WO2013012132A1 - Method for manufacturing porous scaffold of calcium phosphate cement - Google Patents

Abstract

The present invention relates to a method for manufacturing a porous scaffold of a calcium phosphate cement, and more specifically, to a method for manufacturing a porous scaffold of a calcium phosphate cement by preparing a suspension of the calcium phosphate cement and alginate, and injecting the suspension into a mold, which is filled with a calcium ion aqueous solution, and hardening same.

Description

Method for manufacturing porous scaffold of calcium phosphate cement

The present invention relates to a method for producing a porous scaffold of calcium phosphate cement, and more particularly, after preparing a suspension of calcium phosphate cement and alginate, the suspension is put into a mold filled with an aqueous solution of calcium ions, and then cured. It relates to a method of making a scaffold.

Rapid hardening cement is very useful for bone tissue regeneration as a direct filling or injectable material. 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). There are some issues that need to be improved, such as mechanical properties, the introduction of macropores, and the control of the rate of degradation, but CPCs have many attractive properties and are therefore used as a useful alternative to treating bone defects (Brown WE. Et al., J Dent Res , 1986, 63, 200; Bohner M., J Mater Chem , 2007, 17, 3980-6). CPCs are cell- and tissue-friendly, self-curable and useful as injectable materials that require minimally invasive surgery, and they can also contain therapeutic molecules in formulations (Planell JA. Et al., Biomater , 2006, 27 (10), 2171-7; Burger EH. Et al., J Dent Res , 2000, 79, 255).

Scaffolds with three-dimensional (3-D) porous networks provide effective matrix conditions for bone tissue engineering (Xu HHK. Et al., Biomater , 2009, 30, 2675-82; Barlow SK, et al. , 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 natural tissue than materials or cells alone (Hutmacher DW., Biomater , 2000, 21, 2529-43; Vacanti JP. Et al., Science , 1993, 260, 920-6). In in vitro tissue engineering processes, controlled release of therapeutic molecules, such as growth factors, is beneficial to modulate cell function and promote bone formation. In addition, CPC-based materials have been regarded as good candidates for delivery of therapeutic agents transported within these structures, as they self-cure under mild conditions, safely bind the therapeutics, 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 this background, the present inventors have made the present invention by confirming that a porous scaffold of calcium phosphate cement can be prepared by preparing a suspension of calcium phosphate cement and alginate and then curing the suspension into a mold filled with aqueous calcium ion 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 preparing a porous scaffold of calcium phosphate cement.

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

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

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

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

As used herein, the term 'scaffold' refers to a structure that serves to provide an environment suitable for attachment, differentiation, and proliferation and differentiation of cells transferred from and around the seeded cells inside and outside the structure. It is one of the important basic elements in the field.

Step 1 is a step of preparing a suspension of calcium phosphate cement and alginate, a step of preparing a suspension by mixing powdered calcium phosphate cement with an alginate solution.

The term "cement" as used in the present invention means a cured body of a paste obtained by mixing a powdery solid phase and a liquid phase. "Cure" of the cement refers to spontaneous curing of the paste, which is done without artificial treatment at room temperature or body temperature, wherein the paste is obtained as a result of mixing a solid phase and a liquid phase.

As used herein, the term "calcium phosphate cement" means a cement in which the powdered solid phase consists of a calcium phosphate compound or a mixture of calcium and / or phosphate compounds.

The calcium phosphate cement (CPC) is a material consisting of an aqueous solution containing a calcium phosphate particles, the main component of the powder and a substance that promotes hardening, such as phosphate. When applied, a calcium phosphate compound precipitates and hardens by a chemical reaction of two components at the site of application, thereby filling a damaged bone and bone, or an empty space between bone and implant, to fix and stabilize the bone substitute. Form.

The mixing ratio of the calcium phosphate cement and alginate is preferably a calcium phosphate cement: alginate ratio of 20: 1 to 500: 1 by weight.

The suspension may further comprise a biological protein or drug, wherein the biological protein is bovine serum albumin, lysozyme, growth factor and the like, and the drug may be an antibiotic, an anticancer agent, an anti-inflammatory agent, or the like.

In step 2, the suspension is introduced into a mold filled with an aqueous solution of calcium ions and cured. The suspension is introduced into a mold filled with an aqueous solution containing calcium ions to cure the suspension.

The concentration of the calcium ions 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, but is not limited to, tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, dicalcium phosphate, hydroxyapatite or a combination thereof.

Step 3 is a step of mechanically compressing the mechanically compressed scaffold formed by the self-curing to adjust the porosity or to optionally reassemble the shape.

Through the compression, the porosity of the scaffold can be arbitrarily adjusted, and in particular, it can be adjusted with a hand press, a machine that can apply a load.

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

The present invention also provides a material for producing a porous scaffold of calcium phosphate cement composed of calcium phosphate cement powder, alginate solution, and calcium ion aqueous solution, each packaged in a separate container; Provided are a kit for preparing a porous scaffold of calcium phosphate cement comprising a syringe.

The suspension was prepared by mixing calcium phosphate cement powder and alginate solution packaged in each individual container of the kit, and putting it in a syringe, and then injecting the suspension into a mold of a form filled with aqueous calcium ion solution to form a porous scavenger of calcium phosphate cement. Folds can be prepared on the fly.

The mold may be any one that can be easily obtained, but can be used to make the scaffold more conveniently when the mold further includes a mold for scaffold forming.

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

The present invention seeks to improve novel cell scaffold materials produced by using CPCs and sodium alginate in combination. In particular, the composite suspension was directly deposited in Ca-containing solution to form a fibrous network. The deposited suspension rapidly cures to form a gelled network in which alginate is present and crosslinked by Ca ²⁺ ions (Langer R. et al., Curr Top Dev Biol , 2004, 61, 113-34; Asaoka K et al., Biomater , 1995, 16, 527-32). Cured porous scaffolds are cell friendly and useful for bone tissue engineering. In addition, the scaffold has been shown to be able to load and deliver bioactive molecules contained within the structure. The following describes a method for making a CPC-alginate porous scaffold. In addition, in vitro cell responses of rat bone marrow-derived mesenchymal stem cells (MSCs) to the scaffold were examined prior to their application to bone tissue engineering. Furthermore, in vivo pilot studies were performed to assess tissue suitability and the drug delivery potential of the scaffold was investigated using two different model proteins.

CPC suspensions in combination with alginate solutions effectively formed porous scaffolds by direct fibrous deposition into Ca-containing solutions. CPC-alginate scaffolds were self-curing, moldable in a variety of forms, and controlled porosity. The scaffolds have been shown to have advantageous 3-D matrix properties for MSCs adhesion and proliferation as well as their differentiation into osteoblasts. When the scaffold was implanted in the rat cranial defect, tissue conformity and bone regeneration ability through pore shape were confirmed. The scaffolds also showed the ability to safely load biological proteins (BSA and lysozyme) during manufacture and to release them in vitro 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 prepare a porous scaffold of calcium phosphate cement by preparing a suspension of calcium phosphate cement and alginate and then curing the suspension into a mold filled with aqueous calcium ion solution to stimulate bone regeneration during manufacture of the scaffold. There is an effect that can be provided to a tissue engineering construct that is loaded with biological molecules to deliver the biological molecules for stimulating bone regeneration.

1 is a schematic representation of a process tool for producing a calcium phosphate cement (CPC) / alginate composite (CPA) fibrous network.

Figure 2a is a graph showing the change in the diameter of the fiber according to the composition of the needle gauge and suspension.

Figure 2b is the result of investigating the pore structure of the 3-D composite scaffold by μCT.

3A shows the results of investigating the microstructure of the scaffold after various immersion times after immersing the CPC-alginate 3-D pore scaffold in mock body fluids.

3B is an XRD pattern for monitoring phase changes of CPC and CPC-alginate scaffolds during immersion test.

3C shows the results of EDS analysis of CPC and CPC-alginate scaffolds during immersion test.

4 is a result of measuring mesenchymal stem cell growth as the mitochondrial activity of the cells.

5A-5C show the results of observation of the cell morphology on fibrous scaffolds with different resolution during incubation for 7 days and 14 days.

FIG. 6 shows the results of measuring basic phosphatase (ALP) activity as an indicator for in vitro osteogenic differentiation of MSCs in culture on CPC-alginate composite scaffolds.

FIG. 7 shows the results of irradiating with a CT sample of tissue samples collected 6 weeks after transplanting the high porosity CPC-alginate scaffold into the rat.

8 shows the results of H & E (a and b) and MT (c and d) staining at different resolutions.

9 shows the release profile of protein in PBS measured over 28 days.

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

Example 1 Preparation of Calcium Phosphate Cement-Alginate Composite Suspension

α-tricalcium phosphate (α-TCP) -based cement powders were prepared according to known methods (Kim HW. et al., J Mater Sci Mater Med , 2010, 21, 3019-27). Commercial calcium carbonate (Aldrich) and anhydrous dibasic calcium phosphate (Aldrich) were mixed and then 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 μm and then stored under vacuum for use. As measured using a particle size analyzer (Saturn DigiSizer 5200, Micromeritics, USA), the average particle size of α-TCP was 4.79 μm. Sodium alginate solution was prepared using 5% Na ₂ HPO ₄ (distilled deionized water, DDW) as a solvent at a concentration of 2% by weight. Cement powder was mixed with the alginate solution in an appropriate ratio to prepare a composite suspension.

Example 2: Preparation of Scaffolds by Direct Deposit

The possibility of direct deposition of the composite suspension prepared in Example 1 above was investigated by varying the mixing ratio of cement powder / alginate solution to 1.0 to 2.5 weight ratio. Above 2.0 weight ratio, the suspension had too high a viscosity to be difficult to enter through the nozzle. Thus, 1.0-2.0 weight ratio suspension was then used.

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

As the mixture suspension was deposited into a Ca-containing bath, rapid curing occurred due to the reaction of sodium alginate with Ca ²⁺ . Therefore, the deposited form can be maintained during the process, resulting in a 3-D network structure. In fact, rapid curing without alginate in the CPC composite was not possible, causing the scaffold to collapse completely during dosing. Even with the ratio of powder to liquid used in the present invention (1.0-2.0), which is required to be injectable through the nozzle, no hardening was observed after several hours in the CPC suspension without alginate. However, this difficulty could be overcome by using alginate which has a rapid crosslinking reaction with Ca ²⁺ ions in the deposition bath. Therefore, the addition of alginate to the CPC composite and the use of an appropriate ratio of powder to liquid was an essential processing consideration in terms of both injectability and curability.

The diameter of the fiber could be adjusted by changing the composition of the needle gauge and suspension as shown in FIG. 2A. In the present invention, different gauge needles (23, 25, 26 and 27 G) and compositions (CPA20 and CPA15) were able to obtain fiber diameters in the range of 200-600 μm. Fiber diameter decreased as the ratio of CPC powder to alginate liquid increased (1.5-2.0) and needle gauge increased (corresponding to needle internal diameter reduction of 23-27 G, 0.32-0.16 mm). The stacked fibrous network structures were further molded into 3-D scaffolds by applying a compressive load. By varying the degree of compression it was easy to control the porosity of the scaffold. In the present invention, the porosity of the composite scaffold was changed to low, medium, and high levels. In fact, few examples have fabricated porous scaffolds based on CPC composites compared to other types of bioceramic. Recent studies have highlighted the importance as a potential drug delivery system because CPC-based scaffolds have self-curing properties (Mestres G. et al., Acta Biomater , 2010, 6, 2863-73; Montufar EB, et al. Acta Biomater , 2009, 5, 2752-62). Some studies on scaffold fabrication have included biopolymer phases such as chitosan and poly (lactic acid) / alginate in CPC and applied conventional scaffolding methods (Wang Y. et al., J Biomed Mater Res A, 2009 , 89, 980-7). Compared with this previous study, the scaffolds produced in the present invention are characterized in that they are produced by new methods, namely by direct deposition of suspensions and by molding of 3-D structures. Using this process, the pore arrangement, including stem size and porosity, is controllable, and the scaffold can be manufactured into complex shapes by filling the appropriate amount into the desired mold. In the present invention, deposits are arbitrarily stacked to form a 3-D structure, but a clear 3-D shape can be manufactured through a process such as direct writing.

Experimental Example 1: Investigation of the properties of the 3-D porous scaffold

The composite scaffold obtained in Example 2 was thoroughly washed with distilled water, and then simulated body fluids (142.0 mM Na ⁺ , 5 mM K ⁺ , 1.5 mM Mg ²⁺ , 2.5 mM Ca ²⁺ , 147.8) at 37 ° C. for 7 days. immersion in mM Cl ⁻ , 4.2 mM HCO ₃ ⁻ , 1.0 mM HPO ₄ ^2- , 0.5 mM SO ₄ ^2- containing SBF). The sample was washed and dried in vacuo and then morphologically examined by scanning electron microscopy (SEM) (Hitachi S-3000H). Composition changes were monitored via an energy dispersive spectrometer (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 scaffolds with different porosities was analyzed by micro-computed tomography (μCT) (Skyscan model 1172). Discs ( φ 10 × 3 mm) of each sample were placed on the upper and lower surfaces parallel to the scan plane. Scanning was performed with an 11 Mp X-ray camera and 758 files were obtained with an image pixel size of 19.92 μm. The surface charge of the α-TCP particles was investigated by measuring zeta potential (Zetasizer ZEN3600, Malvern Instruments). The α-TCP particles were sieved (45 μm) and dispersed in distilled water at 1 mg ml ⁻¹ , then at room temperature and at room temperature using a disposable capillary cell (DTS1060C) and Zetasizer software (v. 6.20). Zeta potential was measured at pH 7.0. The measurement was repeated three times on different samples.

The modulus of elasticity of the scaffold was measured by a dynamic mechanical analyzer (DMA) (DMA25, Metravib, France). Samples with three different porosities were prepared with an area of 5 mm diameter x 10 mm height and subjected to dynamic compressive load. 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 shown in FIG. 2B. For low porosity scaffolds (porosity ~ 14%), some pores appeared to be blocked by compression. Medium porosity scaffolds (~ 34% porosity) had larger pore spaces and more advanced pore networks. The pores of the high porosity scaffold (porosity of 54%) had large spaces and connected to each other to provide 3-D pore channels suitable for cell migration and tissue irrigation. After the CPC-alginate 3-D pore scaffold was immersed in the simulated body fluids, the microstructure of the scaffold was examined after various immersion times. The surface prior to immersion ('0d') was dense and had CPC particles embedded in the alginate matrix. After soaking for one 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 crystals. After 7 days ('7d') the nanocrystal formation was even larger and layered and merged together to form micron sized islands.

Phase change of the CPC and CPC-alginate scaffolds was monitored during the immersion test (FIG. 3B). Initially only α-TCP peaks appeared (closed circles). HA (asterisk) appeared as a new phase with soaking, and the intensity of the HA peak increased with soaking time. Therefore, it was found that the phase change from α-TCP to HA. After 7 days only HA phase was observed, indicating complete conversion.

The EDS analysis confirmed that the Ca / P ratio increased from 1.517 (α-TCP like) to 1.659 (HA like) to confirm the phase shift from α-TCP to HA again (FIG. 3C). Based on the phase development of CPC-alginate under these simulated humoral conditions, the scaffolds of the present invention are converted to HA mineral-like HA phases in body fluids to maintain good bioactivity and allow bone-related cells to grow and develop into tissues It can be seen that it can provide favorable substrate conditions, and can play a significant role in the physiological response.

On the other hand, in order to investigate the mechanical properties of the CPC-alginate scaffold, the range similar to the trabecular bone (50 ± 500 MPa for the trabecular bone, 96 ± 63 MPa for high porosity, 398 ± 63 MPa for medium porosity, low porosity The elastic modulus of the scaffold at 573 ± 87 MPa) was found to be applicable mainly for bone regeneration in the unloaded support region.

Experimental Example 2: Measurement of in vitro osteoblast proliferation and ALP activity

For cell response testing, CPC-alginate scaffolds with three different porosity levels were prepared (low porosity: 13.6%, medium porosity: 34.0%, high porosity: 53.7%). Mesenchymal stem cells (MSCs) derived 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 the α-minimum essential medium (α-MEM). The injured bone was treated with collagenase and dispase solution for 30 minutes and then the bone marrow was removed and centrifuged at 1500 rpm. The pellets are ground and contain a homeostatic / antifungal solution (10,000 U penicillin, 10,000 μg streptomycin, and 25 μg amphotericin B / m, Gibco) at 37 ° C. under an atmosphere of 5% CO ₂ /95% air. In α-MEM supplemented with 10% fetal calf serum, the cells were cultured under standard culture conditions. After 5 days of culture, non-adherent cells were removed and supplemented with fresh medium. Cells were maintained under standard culture conditions and then passaged three times before use for in vitro analysis.

Scaffold samples were placed in each well of a 24-well plate and each sample was inoculated with a 1 × 10 ⁵ cell suspension. Cells were incubated for 14 days under the influence of osteogenic factors (50 μg ml ⁻¹ ascorbic acid, 100 nM dexamethasone and 10 mM β-glycerophosphate). Cell proliferation was then performed using the 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) method. It measured using. When MTS reagent (tetrazolium salt) is added to living cells, it is reduced to the colored formazan product that is lysed 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 for in vitro osteogenic differentiation of MSCs in culture on CPC-alginate composite scaffolds. After incubation for 7 days and 14 days in osteogenic medium, the cell layers were collected and treated with 0.1% Triton X-100 cell lysis medium followed by further grinding through sequential freezing and thawing. Total protein content was analyzed using a commercial DC protein analysis kit (Bio-Rad) and measured after standardizing the aliquots of reaction samples to total protein content. ALP activity of the cells 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.

Cell test data are expressed as mean ± standard deviation (SD) and statistical analysis was performed via one-way ANOVA. Statistical significance was set to P <0.05.

The results of measuring cell growth as the mitochondrial activity of the cells are shown in FIG. 4. Increasing MTS levels occurred with incubation time, indicating that MSCs proliferated well in all three types of scaffolds. At early 3 days, cell proliferation was significantly higher in the high porosity scaffolds compared to the low porosity scaffolds and the medium porosity scaffolds (P <0.05), which remained until day 7. On day 14, cell proliferation differences between the scaffolds decreased. The cell morphology on the fibrous scaffolds was observed using SEM at different resolutions during 7 and 14 days of incubation. Where a is low porosity, b is medium porosity, and c is high porosity scaffold. On day 7, the cells showed a long elongated form that adhered well to the basal fiber stem. Day 14 cells showed more extensive cytoskeletal progression that completely covered most of the stem surface. Observation of the inner surface of the cross section revealed a large number of cells deep in the pore channel, especially in the highly porous scaffolds. Based on these results, it can be seen that the underlying CPC-alginate matrix is advantageous for good adhesion of cells, active microprogenitor differentiation and increase in cell number with prolonged culture.

ALP activity measurement results are shown in FIG. 6. ALP activity was found to increase with increasing incubation time up to 21 days in all scaffolds. This increase was greater in scaffolds with high porosity. These results indicate that MSCs cultured in 3-D porosity scaffolds are stimulated to differentiate according to the osteogenic lineage, and this stimulation is greater in scaffolds with high porosity.

Through the cell proliferation and ALP activity results, the use of scaffolds with high porosity resulted in better in vitro MSC action in 3-D fibrous network, ultimately making in vitro tissue engineering constructs for bone tissue engineering. It can be useful for. High porosity scaffolds provide space and substrate conditions for cells to migrate and multiply in three dimensions, making cell movement and action easier through open spaces.

Experimental Example 3: In vivo preliminary examination for bone affinity

Ten week old male Sprague Dawley rats were used for in vivo analysis. Surgical protocols followed the guidelines of the Experimental Animal Ethics Committee of Dankook University. Animals were anesthetized by intramuscular injection using ketamine (80 mg kg ⁻¹ ) and xylazine (10 mg kg ⁻¹ ). The frontal region of the cranial tube was incised and a 5 mm diameter critical size penetrating bone defect was formed using a trepin drill under continuous sterile saline irrigation. For in vivo testing, test scaffolds with dimensions of 5 mm diameter × 2 mm height were prepared using different size molds and the amount of composite deposited was also produced with scaffolds with high porosity (53.7%). To adjust. The prepared scaffolds were implanted into cranial defects. Defects without implanted scaffolds were used as negative controls. Soft tissue was sutured for primary closure. Six weeks after transplantation, the animals were killed. Initial surgical defects and surrounding tissue areas were removed in batches, fixed with 10% neutral formalin solution and then demineralized. Tissues were embedded in paraffin blocks and subsequently incised using a microtome (Leica ™). Sections 4-6 μm thick were fixed on the microscope slides. Paraffin was removed from the slides with tissue sections and hydrated through a series of xylenes and alcohols. The tissue slides were stained with hematocillin and eosin (H & E) and Marthon's trichrome (MT) and examined under an optical microscope for histological observation.

6 weeks after the highly porous CPC-alginate scaffolds were implanted into rats, tissue samples were collected and examined by μCT. First, X-ray observation showed a nearly bright image in the empty bone defect (negative control), while a scaffold showed a faint image of the 2-D fibrous reticulum (FIG. 7A). In 2-D μCT images, the control area was hardly filled, whereas for the scaffold, the bone defect area was completely filled with the scaffold sample (FIG. 7B). The reconstructed 3-D μCT image showed bone growth grown within the 3-D structure and bone defect area of the porous scaffold (FIG. 7C). Little bone regeneration occurred in the negative control group, indicating a critical size bone defect (FIG. 7D).

On the other hand, newly grown bones in the scaffold sample could not be surely differentiated from the remaining material. Explants containing porous scaffolds were stained and histologically observed to show tissue characteristics and bone formation 6 weeks after surgery.

8 shows the results of H & E (a and b) and MT (c and d) staining at different resolutions. No inflammatory response or tissue rejection was observed in the implanted scaffold. Connective bone tissue was shown to fill the pore channel of the scaffold through the bone defect area (FIG. 8A). The magnified image showed the newly formed tissue (dark red) aligned with the fibrous stem (light red) of the scaffold (FIG. 88 b). MT staining showed the formation of bone tissue with extracellular matrix that appeared in light blue or dark blue (FIGS. 8C and 8D). MT staining was found in the CPC-alginate scaffold framework structure, indicating that the scaffold can be replaced with cells and tissues. Although most scaffolds did not appear to be biodegradable for 6 weeks after implantation into the rat cranial canal, the results showed that CPC or alginate or a combination of these combinations were biodegradable.

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

Experimental Example 4: Investigation of Protein Delivery Capacity

Protein release from CPC-alginate porous scaffolds was investigated using bovine serum albumin (BSA) and lysozyme as model proteins. Loading of each protein was performed in two different ways. That is, one is adding the protein to the alginate solution and then mixing it with the CPC powder and then depositing it into the protein-containing porous scaffold (“loading method I”), and the other after adding the protein to the CPC suspension After incubation for 1 hour with gentle stirring, the solution was mixed with an alginate solution and then deposited into a porous scaffold ("loading mode II"). The protein content in each scaffold sample was adjusted to 33.3 μg mg scaffold- ¹ . 1 g of protein-containing porous scaffold was used for protein release testing. This was based on preliminary investigations showing that 1 g (± 0.039 g) final scaffold samples could be prepared via 1.4 g suspension deposition. Each sample was immersed in 10 ml of phosphate buffered saline (PBS) at pH 7 and 37 ° C. for 28 days. At each measurement time (1, 2, 3, 6 and 24 hours, and 2, 3, 7, 10, 14, 21 and 28 days), the scaffold was removed and the residual medium was passed through the bicinconic acid (BCA) method. Analyzed. For each measurement, the medium was freshly replaced.

9 shows the release profile of protein in PBS measured over 28 days.

About 20-30% of lysozyme and BSA were released initially (within 12 hours) under all loading conditions where the protein was loosely bound to the surface and allowed direct contact with the solution. After this initial burst, the release of both proteins continued at a slowed rate with time for 28 days. The BSA release profile did not differ significantly between loading modes. However, for lysozyme, the release rate was significantly reduced in loading mode II compared to loading mode I. When loading of lysozyme in loading mode I was higher than BSA, the trend changed in loading mode II. Therefore, the interaction between the protein and the components of the scaffold, especially the CPC, was found to be different. In other words, CPC may better delay lysozyme release than BSA due to strong affinity or chemical binding.

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

Table 1

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, the electrostatic attraction to the CPC powder was found to be higher in the lysozyme compared to the BSA. Although alginates are also negatively charged and thus have some ionic interactions with lysozyme, their hydrogel properties may have an open structure than CPC, thus allowing water permeation and thereby blocking the protein release pathway from the structure. Can provide. In addition, differences 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 transfer of positively charged growth factors in the same basic fibroblast growth factor. In other words, these results for in vitro protein release allow for easy and safe loading of biological molecules into the scaffold and for the release of sustained release proteins, where the porous scaffold can release particularly positively charged biological molecules for at least a month. It could be confirmed that it can be used.

Claims (15)

1. Preparing a suspension of calcium phosphate cement and alginate (step 1); And

   A method for producing a porous scaffold of calcium phosphate cement comprising the step of injecting the suspension into a mold filled with an aqueous solution of calcium ions (step 2).
2. The method of claim 1, further comprising mechanically compressing after step 2).
3. The method of claim 1, wherein the mixing ratio of the calcium phosphate cement and alginate is calcium phosphate cement: alginate ratio of 20: 1 to 500: 1 by weight.
4. The method of claim 1, wherein the concentration of calcium ions is 10 to 200 mM, the method of producing a porous scaffold of calcium phosphate cement.
5. The method of claim 1, wherein the shape of the mold is cylindrical or hexahedral.
6. The method of claim 1, wherein the calcium phosphate compound is tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, dicalcium phosphate, hydroxyapatite or a combination thereof.
7. The method of claim 2, wherein the porosity of the scaffold is adjusted to 10 to 90% through compression.
8. 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.
10. The method of claim 8, wherein the drug is an antibiotic, an anticancer agent, an anti-inflammatory agent, or a combination thereof.
11. A material for producing a porous scaffold of calcium phosphate cement composed of calcium phosphate cement powder, alginate solution, and calcium ion aqueous solution, each packaged in a separate container; Kit for the preparation of porous scaffolds of calcium phosphate cement comprising a syringe.
12. 12. The kit for preparing a porous scaffold of calcium phosphate cement according to claim 11, further comprising a mold for scaffold forming.
13. The kit for preparing a porous scaffold of calcium phosphate cement according to claim 11, further comprising a biological protein, a drug, or a combination thereof.
14. The kit for preparing a porous scaffold of calcium phosphate cement of claim 13, wherein the biological protein is bovine serum albumin, lysozyme, growth factor, or a combination thereof.
15. The kit for preparing a porous scaffold of calcium phosphate cement according to claim 13, wherein the drug is an antibiotic, an anticancer agent, an anti-inflammatory agent or a combination thereof.

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