KR101686683B1

KR101686683B1 - A preparation method of porous scaffolds of calcium phosphate cement

Info

Publication number: KR101686683B1
Application number: KR1020110070630A
Authority: KR
Inventors: 김해원; 이길수
Original assignee: 단국대학교 산학협력단
Priority date: 2011-07-15
Filing date: 2011-07-15
Publication date: 2016-12-15
Also published as: KR20130009477A; WO2013012132A1

Abstract

The present invention relates to a process for producing a porous scaffold of calcium phosphate cement, and more particularly to a process for producing a porous scaffold of calcium phosphate cement, which comprises preparing a suspension of calcium phosphate cement and alginate and then curing the suspension into a mold filled with a calcium ion aqueous solution, To a method for producing a scaffold.

Description

TECHNICAL FIELD The present invention relates to a porous scaffold for calcium phosphate cement,

Rapidly cured cements are very useful for direct filling or implantable materials for bone tissue regeneration. Calcium phosphate cements (CPCs) are one of the most widely studied bioactive ceramics for this purpose (Brown WE, et al., J Dent Res. , 1983, 62, 672; Brown WE. et al., J Dent Res. , 1986, 63, 200). Although there are some problems to be solved, such as mechanical properties, the introduction of macropores and the control of the rate of degradation, CPCs have been used as an alternative means of treating bone defects since they have many attractive properties (Brown WE et al., J Dent Res. , 1986, 63, 200; Bohner M., J Mater Chem. , 2007, 17, 3980-6). CPCs are useful as injectable materials that are cell and tissue friendly, self-curable and require minimal invasive surgery, and they can also contain therapeutic molecules within the formulation (Planell JA et al., Biomater , 2006, 27 (10), 2171-7; Burger EH. Et al., J Dent Res , 2000, 79, 255).

Scaffolds with a three-dimensional (3-D) porous network provide effective matrix conditions for bone tissue engineering (Barr SK, et al., Biomater , 2009, 30, 2675-82 Biotechnology , 1994, 7, 689-93; Hutmacher DW., Biomater , 2000, 21, 2529-43). Tissue cells are cultured in vitro in a scaffold to better mimic the structure and function of the natural tissue than the material or cell alone (Hutmacher DW., Biomater , 2000, 21, 2529-43; Vacanti JP. Et al., Science , 1993, 260, 920-6). In the in vivo tissue engineering process, controlled release of therapeutic molecules such as growth factors is advantageous in regulating cell function and promoting bone formation. In addition, CPC-based materials have been regarded as excellent candidates for delivery of therapeutic agents delivered within these structures because they self-cure under mild conditions, safely bind therapeutic agents, and maintain a sustained release profile (Planell JA et al., Biomater , 2006, 27 (10), 2171-7). In order to apply CPC-based materials to bone tissue engineering, it is necessary to develop them as 3-D scaffolds that support cell proliferation and cell-material composite structures.

Under these circumstances, the present inventors confirmed that it is possible to prepare a porous scaffold of calcium phosphate cement by preparing a suspension of calcium phosphate cement and alginate, then curing the suspension by injecting the suspension into a mold filled with a calcium ion aqueous solution, Completed.

It is an object of the present invention to provide a method for producing a porous scaffold of calcium phosphate cement.

Another object of the present invention is to provide a kit for manufacturing a porous scaffold of calcium phosphate cement.

In order to solve the above problems, the present invention provides a method for manufacturing a porous scaffold of calcium phosphate cement, comprising the steps of:

1) preparing a suspension of calcium phosphate cement and alginate; And

2) putting the suspension into a mold filled with a calcium ion aqueous solution and curing it.

Preferably, the method further comprises mechanically compressing after step 2).

The term " scaffold " used in the present invention means a structure that plays a role in providing an environment suitable for attachment and differentiation of cells seeded in and out of the structure, and proliferation and differentiation of cells moving from around the tissue. It is one of the fundamental elements in the field.

The step 1 is a step of producing a suspension of calcium phosphate cement and alginate by mixing a calcium phosphate cement in powder form with an alginate solution to prepare a suspension.

The term "cement" used in the present invention means a cured product of a paste obtained by mixing powdery solid and liquid phases. "Curing" of the cement means spontaneous curing of the paste performed at room or body temperature without any artificial treatment. The paste is obtained as a result of mixing the solid phase and the liquid phase.

The term "calcium phosphate cement " used in the present invention means a cement in which the powdery solid phase is composed of a calcium phosphate compound or a mixture of calcium and / or phosphate compounds.

The calcium phosphate cement (CPC) is a material composed of an aqueous solution containing calcium phosphate particles as main components and a substance for promoting hardening such as phosphate. In the course of the treatment, the two components are mixed to form a high viscosity liquid state When applied, the calcium phosphate compound is precipitated and cured by the chemical reaction of the two components at the application site, thereby filling the void space between the damaged bone and bone, or the bone and the implant, thereby fixing and stabilizing the two. .

The mixing ratio of the calcium phosphate cement to the alginate is preferably 20: 1 to 500: 1 based on the weight of the calcium phosphate cement: alginate.

The suspension may further include a biological protein or a drug, and the biological protein may be bovine serum albumin, lysozyme, growth factor and the like. Examples of the drug include antibiotics, anticancer agents, and anti-inflammatory agents.

Step 2 is a step of injecting the suspension into a mold filled with a calcium ion aqueous solution and curing the suspension, and introducing the suspension into a mold filled with an aqueous solution containing calcium ions to induce self curing by curing.

The concentration of the calcium ion is preferably 10 to 200 mM.

The shape of the mold may be cylindrical, hexahedral, or the like, but is not limited thereto.

The calcium phosphate compound may be tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, dicalcium phosphate, hydroxyapatite, or a combination thereof, but is not limited thereto.

Step 3 is a step of mechanically compressing the scaffold having a flexible physical property formed by the self-curing and adjusting the porosity or arbitrarily re-assembling the shape.

The porosity of the scaffold can be arbitrarily adjusted through the compression, and can be controlled by a hand press, which is a machine capable of applying a load.

The porosity can be adjusted to 10 to 90%, preferably 14 to 54%.

The present invention also provides a porous scaffold-producing material comprising calcium phosphate cement powder, an alginate solution, and a calcium ion aqueous solution each packaged in a separate container; A kit for manufacturing a porous scaffold of calcium phosphate cement including a syringe is provided.

The calcium phosphate cement powder packed in each individual container of the kit was mixed with an alginate solution to prepare a suspension. The suspension was poured into a syringe, and the suspension was poured into a mold of a certain type filled with a calcium ion aqueous solution, Folds can be produced on the fly.

The template may be any readily available material, but may be more conveniently used in the manufacture of a scaffold if it further comprises a template for forming scaffolds in the kit.

The kit may further comprise a biological protein or a drug, wherein the biological protein is bovine serum albumin, lysozyme, etc., and the drug may be an antibiotic, an anti-cancer agent, or an anti-inflammatory agent.

In the present invention, a new cell scaffold material prepared by using CPCs and sodium alginate in combination is proposed. Particularly, the composite suspension was directly immersed in the Ca-containing solution to form a fibrous network structure. The immersed suspension rapidly cures to form a gelled network in which the alginate is present and is cross-linked by Ca ^& lt ^; ² ^{+ &} gt ^; ions (Langer R. et al., Curr Top Dev Biol , 2004, 61, 113-34; Asaoka K. et al., Biomater , 1995, 16, 527-32). The cured porous scaffold was cell-friendly and useful for bone tissue engineering. It has also been shown that the scaffold can load and transfer bioactive molecules contained within the structure. The following describes a method for preparing CPC-alginate porous scaffolds. In addition, prior to their application to bone tissue engineering, the in vitro cell response of mesenchymal stem cells (MSCs) from rat bone marrow to the scaffold was investigated. Further, in vivo pilot studies were performed to assess tissue compatibility and the drug delivery potential of the scaffolds was investigated using two different model proteins.

The CPC suspension combined with the alginate solution effectively formed the porous scaffold by direct fibrous deposition into the Ca-containing solution. The CPC-alginate scaffold was autocured, moldable in various shapes, and the porosity was adjustable. The scaffolds were shown to have advantageous 3-D matrix properties for MSCs attachment and proliferation as well as their differentiation into osteoblasts. When the scaffold was transplanted into the two open defect regions of the rats, it was possible to confirm the tissue compatibility and the bone regeneration ability through the pore shape. In addition, the scaffolds demonstrated the ability to safely load biological proteins (BSA and lysozyme) during manufacture and their ability to release them into the test tube over a month. Thus, it can be seen that the CPC-alginate scaffold can be provided as a tissue engineering construct that delivers biological molecules to stimulate bone regeneration.

The present invention can produce a porous scaffold of calcium phosphate cement by preparing a suspension of calcium phosphate cement and alginate and then curing the suspension by injecting the suspension into a mold filled with an aqueous solution of calcium ion to stimulate bone regeneration during the manufacture of the scaffold The present invention can be provided as a tissue engineering structure for transferring biological molecules for stimulating bone regeneration by loading biological molecules together.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a process tool for fabricating a calcium phosphate cement (CPC) / alginate composite (CPA) fibrous network.
FIG. 2A is a graph showing the change in diameter of the fiber as the composition of the needle gauge and the suspension is changed. FIG.
Figure 2B shows the pore structure of the 3-D composite scaffold with μCT.
Figure 3a shows the microstructure of the scaffold after various immersion times after immersing the CPC-alginate 3-D pore scaffold in the simulated body fluids.
Figure 3B is an XRD pattern for monitoring the phase change of the CPC and CPC-alginate scaffold during the immersion test.
Figure 3C shows the results of EDS analysis of CPC and CPC-alginate scaffolds during the immersion test.
FIG. 4 shows the result of measuring mesothelial stem cell growth as mitochondrial activity of cells.
Figure 5 shows the results of observing cell morphology on fibrous scaffolds using SEM at different resolutions during 7 and 14 days of culture.
Figure 6 is an index for in vitro bone formation differentiation of MSCs during culture on a CPC-alginate composite scaffold, which is the result of measurement of basic phosphatase (ALP) activity.
FIG. 7 shows the result of a tissue sample collected and subjected to μCT 6 weeks after transplantation of highly porous CPC-alginate scaffolds into rats.
Figure 8 shows H & E (a and b) and MT (c and d) staining results at different resolutions.
Figure 9 shows the release profiles of proteins in PBS measured over 28 days.

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

Example 1: Calcium phosphate cement - Alginate Preparation of Composite Suspension

The α-tricalcium phosphate (α-TCP) -based cement powder was prepared according to a known method (Kim HW. et al., J Mater Sci Mater Med , 2010, 21, 3019-27). Commercial calcium carbonate (Aldrich) and anhydrous calcium phosphate (Aldrich) were mixed and thermally reacted at 1400 ° C for 3 hours, followed by air quenching to complete the reaction to form an α-TCP phase (Kim HW. et al., J Mater Sci Mater Med , 2010, 21, 3019-27). The powder was ball-milled and then sieved to 45 [mu] m, then stored under vacuum for later use. The average particle size of α-TCP was 4.79 μm as measured by a particle size analyzer (Saturn DigiSizer 5200, Micromeritics, USA). The sodium alginate solution was prepared by using 5% Na ₂ HPO ₄ (distilled deionized water, DDW) as a solvent at a concentration of 2% by weight. The cement powder was mixed with the alginate solution in an appropriate ratio to prepare a composite suspension.

Example 2: through direct deposition Scaffold Produce

The possibility of direct deposition of the composite suspension prepared in Example 1 according to the mixing ratio of the cement powder / alginate solution was changed from 1.0 to 2.5 by weight. At a weight ratio of 2.0 or more, the suspension had an excessively large viscosity and was difficult to inject through the nozzle. Therefore, a suspension of 1.0 to 2.0 weight ratio was used thereafter.

The mixed suspension was then placed in a syringe and then poured into a Ca-containing bath (150 mM CaCl ₂ ) as shown diagrammatically in FIG. 1 to rapidly solidify the deposit. The injection pressure was adjusted to 500 kPa using a regulator (IEI, AD2000C). The size was controlled by another needle gauge (23-27 G). The material was poured into 10 ml of Ca-containing solution in a cylindrical mold ( φ = 10 mm), and then the fibrous deposit was further mechanically compressed to produce a disc-shaped scaffold of specific height. The process of immersing the scaffold in the Ca-containing bath took about one minute. The height of the scaffold was varied to vary the porosity level (porosity: 1.2 mm, medium porosity: 1.5 mm, high porosity: 2.0 mm). Similarly, scaffolds with different porosity levels can be fabricated by varying the amount (weight) of scaffold material loaded while maintaining the height of the scaffold constant (3 mm) (porosity: 0.5 g, medium Porosity: 0.4 g, and high porosity: 0.3 g). The cured scaffold was then used for in vitro cell analysis and in vivo animal studies without further treatment, such as immersion in water.

According to Sikkim depositing the mixture in a suspension containing Ca- bath, rapid hardening occurred due to the reaction of the sodium alginate and Ca ⁺ ^2. Thus, the as-deposited form can be maintained during the process, resulting in a 3-D network. In fact, the rapid curing of CPC composites without alginate was impossible, and the scaffold was completely collapsed during injection. Even when the ratio (1.0-2.0) of the liquid contrast powder used in the present invention required to be injectable through the nozzle was used, no curing was observed even after several hours in the alginate-free CPC suspension. However, this difficulty could be overcome by using alginate that undergoes a rapid crosslinking reaction with Ca ² ⁺ ions in the immersion bath. Therefore, the addition of alginate to CPC composites and the use of appropriate proportions of liquid-to-liquid powders were essential processing considerations in terms of both implantability and curability.

The diameter of the fibers could be adjusted by varying the composition of the needle gauge and suspension as shown in Figure 2a. In the present invention, fiber diameters in the range of 200-600 mu m could be obtained with the other gauge needles 23, 25, 26 and 27G and compositions (CPA20 and CPA15). The fiber diameter decreased as the ratio of alginate liquid to CPC powder increased (1.5 to 2.0) and needle gauge increased (23 to 27 G, corresponding to needle inner diameter reduction of 0.32 to 0.16 mm). The piled fibrous network was further formed into a 3-D scaffold by applying a compressive load. By changing the degree of compression, the porosity of the scaffold could be easily controlled. In the present invention, the porosity of the composite scaffold was changed to low, medium, and elevation levels. In fact, compared to other types of biomedical ceramics, few examples have produced porous scaffolds based on CPC composites. Recent studies have highlighted the importance of the CPC-based scaffold as a potential drug delivery system because of its autocuring properties (Mestres G. et al., Acta Biomater , 2010, 6, 2863-73; Montufar EB, et al. Acta Biomater , 2009, 5, 2752-62). In some studies on scaffold production, biopolymer phases such as chitosan and poly (lactic acid) / alginate were included in the CPC and conventional scaffolding methods were applied (Wang Y. et al., J Biomed Mater Res A, 2009, 89, 980-7 ). Compared with this previous study, the scaffold prepared in the present invention is characterized in that it is prepared by a novel method, namely direct deposition of the suspension and molding of a 3-D structure. By using this process, the pore array including the stem size and the porosity can be controlled, and the scaffold can be filled in a desired amount in a desired shape to produce a composite shape. In the present invention, a 3-D structure is optionally formed by depositing deposits, but a definite 3-D shape can be produced through a process such as direct writing.

Experimental Example 1: 3-D Porosity Scaffold Investigate characteristics

Example 2 A thoroughly washing the composite scaffold obtained in with distilled water, simulated body fluid (142.0 mM at 37 ℃ for 7 days Na ^+, 5 mM K ^+, 1.5 mM Mg ² ^+, 2.5 mM Ca ² ^+, 147.8 mM Cl ^- was immersed in a contained _{^{SBF) -, 4.2 mM HCO 3}} -, 1.0 mM HPO 4 2 -, 0.5 mM SO 4 2. The sample was washed and vacuum dried, and then its shape was examined with a scanning electron microscope (SEM) (Hitachi S-3000H). Composition changes were monitored via an energy dispersive spectroscopy (EDS) (Bruker SNE-3000 M) in a scanning electron microscope. The crystal phase change of the scaffold was measured using an X-ray diffractometer (Rigaku Ultima IV). The pore structure of the scaffold with different porosity was analyzed by micro-computed tomography (μCT) (Skyscan model 1172). Disks ( φ 10 × 3 mm) of each sample were placed on the upper and lower surfaces parallel to the scan plane. 11 Mp X-ray camera and 758 files were obtained with an image pixel size of 19.92 ㎛. The surface charge of the α-TCP particles was investigated by measuring the zeta potential (Zetasizer ZEN3600, Malvern Instruments). α-TCP filtering sieve particles (45 ㎛), 1 mg ml - was dispersed in ¹ of distilled water, at room temperature using a disposable capillary cell (DTS1060C) and between zeta low software (Zetasizer software) (. v 6.20 ) and The zeta potential was measured at pH 7.0. The measurements were repeated three times on different samples.

The elastic modulus of the scaffold was measured using a dynamic mechanical analyzer (DMA) (DMA25, Metravib, France). A sample with three different porosities was prepared in an area of 5 mm diameter x 10 mm height and subjected to dynamic compression loading. The dynamic modulus of the sample was recorded. Three samples were tested for each group.

The pore structure of the 3-D composite scaffold was examined by μCT, and the result is shown in FIG. 2B. In the case of a low porosity scaffold (porosity ~ 14%), some of the pores were blocked by compression. The medium porosity scaffold (porosity ~ 34%) had a larger pore space and the pore network was more developed. The high porosity scaffold (porosity ~ 54%) pores had a large space and were connected to each other to provide a 3-D pore channel suitable for cell migration and tissue culture. The microstructure of the scaffold after various immersing time after immersing the CPC-alginate 3-D pore scaffold in the simulated body fluid is shown in FIG. The pre-dipping ('0d') surface was dense and had CPC particles embedded in the alginate matrix. After immersion for 1 day ('1d') some small crystals began to form on the surface. After 3 days ('3d'), the crystal phase grew to form a uniform network of plate-like crystals. After 7 days ('7d'), the formation of nanocrystals became larger, and they layered and merged to form a micron-sized island.

During the immersion test, phase changes of CPC and CPC-alginate scaffold were monitored (FIG. 3B). Initially only α-TCP peaks appeared (closed circles). According to immersion, HA (asterisk) appeared as a new phase and HA intensity increased with increasing immersion time. Thus, we could see the phase transition from α-TCP to HA. After 7 days, only the HA phase was observed, indicating complete conversion.

The EDS analysis confirmed that the Ca / P ratio increased from 1.517 (α-TCP analogue value) to 1.659 (HA analogy value), confirming the phase change from α-TCP to HA (FIG. Based on the phase evolution of CPC-alginate under these simulated body fluids conditions, the scaffold of the present invention is converted into a HA-like bone mineral-like phase in body fluid to maintain good bioactivity, to develop bone-related cells, And can play a significant role in the physiological response.

In order to investigate the mechanical properties of the CPC-alginate scaffold, a range similar to that of the trabecular bone (96-53 MPa for high porosity versus 50-500 MPa for trabecular bone, 398 63 MPa for medium porosity, 573 ± 87 MPa), the elastic modulus of the scaffold was measured and found to be applicable mainly for bone regeneration in the unloaded support region.

Experimental Example 2: In vitro Osteoblast Propagation and ALP Active measurement

For cell reaction tests, CPC-alginate scaffolds with three different porosity levels were prepared (porosity: 13.6%, medium porosity: 34.0%, and high porosity: 53.7%). Mesenchymal stem cells (MSCs) from rat bone marrow were collected from the femur and tibia of 5-week-old male rats. The femur and tibia were quickly dissected and placed in α-minimal essential medium (α-MEM). The incised bone was treated with collagenase and dispase solution for 30 minutes, then the bone marrow was taken out and centrifuged at 1500 rpm. The pellet was pulverized and stored at 37 째 C under an atmosphere of 5% CO ₂ /95% air at all times, containing an antifungal agent solution (10,000 U penicillin, 10,000 ug streptomycin, and 25 ug amphotericin B / m, Gibco) The cells were cultured in α-MEM supplemented with 10% fetal bovine serum under standard culture conditions. After 5 days of culture, unattached cells were removed and replenished with fresh medium. Cells were maintained under standard culture conditions and then subcultured 3 times before use for in vitro analysis.

Insert the scaffold sample to each well of a 24-well plate was inoculated with 1 × 10 ⁵ cell suspension to each sample. Cells were cultured for 14 days under the influence of osteogenic factors (50 [mu] g ml- ¹ ascorbic acid, 100 nM dexamethasone and 10 mM [beta] -glycerophosphate). Cell proliferation was then induced by the 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium . When an MTS reagent (tetrazolium salt) is added to a living cell, it is reduced to a colored formazan product that is dissolved in the culture medium by the cells. The amount of formazan product, directly proportional to the number of living cells, was measured by absorbance at 490 nm using an ELISA plate reader (iMARK, BioRad). MTS analysis was performed with three replicate samples.

Basic phosphatase (ALP) activity was measured as an indicator of in vitro osteogenic differentiation of MSCs during culture on CPC-alginate composite scaffolds. After culturing for 7 days and 14 days in osteogenic medium, the cell layer was collected, treated with 0.1% Triton X-100 cell lysis medium, and further pulverized by sequential freezing and thawing. The total protein content was analyzed using a commercial DC protein assay kit (Bio-Rad) and the aliquot of the reaction sample was normalized to the total protein content and then measured. Cell ALP activity was measured using an ALP assay kit (procedure No. ALP-10, Sigma). The p-nitrophenol produced in the presence of ALP was measured by absorbance at 405 nm. Three replicate samples were tested for ALP activity.

Cellular test data were expressed as mean ± standard deviation (SD) and statistical analysis was performed via one-way analysis of variance (ANOVA). Statistical significance was P <0.05.

The result of measurement of cell growth as mitochondrial activity of the cells is shown in Fig. The increase in MTS level was observed with incubation time, indicating that MSC proliferated well in all three types of scaffolds. At day 3, cell proliferation was significantly higher (P <0.05) in the high porosity scaffold compared to the porosity scaffold and intermediate porosity scaffold, which remained until day 7. On day 14, the difference in cell proliferation between the scaffolds decreased. The cell morphology on the fibrous scaffold was observed using SEM at different resolutions during 7 and 14 days of culturing, as shown in FIG. Here, a represents porosity, b represents medium porosity, and c represents highly porosity scaffold. On day 7, the cells were elongated in shape, well attached to the basal fiber stalk. On day 14, the cells showed more extensive cytoskeletal progression that completely covered most of the stem surface. Observation of the internal surface of the cross section revealed that, especially in highly porous scaffolds, a large number of cells were found deep in the pore channel. Based on these results, it was found that the basal CPC-alginate matrix is advantageous for excellent adhesion of cells, active microprojectile differentiation and cell number increase according to long-term culture.

The results of ALP activity measurement are shown in Fig. ALP activity increased with incubation time from day 21 to day 21 in all scaffolds. This increase was even greater in scaffolds with high porosity. These results indicate that the MSCs cultured in the 3-D porous scaffold are stimulated to differentiate according to the osteogenic lineage and that the stimulation is greater in scaffolds with a high degree of porosity.

Through the cell proliferation and ALP activity results, the MSC action in vitro under the 3-D fibrous network was superior by using a scaffold with a high degree of porosity, and ultimately the production of an in vitro tissue engineering structure for bone tissue engineering Which is useful for. The highly porous scaffolds provide space and substrate conditions for cell migration and three-dimensional propagation, facilitating cell migration and action through open spaces.

Experimental Example 3: For bone affinity In vivo Advance investigation

A 10 week old male Sprague Dawley rats were used for in vivo analysis. The surgical protocol followed the guidelines of the Laboratory Animal Ethics Committee of Dankook University in Korea. Animals were anesthetized with intramuscular injection using ketamine (80 mg kg ^-1 ) and xylazine (10 mg kg ^-1 ). The frontal area of both openings was cut and a 5 mm diameter critical dimension full thickness bone defect was formed using a trapezoidal drill under continuous sterile saline control. For in vivo testing, a test scaffold with a size of 5 mm diameter x 2 mm height was prepared using different sized molds and the amount of composite material deposited was also used to prepare a scaffold with high porosity (53.7%) Respectively. The manufactured scaffold was transplanted into two open-ended defects. Implants without transplanted scaffolds were used as negative control. Soft tissue was sutured for primary closure. Six weeks after transplantation, animals were sacrificed. The initial surgical defect and surrounding tissue area were removed at once, fixed with 10% neutral formalin solution and demineralized. The tissue was embedded in a paraffin block and subsequently incised using a microtome (Leica ™). 4-6 [mu] m thick sections were fixed on a microscope slide. Paraffin was removed from slides with tissue sections and hydrated through a series of xylenes and alcohols. The tissue slides were stained with hematocylin, eosin (H & E), and maltose trichrome (MT) and examined under an optical microscope for histological observation.

Fig. 7 shows the results obtained by collecting tissue samples after 6 weeks of transplantation of highly porous CPC-alginate scaffolds into rats and irradiating them with μCT. First, X-ray observations showed a brighter image of the 2-D fibrous network in the scaffold (FIG. 7a), whereas a brighter image was seen in the empty bone defect (negative control). In the 2-D μCT image, the control was almost completely unfilled, whereas the scaffolds showed that the bone defect area was completely filled with the scaffold sample (FIG. 7b). The reconstructed 3-D μCT image showed the 3-D structure of the porous scaffold and the bone morphology grown in the bone defect region (FIG. 7c). In the negative control group, bone regeneration was scarcely occurred, indicating that this was a critical size bone defect (Fig. 7d).

On the other hand, the newly-grown bone in the scaffold sample could not be reliably differentiated from the remaining material. The porous scaffold containing the outer scaffold was stained with tissue engineering and observed. After 6 weeks of operation, tissue characteristics and bone formation were observed.

Figure 8 shows H & E (a and b) and MT (c and d) staining results at different resolutions. No inflammatory response or tissue rejection was observed in the transplanted scaffolds. And the connective bone tissue filled the pore channels of the scaffold through the bone defect region (Fig. 8A). The enlarged image showed newly formed tissue (dark red) aligned with the fiber stem (dark red) of the scaffold (Fig. 88B). MT staining showed the formation of bone tissue with an extracellular matrix appearing as blue or dark blue (Figs. 8c and 8d). In the tissue engineering images, MT staining was found in the CPC-alginate scaffold frame structure, indicating that the scaffold could be replaced by cells and tissues. Although the majority of scaffolds appeared to be biodegradable for 6 weeks after implantation in both rats, the results showed that CPC or alginate or a combination of these composites could be degraded in vivo.

Therefore, it was confirmed that the CPC-alginate porous scaffold has excellent tissue adherence and reproducibility of bone tissue through the in vivo tissue reaction, and thus it can be used as an implantable material for bone regeneration.

Experimental Example 4: Investigation of protein transfer capacity

Protein release from the CPC-alginate porous scaffold was investigated using bovine serum albumin (BSA) and lysozyme as model proteins. Loading of each protein was performed in two different ways. One is a method in which the protein is added to the alginate solution and then mixed with the CPC powder and then immersed in a protein-containing porous scaffold (the "loading scheme I") and the other is after the protein is added to the CPC suspension , Followed by culturing for 1 hour with gentle stirring, then mixing the solution with an alginate solution, and immersing the solution in a porous scaffold ("Loading Method II"). The protein content in each scaffold sample was adjusted to 33.3 mg mg scaffold- ¹ . One gram of protein-containing porous scaffold was used for the protein release test. This was based on a preliminary study showing that a final scaffold sample of 1 g (0.039 g) can be prepared through suspension deposition of 1.4 g. Each sample was immersed in 10 ml of phosphate buffered saline (PBS) at pH 7 and 37 ° C for 28 days. Scaffolds were removed at each measurement time (1, 2, 3, 6, and 24 hours, and 2, 3, 7, 10, 14, 21, and 28 days) and the residual medium was removed via bicinchoninic acid Respectively. For each measurement, the medium was replaced freshly.

Figure 9 shows the release profiles of proteins in PBS measured over 28 days.

About 20-30% of lysozyme and BSA were released in the early (within 12 hours) under all loading conditions where the proteins loosely bound to the surface and were in direct contact with the solution. After this initial burst, the release of both proteins lasted for 28 days at a reduced rate over time. There was no significant difference in BSA release profile between loading methods. However, the release rate of lysozyme significantly decreased in loading method II compared to loading method I. When the release of lysozyme is higher than BSA in loading mode I, this tendency has changed in loading mode II. Therefore, the interaction between protein and scaffold components, especially CPC, was found to differ. That is, CPC can delay the release of lysozyme more than BSA due to strong affinity or chemical binding.

To investigate the reason for this phenomenon, surface charge of α-TCP powder and protein was measured by zeta potential measurement, and the results are shown in Table 1 below.

sample CPC powder Lysozyme BSA Zeta potential (pH 7) -18.14 2.53 -16.84

At pH 7, α-TCP and BSA were negatively charged while lysozyme was positively charged. Based on this, it was found that the electrostatic attraction for CPC powder was higher in lysozyme compared to BSA. Even though the alginate is also negatively charged and has a somewhat ionic interaction with lysozyme, its hydrogel properties may have an open structure than CPC, and thus water permeation is possible and thereby the protein release pathway from the structure . In addition, the difference in degradation behavior of alginate and CPC can also affect the release rate of lysozyme. These results indicate that the CPC-alginate scaffold is effective for the delivery of the charged growth factor in the same amount as the basic fibroblast growth factor. That is, these results on in vitro protein release suggest that biological molecules can be loaded easily and safely into the scaffold and that the porous scaffold can release biologically charged molecules, particularly positively, for at least one month, It was confirmed that it is usable.

Claims

Preparing a suspension of calcium phosphate cement and alginate (step 1);
Introducing the suspension into a mold filled with a calcium ion aqueous solution to cure (step 2); And
Mechanically compressing the cured product after the curing to adjust the porosity of the scaffold to 10 to 90%;
&Lt; / RTI > wherein the porous scaffold is a porous calcium phosphate cement.

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The method of claim 1, wherein the mixing ratio of the calcium phosphate cement to the alginate is 20: 1 to 500: 1 by weight based on the weight of calcium phosphate cement: alginate.

The method of claim 1, wherein the calcium ion concentration is from 10 to 200 mM.

The method of claim 1, wherein the shape of the mold is cylindrical or hexahedral.

The method of claim 1, wherein the calcium phosphate compound is tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, dicalcium phosphate, hydroxyapatite or a combination thereof.

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3. The method of claim 1, wherein the suspension further comprises a biological protein, a drug or a combination thereof.

9. The method of claim 8, wherein the biological protein is bovine serum albumin, lysozyme, growth factor or a combination thereof.

9. The method of claim 8, wherein the drug is an antibiotic, an anti-cancer agent, an anti-inflammatory agent or a combination thereof.

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