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WO2007129828A1 - Procédé permettant de préparer une éponge poreuse d'acide hyaluronique pour système de fourniture de cellules - Google Patents

Procédé permettant de préparer une éponge poreuse d'acide hyaluronique pour système de fourniture de cellules Download PDF

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Publication number
WO2007129828A1
WO2007129828A1 PCT/KR2007/002162 KR2007002162W WO2007129828A1 WO 2007129828 A1 WO2007129828 A1 WO 2007129828A1 KR 2007002162 W KR2007002162 W KR 2007002162W WO 2007129828 A1 WO2007129828 A1 WO 2007129828A1
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Prior art keywords
hyaluronic acid
sponge
porous
cells
acid sponge
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PCT/KR2007/002162
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English (en)
Inventor
Dae Duk Kim
Jeong Yeon Kang
Chung Wook Chung
In Soo Yoon
Sun Young Kim
Byung Soon Park
Jong Hyuk Sung
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Seoul National University Industry Foundation
Prostemics Co Ltd
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Seoul National University Industry Foundation
Prostemics Co Ltd
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Priority to US12/299,496 priority Critical patent/US8193340B2/en
Priority claimed from KR1020070042611A external-priority patent/KR100891373B1/ko
Publication of WO2007129828A1 publication Critical patent/WO2007129828A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • 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
    • A61L27/20—Polysaccharides
    • 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

Definitions

  • the present invention relates to preparation method of a porous hyaluronic acid sponge, more particularly to a preparation method of a porous hyaluronic acid sponge which supports cell growth inside the sponge, serves as scaffold for cell delivery and can be administrated through various administration routes.
  • Hyaluronic acid is a naturally occurring linear polysaccharide. It is a polymer of disaccharide units composed of ⁇ -l,4-D-glucuronic acid and N-acetyl-D-glucosamine linked together by ⁇ -l,3-glycosidic bonds. Hyaluronic acid has a molecular weight ranging from 1,000 to 10,000,000 daltons.
  • Hyaluronic acid is the only non-sulfated glucosaminoglycan (GAG) found in the extracellular matrix (ECM) of higher animals.
  • Biomaterials derived from hyaluronic acid are used, for example, in regeneration of cartilage and skin. Also, they play key roles in drug delivery or surgery. Chemically unmodified hyaluronic acid is utilized as the aid for the delivery of ophthalmologic drug to improve the absorption of drugs and proteins at the mucosal tissue.
  • hyaluronic acid improves lubrication at the joint surface and thus reduces pain.
  • Arthritis reduces the production of hyaluronic acid and the increased breakdown by proteases further reduces hyaluronic acid in the joint.
  • the articular damage can be aggravated because external impact cannot be adequately absorbed or distributed at the joint.
  • hyaluronic acid solution With compositions similar to those of the joint fluid, hyaluronic acid solution regains viscosity and elasticity when injected into the joint and improves lubrication and absorbs impact, thereby protecting the joint cartilage and preventing further damage.
  • hyaluronic acid can be utilized as scaffold for cell delivery in the process of cell transplantation therapy of chondrocytes for cartilage tissue regeneration.
  • Hyaluronic acid synthase is an integral membrane enzyme, which synthesizes hyaluronic acid polymers and excretes them out of the cell. In mammals, HASl, HAS2 and HAS3 isoenzymes are found.
  • Hyaluronidase is an enzyme that breaks down hyaluronic acid. Depending on the activation pH, it can be classified into neutral hyaluronidase and acidic hyaluronidase. Neutral hyaluronidase (PH-20) is specifically found in the testis (sperm) and shows activity in the physiological pH.
  • Acidic hyaluronidase (Hyall-4) is found in body fluids as well as in a variety of human organs, including spleen, cartilage, skin, eyes, liver, kidney, bladder, placenta, etc., and shows activity around at pH 3. Since this enzyme is present in the lysosome, hyaluronic acid is transported into the cytoplasm by endocytosis and degraded.
  • CD44 The primary cell surface receptor for HA is CD44.
  • CD44 is a transmembrane glycoprotein and binds primarily to hyaluronic acid. It is expressed in most of human cell membranes. The hyaluronic acid fragments degraded at the lysosome induce different signal transfers depending on their size, affecting cell proliferation and differentiation.
  • chondrocytes cultured in 3D condition maintain their characters and functions, resulting in spherical cell shape. That is, a 3-dimensional scaffold helps the cell attachment and cartilage formation in vitro and in vivo.
  • porous scaffolds are very useful in that chondrocytes can be cultured under a restricted condition with significantly reduced necrosis of the tissue.
  • a scaffold in addition to cell delivery, can serve as mold to fill lesion site of tissue.
  • An ideal scaffold is required to be non-immunogenic, non-toxic, biocompatible, biodegradable and easy to manipulate.
  • the pore size of the scaffold is important. An inadequate pore size may result in the restricted nutrient supply to the cells being cultured in the scaffold or ineffective removal of the wastes from the cells.
  • the pore size is too small, the cells may not be completely filled inside the scaffold, which negatively affects the generation of the tissue. In contrast, if the pore size is too large, not all the extracellular matrix proteins synthesized by cells are ac- cumulated. That is, the proteoglycan and collagen accumulated in the scaffold can be found only a part of the total degree of actually synthesized ECM.
  • the scaffold may deem as inadequate for the growth of chondrocytes.
  • control of the pore size is important in improving the retention rate of newly synthesized extracellular matrix molecules inside the scaffold.
  • the present inventors completed the present invention by developing a porous hyaluronic acid sponge which enables stable cell cultivation, offers controllable mi- croenvironment for cell culture through controlling the pore size of the sponge and allows cell delivery through various administration routes including injection.
  • an object of the present invention is to provide a preparation method of a porous hyaluronic acid sponge the pore size of which can be controlled to ensure cell growth.
  • Another object of the present invention is to provide a preparation method of a gel- state porous hyaluronic acid sponge comprising hyaluronic acid, an aqueous sodium hydroxide solution and an epoxy-based crosslinking agent with given compositions, which allows the delivery of the entire scaffold including cells to a disease site through various administration routes other than surgical operation.
  • Still another object of the present invention is to provide a porous hyaluronic acid sponge prepared by the aforementioned methods and a cell delivery system using the same.
  • the present invention provides a preparation method of a porous hyaluronic acid sponge comprising the steps of: dissolving hyaluronic acid in an aqueous sodium hydroxide solution; adding an epoxy-based crosslinking agent to the aqueous sodium hydroxide solution in which hyaluronic acid is dissolved and homogenizing the hyaluronic acid solution; hydrogelating the homogenized hyaluronic acid solution; washing the hydrogelated hyaluronic acid hydrogel with ultrapure water; swelling the washed hyaluronic acid hydrogel to obtain porosity; and freeze-drying the hyaluronic acid hydrogel to obtain a porous hyaluronic acid sponge.
  • the preparation method of a porous hyaluronic acid sponge in accordance with the present invention offers a scaffold in which cells can be cultured stably, provides pores which are essential for the cell survival through swelling and allows the control of the microenvironment for cell culture through the control of the pore size of the porous hyaluronic acid sponge with the swelling time.
  • the sponge can be prepared not only into solid but also into gel, cell delivery to the disease site can be accomplished through various administration routes, not only by surgical operation but also by direct injection, application on skin, and so forth.
  • FIG. 1 illustrates a flowchart for the preparation method of a porous hyaluronic acid sponge according to the present invention.
  • Fig. 2 shows the photograph of the porous hyaluronic acid sponge prepared by the preparation method of a porous hyaluronic acid sponge according to the present invention.
  • Fig. 3 is the scanning electron micrograph (SEM) of the porous hyaluronic acid sponge shown in Fig. 2.
  • SEM scanning electron micrograph
  • FIG. 4 shows the photograph of the gellable porous hyaluronic acid sponges according to the present invention after 2 days of swelling (each of 1.25 g and 2.50 g of hyaluronic acid was added to each of upper and lower rows; the PEGDG contents were 1.0 mL, 2.5 mL and 5.0 mL from left to right).
  • Fig. 5 shows the photograph of taking cells and the gellable porous hyaluronic acid sponge of the present invention after culturing the cells for 4 weeks in the sponge with a syringe.
  • Fig. 6 shows the scanning electron micrograph of the porous hyaluronic acid sponge prepared in Example 1.
  • Fig. 6 shows the scanning electron micrograph of the porous hyaluronic acid sponge prepared in Example 1.
  • FIG. 7 shows the scanning electron micrograph of the porous hyaluronic acid sponge prepared in Example 2.
  • Fig. 8 shows the scanning electron micrograph of the porous hyaluronic acid sponge prepared in Example 3.
  • Fig. 9 shows the scanning electron micrograph of the porous hyaluronic acid sponge prepared in Example 4.
  • Fig. 10 shows the scanning electron micrograph of the porous hyaluronic acid sponge prepared in Example 5.
  • Fig. 11 shows the scanning electron micrograph of the chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 2.
  • Fig. 11 shows the scanning electron micrograph of the chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 2.
  • FIG. 12 shows the optical micrograph of the chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 2 after the MTT staining.
  • Fig. 13 is a graph showing the change of the number of living chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 2 with time.
  • Fig. 14 shows the RT-PCR result for the aggrecan (a) and type II collagen (b) in the stem cells to be differentiated into chondrocytes in the porous hyaluronic acid sponge prepared in Example 2.
  • Fig. 15 shows the inside of the Alcian blue-stained porous hyaluronic acid sponge prepared in Example 2 [45] Fig.
  • FIG. 16 shows the graphs showing the change of the number of living chondrocytes cultured in the porous hyaluronic acid sponges prepared in Examples 6 to 8 with time.
  • Fig. 17 shows the RT-PCR result for the aggrecan (a) and type II collagen (b) in the stem cells to be differentiated into chondrocytes in the porous hyaluronic acid sponges prepared in Examples 6 to 8.
  • Fig. 18 is a graph showing the change of the number of living chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 9 with time.
  • Fig. 19 shows the scanning electron micrograph of the hyaluronic acid sponge prepared in Example 10.
  • Fig. 20 shows the graphs showing the change of the number of living chondrocytes after being cultured in the gellable porous hyaluronic acid sponges prepared in
  • the present invention provides a preparation method of a porous hyaluronic acid sponge comprising the steps of: dissolving hyaluronic acid in an aqueous sodium hydroxide solution; adding an epoxy-based crosslinking agent to the aqueous sodium hydroxide solution in which hyaluronic acid is dissolved and homogenizing the hyaluronic acid solution; hydrogelating the homogenized hyaluronic acid solution; washing the hydrogelated hyaluronic acid hydrogel with ultrapure water; swelling the washed hyaluronic acid hydrogel to obtain porosity; and freeze-drying the hyaluronic acid hydrogel to obtain a porous hyaluronic acid sponge.
  • hyaluronic acid having a number- average molecular weight ranging from 1,000,000 to 5,000,000 daltons (Da) is used.
  • the concentration of the aqueous sodium hydroxide solution used in the dissolution step is preferably from ⁇ .05 to 1.0 N, more preferably from 0.1 to 0.3 N.
  • the hydrogelation of the homogenized hyaluronic acid solution is preferably performed at 40 to 80 0 C for 1 to 6 hours, more preferably at 55 to 65 0 C for 1 to 3 hours.
  • the swelling of the hyaluronic acid hydrogel is preferably performed for 1 to 7 days using ultrapure water.
  • the epoxy-based crosslinking agent used in the homogenization step is selected from the group consisting of polyethylene glycol diglycidyl ether, epichlorohydrin, methyl glycidyl ether, phenyl glycidyl ether, lauryl alcohol glycidyl ether, ethylene glycol dimethacrylate, 1,4-butanediol diglycidyl ether and ethylene glycol diglycidyl ether.
  • the present invention also provides a porous hyaluronic acid sponge which is prepared by the aforesaid preparation method and has a pore size of 30 to 500 D.
  • the present invention further provides a cell delivery system using the aforesaid porous hyaluronic acid sponge as scaffold for delivering cells.
  • the cells are selected from the group consisting of chondrocytes, stem cells, neurocytes, brain cells, myocytes, sensory cells and blood cells.
  • the preparation method of a porous hyaluronic acid sponge according to the present invention comprises a dissolution step (SlOl), a homogenization step (S 102), a hy- drogelation step (S 103), a washing step (S 104), a swelling step (S 105) and a freeze- drying step (S106).
  • the dissolution step (SlOl) is the step of dissolving hyaluronic acid in a solvent to obtain a hyaluronic acid solution.
  • the solvent may be an aqueous sodium hydroxide solution having a concentration of 0.05 N to 1.0 N, preferably an aqueous sodium hydroxide solution having a concentration of 0.1 N to 0.3 N.
  • the pore size of the porous hyaluronic acid sponge prepared according to the present invention can be controlled by changing the concentration of the sodium hydroxide solution.
  • concentration of the aqueous sodium hydroxide solution is below 0.05 N, the sponge becomes too soft, resulting in problems with regard to cell injection and cultivation. In contrast, if the concentration of the aqueous sodium hydroxide solution exceeds 1.0 N, the sponge becomes too stiff, resulting in a scaffold that cannot be seen as sponge.
  • the homogenization step (S 102) is the step in which the dissolved hyaluronic acid solution is homogenized after adding a crosslinking agent. The homogenization is performed for 5 to 20 minutes using a mechanical stirrer at 800 to 1200 rpm.
  • a porous hyaluronic acid sponge is prepared without adding a crosslinking agent, a porous hyaluronic acid sponge which is readily soluble in water is attained.
  • a porous hyaluronic acid sponge cannot serve as stable scaffold for cell cultivation. Accordingly, in the preparation method of a porous hyaluronic acid sponge according to the present invention, the step of adding a crosslinking agent is performed to improve the water insolubility of the porous hyaluronic acid sponge.
  • a non-cytotoxic and economical epoxy compound for example, selected from the group consisting of polyethylene glycol diglycidyl ether, epichlorohydrin, methyl glycidyl ether, phenyl glycidyl ether, lauryl alcohol glycidyl ether, ethylene glycol dimethacrylate, 1,4-butanediol diglycidyl ether and ethylene glycol diglycidyl ether is used.
  • the epoxy-based crosslinking agent is preferably used in 1 to 50 parts by weight per 100 parts by weight of the hyaluronic acid solution. If the epoxy-based crosslinking agent is used less than the aforesaid range, the sponge may become useless because of inadequate crosslinking.
  • a porous hyaluronic acid sponge that transforms into a gel phase after a given period of the cell cultivation is attained when 3 to 10 parts by weight of an epoxy-based crosslinking agent is used per 100 parts by weight of the hyaluronic acid solution.
  • the hydrogelation step (S 103) is the step in which the solution homogenized in the homogenization step (S 102) is hydrogelated by crosslinking.
  • the hydrogelation step (S 103) is performed preferably at 40 to 80 0 C for 1 to 6 hours, more preferably at 55 to 65 0 C for 1 - 3 hours.
  • the washing step (S 104) is the step in which the hydrogelated hyaluronic acid hydrogel is washed.
  • unreacted residues of the hyaluronic acid hydrogel for example, excess crosslinking agent and sodium hydroxide, are removed to provide an environment adequate for cell cultivation.
  • ultrapure water is used for the washing.
  • the swelling step (S 105) is the step in which the washed hyaluronic acid hydrogel is swollen.
  • the swelling step (S 105) is performed by swelling the hyaluronic acid hydrogel in ultrapure water for 1 to 7 days.
  • the solution used in the washing step (S 104) and the swelling step (S 105) is not limited to ultrapure water.
  • Various buffer solutions may be used, too.
  • a hyaluronic acid sponge When a hyaluronic acid sponge is prepared by swelling the hyaluronic acid hydrogel for less than 1 day, the pore size may change as the hyaluronic acid sponge swells further by the medium used for cell cultivation.
  • the swelling step is omitted, the pore size becomes so small that the cells cannot be held inside the scaffold. Further, the supply of nutrients to cells or the removal of wastes may be ineffective.
  • Fig. 19 shows the scanning electron micrograph of the hyaluronic acid sponge prepared without the swelling step
  • Fig. 20 shows the graphs showing the change of the number of living chondrocytes after being cultured in the gellable porous hyaluronic acid sponges [(a), (b)] and the porous hyaluronic acid sponges (c) prepared without the swelling step for 1 day.
  • the freeze-drying step (S 106) is the step in which the hyaluronic acid hydrogel is freeze-dried to obtain a porous hyaluronic acid sponge.
  • the freeze drying is performed in a freezer of -20 to -152 0 C for about 24 hours and then at -50 to -80 0 C and 8 to 15 DHg for at least 48 to 72 hours.
  • Fig. 2 The resultant hyaluronic acid sponge is shown in Fig. 2.
  • Fig. 3 shows the scanning electron micrograph of the hyaluronic acid sponge.
  • the porous hyaluronic acid sponge (100) has a plurality of pores (101) inside.
  • porous hyaluronic acid sponge according to the present invention is generally in solid state
  • a solid porous hyaluronic acid sponge prepared with 3 to 10 parts by weight of an epoxy-based crosslinking agent per 100 parts by weight of the hyaluronic acid solution obtained by dissolving hyaluronic acid in an aqueous sodium hydroxide solution may be transformed into a fluid, gel-state porous hyaluronic acid sponge by cultivation for a given time.
  • the phase transformation of the sponge can be attained by optimizing the proportion of the hyaluronic acid, the aqueous sodium hydroxide solution and the epoxy-based crosslinking agent.
  • a required fluidity may be attained at the time of administration.
  • Fig. 4 shows the photograph of the porous hyaluronic acid sponge after 2 days of swelling.
  • Fig. 5 shows the photograph of taking cells and the porous hyaluronic acid sponge after culturing the cells for 4 weeks with a 17 gauge syringe.
  • the gellable porous hyaluronic acid sponge according to the present invention is fluid and thus can be taken with a syringe. Consequently, it can be delivered to a disease site using a cell-scaffold structure without surgical operation or directly applied to skin or mucosa. As a result, the porous hyaluronic acid sponge can be utilized widely.
  • the porous hyaluronic acid sponge (100) of the present invention has a pores (101) ranging from 30 to 500 D.
  • chondrocytes, stem cells, neurocytes, brain cells, myocytes, sensory cells or blood cells may be proliferated, differentiated and injected to the disease site for treatment.
  • chondrocytes may be proliferated and differentiated using the porous hyaluronic acid sponge of the present invention as scaffold and then injected to a damaged cartilage to treat arthritis.
  • a preferable pore size of the pores (101) is dependent on the cells, but 30 to 500 D is preferable. If the pore size of the pores (101) is smaller than 30 D, there may be a problem in supplying nutrients to the cells or removing wastes.
  • the pore size of the pores (101) is larger than 500 D, the cells are not properly attached to the hyaluronic acid sponge (100) and the cells may not be differentiated properly even if the cells cultured in the sponge are in metabolically activated state.
  • the swollen hyaluronic acid hydrogels were frozen in a -80 0 C freezer for 24 hours and then freeze- dried at -50 0 C and 10 DHg for 48 hours to obtain hyaluronic acid sponges.
  • the porous hyaluronic acid sponge prepared in Example 1 is shown in Fig. 6 and the porous hyaluronic acid sponge prepared in Example 2 is shown in Fig. 7.
  • pores (201) are formed inside the porous hyaluronic acid sponge (200) prepared in Example 1.
  • pores (301) are formed inside the porous hyaluronic acid sponge (300) prepared in Example 2.
  • Example 5 The hyaluronic acid hydrogels prepared in Example 3, Example 4 and Example 5 were frozen in a -80 0 C freezer for 24 hours and then freeze-dried at -50 °C and 10 DHg for 48 hours to obtain hyaluronic acid sponges.
  • pores (410) are formed inside the porous hyaluronic acid sponge (400) prepared in Example 3. Pores (510) are also formed inside the porous hyaluronic acid sponge (500) prepared in Example 4. Pores (610) are formed inside the porous hyaluronic acid sponge (600) prepared in Example 5, too.
  • Example 3 Example 4 and Example 5, PBS solution was used in the swelling step.
  • the use of the PBS solution resulted in less swelling and increased strength of the porous hyaluronic acid sponge obtained following the freeze drying than when ultrapure water was used.
  • the chondrocytes were cell lines obtained from the chondrocytes of the human rib and had been distributed from the Biochemical Lab of Kyungpook National University.
  • the Karnovsky's fixative was prepared by dissolving 2 wt% of glu- taraldehyde, 2 wt% of paraformaldehyde and 0.5 wt% of calcium chloride (CaCl ) in a 0.05 M cacodylate buffer (pH 7.4).
  • FIG. 11 shows the scanning electron micrograph (SEM) of the chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 2. It can be seen that there are numbers of chondrocytes (302) in the pores of the porous hyaluronic acid sponge (300).
  • MTT stains living cells with deep purple color.
  • Fig. 12 shows the optical micrograph of the chondrocytes cultured in the porous hyaluronic acid sponge seen in Fig. 7. Referring to Fig. 12, it can be seen that there are numbers of living chondrocytes (302) inside the pores of the porous hyaluronic acid sponge (300).
  • an MTT solution (0.5 mg/mL) was added to the sample. Then, the sample was kept at 37 0 C and 5 % CO for 4 hours to stain the living cells.
  • Fig. 13 is a graph showing the change of the number of living chondrocytes cultured in the porous hyaluronic acid sponge prepared in Example 2.
  • the abscissa is the incubation time and the ordinate is the absorbance of samples at 560nm.
  • ADSCs stem cells obtained from the human fat tissue (processed lipoaspirate, PLA) are differentiated into chondrocytes inside the pores of the porous hyaluronic acid sponge.
  • the stem cells were undifferentiated cell lines obtained from the human fat tissue through liposuction and had been distributed from the Prostemics.
  • the samples for the differentiation test were prepared by seeding the stem cells inside the porous hyaluronic acid sponge, culturing in a cell incubator at 5 % CO and 37 0 C for 7 days, 10 days and 14 days using a DMEM medium containing 1 % serum, TGF-Bl (10 ng/ mL), ascorbate (5OnM), ITS (5D/mL) and dexamethasone (100 nM), transferring the cell-sponge system into a 1.5 niL Eppendorf tube, washing off the excess medium with PBS and cooling at -80 0 C.
  • a lysis buffer was added to the samples to extract RNAs from the cells.
  • a reaction solution containing PCR buffer, dNTP mixture, RNase inhibitor and oligo dT primer was prepared. After adding the RNAs to the reaction solution, reverse transcription of synthesizing DNA from RNA was performed for 30 minutes at 60 0 C, for 5 minutes at 99 0 C and for 5 minutes at 5 0 C.
  • ADSCs stem cells derived from the human fat cells were differentiated into chondrocytes inside the hyaluronic acid sponge can be confirmed by the presence of type II collagen gene or aggrecan gene.
  • RT-PCR was performed using the pair of the nucleotide sequences of SEQ ID NO:
  • SEQ ID NO: 1 and SEQ ID NO: 2 as a primer in order to utilize the aggrecan gene as chondrogenic differentiation marker and using the pair of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 as another primer in order to utilize the type II collagen gene as differentiation marker.
  • the RT-PCR result is shown in Fig. 14.
  • Fig. 14 shows the RT-PCR result for the stem cells to be differentiated into chondrocytes in the porous hyaluronic acid sponge prepared in Example 2.
  • lane 1 is for 100 bp marker and lane 2 is the RT-PCR result for the
  • Lanes 3 and 4 in Fig. 14 are the RT-PCR for the RNAs extracted from the stem cells that have been cultured for 10 days and 14 days, respectively.
  • the marker for chondrogenic differentiation was stained with Alcian blue to investigate if the chondrocytes maintain their characteristics inside the pores of the porous hyaluronic acid sponge. Alcian blue selectively stains s-GAG (sulfate-glucosaminoglycan), the marker for chondrogenic differentiation.
  • s-GAG sulfate-glucosaminoglycan
  • Chondrocytes were cultured inside the pores of the porous hyaluronic acid sponge and were fixed using 4 % paraformaldehyde at room temperature for 15 minutes. Subsequently, the sample was washed with a PBS solution and stained with Alcian blue at room temperature for 30 minutes.
  • the Alcian blue dye was prepared by dissolving Alcian blue in 0.1 N hydrochloric acid (HCl) to 1 %. After staining, the sample was washed with 0.1 N hydrochloric acid for 5 minutes and observed with an optical microscope.
  • Fig. 15 shows the inside of the Alcian blue-stained porous hyaluronic acid sponge prepared in Example 2.
  • the stem cells are differentiated into chondrocytes inside the pores of the porous hyaluronic acid sponge and maintain their characteristics.
  • the pores of the porous hyaluronic acid sponge provide the environment in which the cells are differentiated into chondrocytes and maintain chondrocytic characteristics.
  • FIG. 5 shows the photograph of taking cells and the gellable porous hyaluronic acid sponge of prepared in Example 6 after culturing chondrocytes for 4 weeks in the sponge with a syringe. It was confirmed that the gellable porous hyaluronic acid sponge turns into fluid gel state when the cells are cultured for a given period of time.
  • Example 6 except that the addition amount of the PEGDG crosslinking agent was changed to 2.5 mL and 5.0 mL.
  • FIG. 4 shows the degree of swelling of the gellable porous hyaluronic acid sponges prepared in Examples 6 to 8. It can be seen that the degree of swelling decreases as the addition amount of the crosslinking agent increases.
  • the bottom row in Fig. 4 shows the degree of swelling of the gellable porous hyaluronic acid sponges prepared by adding each 1.0 mL, 2.5 mL and 5.0 mL of the PEGDG crosslinking agent to 14 mL of a solution in which 2.50 g of hyaluronic acid is dissolved in a 0.3 N aqueous sodium hydroxide solution.
  • the degree of swelling decreases as the addition amount of the crosslinking agent increases.
  • chondrocytes were grown in the gellable porous hyaluronic acid sponges prepared in Examples 6 to 8 for 3weeks and samples were taken.
  • the chondrocytes were cell lines obtained from the joints of New Zeeland white rabbits.
  • stem cells were differentiated into chondrocytes in the gellable porous hyaluronic acid sponge. Also, it can be seen that the chondrogenic differentiation of stem cells occurs actively and constantly since the bands for the aggrecan and type II collagen were thickened with cultivation time.
  • lane 1 is for 100 bp marker and lanes 2 and 3 are the RT-PCR results for the RNAs extracted from the stem cells that have been cultured for 7 days and 14 days, respectively.
  • a hyaluronic acid sponge was prepared in the same manner as in Example 6, except for omitting the swelling step.
  • FIG. 19 shows the scanning electron micrograph of the hyaluronic acid sponge prepared in Example 10. As seen in the figure, pores were hardly formed or, even if they were, the pore size of them was significantly smaller than that that of pores seen in Fig. 3, Fig. 6, Fig. 7, Fig. 8, Fig. 9 and Fig. 10.
  • FIG. 20 shows the graphs showing the change of the number of living chondrocytes after being cultured in the gellable porous hyaluronic acid sponges prepared in Examples 6(a) and 7(b) and the hyaluronic acid sponge prepared in Example 10(c) for 1 day. The number of living chondrocytes was counted in the same manner as in Testing Example 2.
  • the porous hyaluronic acid sponge prepared in accordance with the present invention serves as the scaffold in which cells can be cultured stably. Since the pore size of the porous hyaluronic acid sponge can be controlled, the microenvironment for cell culture can be adjusted. Particularly, cells can be cultured and delivered to the wanted disease site through various administration routes since the porous hyaluronic acid sponge can be prepared into not only solid but also fluid gel form.

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Abstract

L'invention concerne un procédé permettant de préparer une éponge poreuse d'acide hyaluronique, qui comprend les étapes consistant : à dissoudre de l'acide hyaluronique dans une solution aqueuse d'hydroxyde de sodium; à ajouter un agent de réticulation à base d'époxyde à la solution aqueuse d'hydroxyde de sodium obtenue, dans laquelle l'acide hyaluronique est dissous, et à homogénéiser la solution d'acide hyaluronique; à procéder à l'hydrogélification de la solution d'acide hyaluronique homogénéisée; à rincer l'hydrogel d'acide hyaluronique hydrogélifié avec de l'eau ultrapure; à faire gonfler l'hydrogel d'acide hyaluronique rincé jusqu'à ce qu'il atteigne l'état poreux; et à lyophiliser l'hydrogel d'acide hyaluronique afin d'obtenir une éponge poreuse d'acide hyaluronique.
PCT/KR2007/002162 2006-05-04 2007-05-02 Procédé permettant de préparer une éponge poreuse d'acide hyaluronique pour système de fourniture de cellules Ceased WO2007129828A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/299,496 US8193340B2 (en) 2006-05-04 2007-05-02 Preparation method of porous hyaluronic acid sponge for cell delivery system

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20060040652 2006-05-04
KR10-2006-0040652 2006-05-04
KR1020070042611A KR100891373B1 (ko) 2006-05-04 2007-05-02 세포 전달 시스템용 다공성 히아루론산 스펀지의 제조방법
KR10-2007-0042611 2007-05-02

Publications (1)

Publication Number Publication Date
WO2007129828A1 true WO2007129828A1 (fr) 2007-11-15

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PCT/KR2007/002162 Ceased WO2007129828A1 (fr) 2006-05-04 2007-05-02 Procédé permettant de préparer une éponge poreuse d'acide hyaluronique pour système de fourniture de cellules

Country Status (1)

Country Link
WO (1) WO2007129828A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010051783A1 (fr) 2008-11-06 2010-05-14 Cpn S.R.O. Procédé de préparation de dérivés de l'acide hyaluronique réticulé par du dtpa et modification desdits dérivés
US20120095206A1 (en) * 2009-04-09 2012-04-19 Scivision Biotech Inc. Method for producing cross-linked hyaluronic acid
WO2012068248A3 (fr) * 2010-11-16 2012-06-21 Ingeneron, Inc. Procédés de conservation de cellules cibles
US8288129B2 (en) * 2007-11-26 2012-10-16 Body Organ Biomedical Corp. Method for producing hyaluronic acid
WO2014013121A1 (fr) * 2012-07-19 2014-01-23 Universitat Politècnica De València Matériau composé d'acide hyaluronique et d'au moins un polymère acrylique pour applications biomédicales
EP2783702A4 (fr) * 2011-11-24 2015-08-12 Amorepacific Corp Composition de gel hydro-insoluble et procédé de préparation correspondant
CN115246958A (zh) * 2022-07-28 2022-10-28 爱博诺德(北京)医疗科技股份有限公司 具有与组织、细胞生长相适应的孔壁结构的交联透明质酸凝胶

Citations (4)

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KR20000025222A (ko) * 1998-10-09 2000-05-06 서경배 하이아론산 스펀지의 제조방법
JP2000230003A (ja) * 1999-02-12 2000-08-22 Denki Kagaku Kogyo Kk 溶解速度が制御されたヒアルロン酸ゲル及びその製造方法
JP2003055402A (ja) * 2001-08-13 2003-02-26 Shiseido Co Ltd 架橋ヒアルロン酸スポンジの製造方法
KR20050042517A (ko) * 2003-11-03 2005-05-10 박성영 히알루론산을 주재로 한 다공성 스폰지의 제조방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000025222A (ko) * 1998-10-09 2000-05-06 서경배 하이아론산 스펀지의 제조방법
JP2000230003A (ja) * 1999-02-12 2000-08-22 Denki Kagaku Kogyo Kk 溶解速度が制御されたヒアルロン酸ゲル及びその製造方法
JP2003055402A (ja) * 2001-08-13 2003-02-26 Shiseido Co Ltd 架橋ヒアルロン酸スポンジの製造方法
KR20050042517A (ko) * 2003-11-03 2005-05-10 박성영 히알루론산을 주재로 한 다공성 스폰지의 제조방법

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8288129B2 (en) * 2007-11-26 2012-10-16 Body Organ Biomedical Corp. Method for producing hyaluronic acid
WO2010051783A1 (fr) 2008-11-06 2010-05-14 Cpn S.R.O. Procédé de préparation de dérivés de l'acide hyaluronique réticulé par du dtpa et modification desdits dérivés
US20120095206A1 (en) * 2009-04-09 2012-04-19 Scivision Biotech Inc. Method for producing cross-linked hyaluronic acid
US9371402B2 (en) * 2009-04-09 2016-06-21 Scivision Biotech Inc. Method for producing cross-linked hyaluronic acid
WO2012068248A3 (fr) * 2010-11-16 2012-06-21 Ingeneron, Inc. Procédés de conservation de cellules cibles
EP2783702A4 (fr) * 2011-11-24 2015-08-12 Amorepacific Corp Composition de gel hydro-insoluble et procédé de préparation correspondant
WO2014013121A1 (fr) * 2012-07-19 2014-01-23 Universitat Politècnica De València Matériau composé d'acide hyaluronique et d'au moins un polymère acrylique pour applications biomédicales
ES2443544A1 (es) * 2012-07-19 2014-02-19 Universidad Politécnica De Valencia Material compuesto de ácido hialurónico y al menos un polímero acrílico para aplicaciones biomédicas
CN115246958A (zh) * 2022-07-28 2022-10-28 爱博诺德(北京)医疗科技股份有限公司 具有与组织、细胞生长相适应的孔壁结构的交联透明质酸凝胶

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