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WO2013012132A1 - Procédé de fabrication d'un échafaudage poreux en ciment de phosphate de calcium - Google Patents

Procédé de fabrication d'un échafaudage poreux en ciment de phosphate de calcium Download PDF

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WO2013012132A1
WO2013012132A1 PCT/KR2011/008095 KR2011008095W WO2013012132A1 WO 2013012132 A1 WO2013012132 A1 WO 2013012132A1 KR 2011008095 W KR2011008095 W KR 2011008095W WO 2013012132 A1 WO2013012132 A1 WO 2013012132A1
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calcium phosphate
scaffold
phosphate cement
alginate
porous scaffold
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PCT/KR2011/008095
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English (en)
Korean (ko)
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김해원
이길수
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단국대학교 산학협력단
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Publication of WO2013012132A1 publication Critical patent/WO2013012132A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/01—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products based on oxide ceramics
    • C04B35/447—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products based on oxide ceramics based on phosphates, e.g. hydroxyapatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • 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 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).
  • CPCs Calcium phosphate cements
  • 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; Hutraum 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 (Hutraum DW., Biomater , 2000, 21, 2529-43; Vacanti JP. Et al., Science , 1993, 260, 920-6).
  • controlled release of therapeutic molecules, such as growth factors is beneficial to modulate cell function and promote bone formation.
  • 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.
  • 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.
  • Another object of the present invention is to provide a kit for preparing a porous scaffold of calcium phosphate cement.
  • the present invention provides a method for producing a porous scaffold of calcium phosphate cement comprising the following steps.
  • the method further comprises the step of mechanically compressing after step 2).
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the biological protein is bovine serum albumin, lysozyme, growth factor and the like
  • the drug may be an antibiotic, an anticancer agent, an anti-inflammatory agent, or the like.
  • 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.
  • 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 biological protein is bovine serum albumin, lysozyme and the like
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BSA and lysozyme biological proteins
  • 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.
  • CPC calcium phosphate cement
  • CPA alginate composite
  • 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 ⁇ CT.
  • 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.
  • 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.
  • ALP basic phosphatase
  • 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.
  • ⁇ -tricalcium phosphate ( ⁇ -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 ⁇ -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 ⁇ m and then stored under vacuum for use.
  • ⁇ -TCP As measured using a particle size analyzer (Saturn DigiSizer 5200, Micromeritics, USA), the average particle size of ⁇ -TCP was 4.79 ⁇ m.
  • 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 1 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 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).
  • 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.
  • the diameter of the fiber could be adjusted by changing the composition of the needle gauge and suspension as shown in FIG. 2A.
  • different gauge needles 23, 25, 26 and 27 G
  • compositions CPA20 and CPA15
  • 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.
  • 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.
  • 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).
  • SEM scanning electron microscopy
  • composition changes were monitored via an energy dispersive spectrometer (EDS) (Bruker SNE-3000 M) in a scanning electron microscope.
  • EDS energy dispersive spectrometer
  • 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 ( ⁇ CT) (Skyscan model 1172). Discs ( ⁇ 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 ⁇ m.
  • the surface charge of the ⁇ -TCP particles was investigated by measuring zeta potential (Zetasizer ZEN3600, Malvern Instruments).
  • the ⁇ -TCP particles were sieved (45 ⁇ m) 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.
  • DMA dynamic mechanical analyzer
  • the pore structure of the 3-D composite scaffold was examined by ⁇ CT and shown in FIG. 2B.
  • low porosity scaffolds porosity ⁇ 14%), some pores appeared to be blocked by compression.
  • Medium porosity scaffolds ⁇ 34% porosity
  • 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.
  • 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.
  • Phase change of the CPC and CPC-alginate scaffolds was monitored during the immersion test (FIG. 3B). Initially only ⁇ -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 ⁇ -TCP to HA. After 7 days only HA phase was observed, indicating complete conversion.
  • 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.
  • 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.
  • 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 ⁇ -minimum essential medium ( ⁇ -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.
  • MSCs Mesenchymal stem cells
  • the pellets are ground and contain a homeostatic / antifungal solution (10,000 U penicillin, 10,000 ⁇ g streptomycin, and 25 ⁇ g amphotericin B / m, Gibco) at 37 ° C. under an atmosphere of 5% CO 2 /95% air.
  • a homeostatic / antifungal solution 10,000 U penicillin, 10,000 ⁇ g streptomycin, and 25 ⁇ g amphotericin B / m, Gibco
  • ⁇ -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.
  • MTS reagent tetrazolium salt
  • MTS tetrazolium salt
  • 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.
  • 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.
  • 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.
  • 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 TM). Sections 4-6 ⁇ m 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.
  • H & E hematocillin and eosin
  • MT Marthon's trichrome
  • FIG. 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.
  • 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.
  • 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 ⁇ g mg scaffold- 1 . 1 g of protein-containing porous scaffold was used for protein release testing.
  • BSA bovine serum albumin
  • 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.

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Abstract

La présente invention concerne un procédé de fabrication d'un échafaudage poreux en ciment de phosphate de calcium (CPC) et, plus spécifiquement, un procédé de fabrication d'un échafaudage poreux constitué de ciment de phosphate de calcium par préparation d'une suspension du ciment de phosphate de calcium et d'alginate, et injection de la suspension dans un moule qui est rempli d'une solution aqueuse d'ions de calcium, et durcissement de celle-ci.
PCT/KR2011/008095 2011-07-15 2011-10-27 Procédé de fabrication d'un échafaudage poreux en ciment de phosphate de calcium WO2013012132A1 (fr)

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KR1020110070630A KR101686683B1 (ko) 2011-07-15 2011-07-15 인산 칼슘 시멘트의 다공성 스캐폴드 제조방법
KR10-2011-0070630 2011-07-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836594A (zh) * 2017-11-24 2019-06-04 杭州德柯医疗科技有限公司 一种水凝胶、水凝胶的制备方法及其应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101712555B1 (ko) 2015-10-27 2017-03-07 주식회사 썬메디칼 조직공학용 다공성 스캐폴드 조성물 및 그 제조방법
KR20180062132A (ko) * 2016-11-30 2018-06-08 안동대학교 산학협력단 이중 공극이 형성된 3차원 세라믹 인공 지지체용 조성물
KR102422432B1 (ko) * 2019-11-29 2022-07-19 단국대학교 천안캠퍼스 산학협력단 실리케이트-쉘화된 하이드로겔 섬유상 스케폴드 및 이의 제조 방법
WO2024136602A1 (fr) * 2022-12-22 2024-06-27 주식회사 바이오트리 Suspension pour durcissement à basse température de corps poreux en céramique, et procédé de fabrication de corps poreux en céramique

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030082808A1 (en) * 1998-11-13 2003-05-01 Limin Guan Composite biodegradable polymer scaffold
US20090048145A1 (en) * 2004-06-09 2009-02-19 Scil Technology Gmbh In situ hardening paste, its manufacturing and use
WO2010021601A1 (fr) * 2008-08-22 2010-02-25 Agency For Science, Technology And Research Fabrication et utilisation d'échafaudages composites
US20110066242A1 (en) * 2007-02-12 2011-03-17 The Trustees Of Columbia University In The City Of New York Biomimmetic nanofiber scaffold for soft tissue and soft tissue-to-bone repair, augmentation and replacement

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030082808A1 (en) * 1998-11-13 2003-05-01 Limin Guan Composite biodegradable polymer scaffold
US20090048145A1 (en) * 2004-06-09 2009-02-19 Scil Technology Gmbh In situ hardening paste, its manufacturing and use
US20110066242A1 (en) * 2007-02-12 2011-03-17 The Trustees Of Columbia University In The City Of New York Biomimmetic nanofiber scaffold for soft tissue and soft tissue-to-bone repair, augmentation and replacement
WO2010021601A1 (fr) * 2008-08-22 2010-02-25 Agency For Science, Technology And Research Fabrication et utilisation d'échafaudages composites

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GIL-SU LEE ET AL.: "Alginate combined calcium phosphate cements: mechanical properties and in vitro rat bone marrow stromal cell responeses", J MATER SCI: MATER MED, vol. 22, 2011, pages 1257 - 1268 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836594A (zh) * 2017-11-24 2019-06-04 杭州德柯医疗科技有限公司 一种水凝胶、水凝胶的制备方法及其应用

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