized without any pretreatment, and then sutured to the bone graft.

As a result, as shown in Figure 7, the freeze-dried bone graft without pretreatment was unable to maintain its structure during the suture process, while the freeze-dried bone graft after immersion in glycerol was confirmed that the suture is properly inserted and fixed. In other words, these results indicate that the use of glycerol in the freeze-drying process confers physical properties in a state suitable for bone graft surgery.

Experimental Example 4. Evaluation of efficacy of bone graft containing growth factor

4-1. Changes in Cell Activity with Growth Factors

Insulin-Transferrin-Sele (ITS), which contains 1 × 10 ⁷ cells / ml of cells in a bone graft prepared using a DAS-COL-HEP mixed solution, contains 50 mg / ml of TGF-beta3. Incubated for 21 days in supplemented DMEM medium. Thereafter, proteoglycans produced by the cells supported on the bone graft were observed through safranin-O staining.

As a result, as shown in FIG. 8, the cells in the bone graft cultured in the medium containing TGF-beta3 showed enhanced production of proteoglycans compared to the control group, indicating that the growth of the cells was more active. it means. In other words, these experimental results indicate that by adding or containing a growth factor in the bone graft material, it is possible to improve the growth and proliferation of cells present in the transplant site.

4-2. Cell Mobility Changes by Growth Factors

Cells were plated in a 10 cm dish, and when cells present in the medium were more than 90% of the area of the medium, cells in the center of the medium were removed with a cell scratch tool (creation of scratched zones). Thereafter, as shown in FIG. 9A, one side of the bone graft containing DAS-COL-HEP and PDGF-BB, and the other side of the scratched glass slide containing the bone graft containing DAS-COL-HEP Located in The slide glass was covered with a cover slip and fixed so as not to move in the flat medium. After 3 days, the number of cells moved to the scratched zone was observed.

As a result, as shown in B and C of FIG. 9, a remarkably large number of cells were observed in the region where the bone graft including DAS-COL-HEP and PDGF-BB is located. In other words, these experimental results indicate that by adding or containing a growth factor to the bone graft material, it is possible to improve the mobility of the cells around the transplant site.

4-3. Evaluation of efficacy of bone graft containing growth factor

The bone graft prepared in Example 1 was immersed in a PBS solution containing growth factor (TGF-beta3), and then left at 4 ° C. for 12 hours. Through this process, by combining heparin and growth factors present on the surface of the bone graft, a bone graft containing growth factors, that is, DAS-COL-HEP was prepared. Thereafter, a 3 mm diameter defect was made in the bovine bone, and then a bone graft containing DAS-COL-HEP and 50 mg / ml TGF-beta3, or a bone graft containing DAS-COL-HEP was filled into the bone defect. Three weeks later, proteoglycans produced by cells present around the region filled with the graft were observed via safranin-O staining.

As a result, as shown in Fig. 10, when the bone graft containing TGF-beta3 was filled, the production of proteoglycans was more enhanced than that of the control group, indicating that the repair of bone defects was more actively performed. will be.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (15)

Dialdehyde starch (DAS) solution at 16-25% (w / v) concentration, collagen (COL) solution at 6-8% (w / v) concentration, and Heparin solution Preparing a mixed solution;
Adding a basic solution of 0.5 to 1N concentration to the mixed solution to form a gel; And
Immersing the formed gel in glycerol and freeze-drying;
The dialdehyde starch solution and the collagen solution is mixed in a volume ratio of 1: 6 to 1:12, a method for producing a porous bone graft. The method of claim 1, wherein the mixing comprises: preparing a dialdehyde starch-collagen mixed solution by mixing the dialdehyde starch solution and the collagen solution; And
Heparin solution by adding to the prepared dialdehyde starch-collagen mixture solution, and mixing. delete delete The method of claim 2, wherein the heparin solution comprises 0.01 to 1% (w / v) heparin or a salt thereof. delete The method of claim 1, wherein the basic solution comprises sodium hydroxide at a concentration of 0.5-1N. The method of claim 1, wherein the forming of the gel is carried out for 1 to 5 minutes at conditions of 30 to 37 ° C. 3. The method of claim 1, wherein the forming of the gel induces a cross linking reaction, wherein the collagen forms an imine bond with dialdehyde starch, and the heparin forms an amide bond with collagen. . delete The method of claim 1, further comprising immersing the porous bone graft in a solution containing growth factors. Porous bone graft prepared by the method of claim 1. delete The porous bone graft of claim 12, wherein the bone graft is a homogeneous distribution of heparin. The porous bone graft of claim 12, wherein the bone graft further comprises a growth factor.

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