WO2009086743A1 - 配制生物材料的方法 - Google Patents

配制生物材料的方法 Download PDF

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Publication number
WO2009086743A1
WO2009086743A1 PCT/CN2008/070309 CN2008070309W WO2009086743A1 WO 2009086743 A1 WO2009086743 A1 WO 2009086743A1 CN 2008070309 W CN2008070309 W CN 2008070309W WO 2009086743 A1 WO2009086743 A1 WO 2009086743A1
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WIPO (PCT)
Prior art keywords
fish scale
biological material
formulating
material according
fish
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/CN2008/070309
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English (en)
French (fr)
Inventor
Horngji Lai
Chiencheng Lin
Shangming Lin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Body Organ Biomedical Corp
Original Assignee
Body Organ Biomedical Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Body Organ Biomedical Corp filed Critical Body Organ Biomedical Corp
Priority to EP18150067.9A priority Critical patent/EP3329944B1/en
Priority to ES08706682.5T priority patent/ES2692537T3/es
Priority to JP2010541677A priority patent/JP5301565B2/ja
Priority to EP08706682.5A priority patent/EP2241606B1/en
Priority to BRPI0819960A priority patent/BRPI0819960B8/pt
Priority to KR1020107017516A priority patent/KR101816139B1/ko
Publication of WO2009086743A1 publication Critical patent/WO2009086743A1/zh
Priority to IL206873A priority patent/IL206873A/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • 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
    • A61L27/3641—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 characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention relates to a biological material and a method of preparing the same, and more particularly to a method of formulating a biological material from a decellularized fish scale, which can be used for tissue repair and transplantation. Background technique
  • Tissue engineering is closely related to biological and pharmaceutical biomaterials, which can enhance angiography, tissue integration, and tissue reconstruction in patients undergoing transplant surgery; biomaterials are artificially synthesized materials used to reconstruct artificial organs, repair or repair, Even replace human tissue.
  • HAP hydroxyapatite
  • TCP tricalcium phosphate
  • Another object of the present invention is to provide a biomaterial as described above for repairing tissue and tissue transplantation.
  • Another object of the present invention is to provide a method of formulating a biological material as described above.
  • the present invention provides a method of formulating a biological material from fish scales, the method comprising the steps of: decellularizing fish scales and then grinding them to a quantity of particles, wherein the particles comprise sea A mixture of a matrix and a powder.
  • the method of formulating a biological material of the present invention may further comprise: extruding the sponge matrix and powder to form a sheet or a block at a temperature of 200 ° C or lower or a specific shape by a specific mold.
  • the step of dehydrating the fish scale may be included before the fish scale is ground.
  • the dewatering step can include dehydrating the fish scale until the water content is less than 50%.
  • the step of washing the scales may also be included prior to the step of dewatering the scales.
  • the step of decellating the fish scale may be before the step of washing the fish scale.
  • the step of decellularizing the fish scales may also be prior to the step of dewatering the scales.
  • the method of formulating a biological material of the present invention may further comprise the step of separating the sponge matrix and the powder from the particles.
  • the raw fish scales in the present invention may be fish scales having an average diameter of less than 20 cm, such as fish scales of a large size (10-20 cm), or smaller scale fish scales.
  • the focus is on retaining the natural bond and the three-dimensional structure of the fish scale biological material.
  • the degree of grinding of the fish scale is not particularly limited, and the fish scale is generally ground to an average particle diameter of less than 10,000 ⁇ m. If the fish scale material used has an average diameter of less than 10,000 ⁇ m, it needs to be properly ground and ground to a smaller particle size, for example, it can be ground to an average particle diameter of less than 10 ⁇ m.
  • the present invention also provides another method of formulating a biological material from a fish scale, the method comprising the steps of: decellularizing the fish scale, and then subjecting it to a pressing process to form a sheet or a block or obtaining a material of a specific shape through a specific mold. .
  • the extrusion process is carried out at a temperature of 200 ° C or lower.
  • the step of washing the scales may be included before the fish scale extrusion process.
  • the step of dehydrating the scales may also be included after the washing step is completed and before the fish scale extrusion process.
  • the dewatering step comprises dehydrating the fish scale until the water content is less than 50%.
  • the step of decellularizing the fish scale may be before the step of washing the fish scale.
  • the step of decellularizing the fish scales may also be prior to the step of dewatering the fish scales. It may also include soaking fish scales before the fish scale extrusion process The steps in the water.
  • the formulating process comprises dehydrating the decellularized fish scale until the water content is less than 50%, and grinding the dehydrated fish scale until the ground particle diameter
  • the average size is less than 10,000 ⁇ m, wherein each of the ground particles comprises a sponge matrix and a mixture of powders.
  • the fish scale is dewatered to a moisture content of less than 25% and the average diameter of the ground particles is less than 5000 ⁇ m.
  • the process of formulating a biomaterial from fish scales of the present invention includes treating at a temperature below 200 °C.
  • the invention also provides a biomaterial prepared from fish scales using the method.
  • the biological material prepared by the method of the invention retains the natural bond and the three-dimensional structure of the fish scale biological material, and the biological material has better mechanical support force, can be used for repairing tissue and tissue transplantation, and is repaired and transplanted in tissue. In the process, the probability of infection of common diseases between humans and animals has been greatly reduced.
  • FIG. 1A is a first schematic flow chart of a method for preparing a biological material of the present invention
  • FIG. 1B is a schematic view showing a second flow of a method for preparing a biological material according to the present invention
  • FIG. 1C is a first schematic flow chart showing a method for preparing a biological material according to a first embodiment and a second embodiment of the present invention
  • 1D is a second schematic flowchart of a method for preparing a biological material according to a first embodiment and a second embodiment of the present invention
  • FIG. 1E is a first schematic flow chart of a method for preparing a biological material according to a third embodiment of the present invention
  • FIG. 1F is a second schematic flowchart of a method for preparing a biological material according to a third embodiment of the present invention
  • FIG. 2A is a 3T3 of biological material culture according to the present invention
  • FIG. 2B is an SEM image of the biomaterial cultured osteoblasts of the present invention for five days
  • 3A is a confocal microscope image of a three-day culture of 3T3 (fibrous cell) cells of the biomaterial of the present invention
  • Fig. 3B is a confocal microscopic view of the biomaterial cultured osteoblasts of the present invention for five days;
  • Fig. 4 is a H&E staining diagram of the biomaterial cultured osteoblasts of the present invention for five days. detailed description
  • the biological material preparation process of the present invention comprises: decellularizing fish scale 9 (step S10) and dehydrating (step S12) until the fish scale 9 contains water.
  • the amount is less than 50%; the average size of the fish scale 9 after the dewatering is less than 10,000 ⁇ m, preferably, the scale 9 is dehydrated to a water content of less than 25%, and is ground to an average diameter of less than 5000 ⁇ m. .
  • the fish scale 9 is prepared by providing the biological material in a refrigerated or frozen manner. In the present invention, the fish scale 9 having an average diameter of less than 20 cm is selected. First, the fish scale 9 is decellularized (step S10), and decellularization is performed using hypotonicity. Hypotonic solution, detergent, Triton X-100, sodium dodecyl sulfate (SDS), protease inhibitor, deoxyribonuclease (DNase), ribonuclease (RNase), etc.
  • hypotonic solution detergent, Triton X-100, sodium dodecyl sulfate (SDS), protease inhibitor, deoxyribonuclease (DNase), ribonuclease (RNase), etc.
  • the reagent removes the cells on the fish scale 9 in order to prevent the biological material prepared by the fish scale 9 from affecting the tissue repair and transplantation due to the cells attached thereto, and may even cause the host immune reaction, thereby causing the rejection phenomenon;
  • the fish scales 9 must be washed with other detergents (step S1 l).
  • These lotions may be, for example, surfactants, detergents, warm water and polar solvents, and polar solvents such as about 60. Ethanol in °C, but not limited to this.
  • the present invention is not limited to any particular washing step, but the fish scale 9 must be washed to a limulus amebocyte lysate (LAL test), and the washed fish scale 9 is tested in the LAL identification. Must be less than 1000 Eu/ml.
  • FIG. 1B is a second schematic flowchart of a method for preparing a biological material according to the present invention.
  • the steps are substantially the same as those described in the first schematic flow diagram, the only difference being that the cells are removed.
  • the step of the step (step S10) is after the step of washing (step S11), whether the cell is decellularized (step S10) before the fish scale 9 is washed (step S11), or the fish scale 9 is washed ( After step S11), decellularization (step S10) is performed, and both can proceed to the following steps as they are.
  • the fish scale 9 is dehydrated (step S12), and the dehydration method may use a compressed air dehydrator (air blower), an oven, freeze drying or other conventional dehydration methods, and in addition, the fish scale 9 may be immersed in ethanol or other polarities.
  • a compressed air dehydrator air blower
  • the fish scale 9 may be immersed in ethanol or other polarities.
  • the fish scale 9 is dried in an organic solvent for the purpose of dehydrating the fish scale 9 until the water content is less than 50%, and the optimum condition is that the water content is less than 25%; then the dehydrated fish scale 9 is ground until the average diameter of the particles is smaller than the average size 10,000 ⁇ m, the optimum state of the particles is about 5000 ⁇ m in diameter, and after the grinding, the particles comprise a mixture of the sponge matrix 14 and the powder 15, and therefore, the present invention sieves the milled particles using a sifter such as a sieve.
  • a vibrating tool can be used to enhance the sieving effect, and the granules sifted by the sifter provide the biological material in the form of powder, and after sieving
  • the remaining particles are provided in the form of a matrix for the use of the biomaterial; therefore, the equipment for grinding the scales 9 is not limited to a specific grinder, as long as That the average particle size after grinding is not more than ⁇ , ⁇ ⁇ , best is smaller than the diameter 5000 ⁇ , mill scale, or any reduction in the size of the device 9 can be.
  • step S16 The step of squeezing (step S16), sieving, whole or partial drying, and sterilization may also be included in the formulation to produce a sterile biomaterial. In the preferred embodiment, these steps may be performed regardless of heating conditions. Can be carried out.
  • the biomaterial can be used to bind to different types of repaired tissue or to activated and non-activated compositions.
  • the dehydrated fish scale 9 may be subjected to a pressure treatment to produce a sheet or a block or a specific shape of the biological material by a specific mold, wherein the pressurization treatment may include a temperature change (may be warming or low temperature) Even freezing), but not higher than 200 °C.
  • the pressurization treatment may include a temperature change (may be warming or low temperature) Even freezing), but not higher than 200 °C.
  • the fish scale 9 is placed at a temperature below 200 ° C for an extrusion process (step S16) to prepare a sheet or block or obtain a specific shape form 17 of the biological material through a specific mold. , and the dehydrated fish scale 9 is easy to squeeze.
  • a step of immersing the fish scale 9 in the water may be optionally applied (step S18), but the step is required to be washed.
  • Step S1 l is performed; further, in another embodiment of the present invention, the biological material, whether in a matrix or a powder form, can be converted into a sheet or by an extrusion process (step S16).
  • the specific shape form 17 is obtained in a block shape or by a specific mold, and the extrusion process is carried out at a temperature of 200 ° C or lower.
  • the heat treatment of the present invention is not limited to the extrusion process as described above, and any person skilled in the art may employ other heat treatment methods, such as any form of hot extrusion, hot pressing, molding, to The sheet or block is formulated or a specific shape of biological material is obtained by a specific mold.
  • the biomaterial of the present invention comprises a tissue repair factor and can produce a tissue repair material for repairing different tissue damage and tissue defect sites; for example, the biomaterial of the present invention can be used to prepare an injection to a nearby bone defect site. , a cartilage repair site, a cove repair site, or other soft tissue defect site; another embodiment is to make the biomaterial for surgical implantation or implantation adjacent to a bone defect site, a cartilage repair site, or other tissue defect site
  • the covering material therefore, can be used for obtaining a surgically transplanted connected tissue having a tissue repair material from a fish scale, particularly for joining a tissue defect site.
  • the present invention relates to a biological material prepared from fish scales 9, and the biological material is in the form of a powder, a matrix or a sheet or a block or a specific shape obtained by a specific mold, and is applicable.
  • the biological material is in the form of a powder, a matrix or a sheet or a block or a specific shape obtained by a specific mold, and is applicable.
  • These materials obtained from fish scales 9 also indicate that the biomaterials, whether in powder, matrix or in the form of flakes or lumps or through specific moulds, contain tissue repair factors and may even have different rules (different shapes may Different repair methods are produced, such as repairing bones and repairing skins.)
  • FIG. 1C is a first flow diagram of a method for preparing a biological material according to a first embodiment of the present invention, the process starting from a step of decellularizing fish scales (step S20), followed by washing the scales (steps) S19), and the test value of the washed fish scale in the LAL identification must be less than lOOOEu/ml; please refer to FIG. 1D, which is the second flow of formulating the biological material according to the first embodiment of the present invention.
  • FIG. 1D which is the second flow of formulating the biological material according to the first embodiment of the present invention.
  • Schematic diagram the process starts from washing the fish scales (step S19), and the washed fish scales must have a test value of less than 1000 Eu/ml in the LAL identification, and then the fish scales are decellularized ( Step S20).
  • step S20 Whether the cell is decellularized (step S20) before the fish scale is washed (step S19), or the fish scale is washed (step S19), and decellularization (step S20) is performed, both of which can enter the order. The following steps.
  • Step S21 the scales will be dehydrated until the water content is less than 50%, followed by step S22, the dehydrated fish scales will be ground until the average diameter of the particles is smaller than ⁇ , and the particles comprise a sponge matrix and a mixture of powders;
  • Step S23 is a sieving step in which the substrate is separated from the mixture by means of a sifter.
  • FIG. 1C and FIG. 1D are respectively a first flow chart for preparing a biological material according to a second embodiment of the present invention and a second flow chart for preparing a biological material according to a second embodiment of the present invention, and a process for preparing a biological material in a powder form.
  • the ground particles comprise a sponge matrix and a mixture of powders;
  • Step S23 is a sieving step, and the preparation of the biological material in powder form differs in that The sifter is used to separate the powder from the mixture, so the powder form has an exact mechanical structure (the mechanical structure mentioned here means that the biological material is not treated with enzymes or enzymes, so the biological material is not The original mechanical structure is preserved intact, that is, not only the components such as collagen and hydroxyapatite are retained, but even the natural three-dimensional structure and the cross-linking of the material are completely retained, thereby making it different from the matrix form.
  • the diameter of the powder particles is less than 5000 ⁇ m.
  • Third embodiment a sheet shape or a block shape or a specific shape form by a specific mold
  • FIG. 1A is a schematic diagram of a first process for preparing a biological material according to a third embodiment of the present invention.
  • the purpose is to use fish scales to prepare flakes or blocks or to obtain specific shapes of biological materials through specific molds.
  • the process starts from the step of decellularizing the fish scales (step S30), and then the fish scales are washed (step S29), and the washed fish scales must have a test value of less than 1000 Eu/ml in the LAL identification.
  • FIG. 1F which is a schematic diagram of a second process for preparing a biological material according to a third embodiment of the present invention.
  • the purpose is to use a fish scale to prepare a sheet or a block or obtain a specific shape of a biological material through a specific mold, and the process starts from The fish scale is subjected to a washing operation (step S29), and the washed fish scale is required to have a test value of less than 1000 Eu/ml in the LAL identification, and then the fish scale decellularization step is performed (step S30).
  • step S30 Whether the cell is decellularized (step S30) before the fish scale is washed (step S29), or the fish scale is washed (step S29), and decellularization (step S30) is performed, both of which can enter the order. The following steps.
  • step S31 the fish scale will be dehydrated until the water content is less than 25%, followed by step S32, the dehydrated fish scale will be ground until the average diameter of the particle diameter is less than 5000 ⁇ m, and the particles contain the sponge state.
  • a mixture of the substrate and the powder Step S33 is a sieving step of separating the mixture into a matrix and a powder using a sifter; and step S34 is squeezing the substrate and the powder.
  • the matrix or powder may be separately extruded, or the matrix may be mixed with the powder and extruded.
  • the matrix and powder can be mixed at different required ratios.
  • the first method is to directly perform the subsequent action after the washing step (step S29); the second method is shown in FIG. 1E and FIG. 1F. Before the extrusion step, the scales are washed and dehydrated.
  • the extrusion step is carried out to produce flakes or blocks or by specific
  • the mold obtains a specific shape of the biological material; using a complete fish scale or a different type of biological material obtained from the fish scale, first pressing it at a low temperature at a pressure greater than 100 g / 2.5 cm 3 , and the optimum pressure is lkg / 2.5cm 3 , then send it to a temperature less than 200 °C Extrusion of the stamper (the focus of this step is the extrusion action, the extrusion temperature is only an illustrative example, can be extruded at low temperatures (eg -18 ° C to 4 ° C), or high temperature (eg about 30 -60 ° C) Under extrusion, preferably should not exceed 200 ° C, depending on the temperature, the specific extrusion force and extrusion time will be different, in order to obtain a sheet or block or
  • decellularized biomaterials can provide a natural structure for cell migration, which in turn has a fairly good biocompatibility.
  • FIG. 2A is an SEM image of culturing 3T3 (fibrous cell 22) cells for five days using the biomaterial 20 of the present invention
  • FIG. 2B is an SEM image of culturing osteoblasts 24 cells for 5 days using the biomaterial 20 of the present invention, and these figures show that Whether the fibroblasts 22 or the osteoblasts 24 are capable of growing substantially intact using the biomaterial 20 of the present invention.
  • FIG. 3A is a confocol microscope image in which 3T3 (fibroblast) cells are cultured using the biomaterial 20 of the present invention for five days
  • FIG. 3B is a confocal microscope in which osteoblasts are cultured for five days using the biomaterial 20 of the present invention ( Confocol microscopes, which show that both the fibroblasts and osteoblasts can grow quite intact using the biomaterial 20 of the present invention.
  • Figure 4 is a five-day H&E staining of cultured osteoblasts 24 using the biomaterial 20 of the present invention. These figures show that osteoblasts 24 can be grown quite intact using the biomaterial 20 of the present invention.

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Description

配制生物材料的方法 技术领域
本发明是关于一种生物材料及其配制方法， 特别是关于一种由去细胞 化的鱼鳞配制生物材料的方法， 所配制的生物材料可以用于组织修复与移 植方面。 背景技术
组织工程与生物以及药物的生物材料息息相关， 其能够使进行移植手 术的病人增强血管造影、 组织整合， 以及组织重建； 生物材料是经由人工 所合成的材料， 应用于重建人造器官、 修复或修补， 甚至取代人体组织。
数十年来， 胶原纤维、 羟基磷灰石 (HAP)以及三钙磷酸盐 (TCP)皆是经 由许多数据证实且可安全用于人体移植组织的生物材料， 然而， 这些生物 材料具有一些缺点， 如机械支撑力不足、 在交联过程有化学物残留， 以及 人畜共通疾病传染等缺点。
因此， 需要提出一种新的生物材料， 增强机械支撑力， 且于组织修复 以及移植过程中减低人畜共通疾病传染机率。 发明内容
本发明的目的在于提供一种新的生物材料， 以增强机械支撑力， 且于 组织修复以及移植过程中减低人畜共通疾病传染机率。
本发明的另一目的在于提供如上所述的生物材料以用以修复组织以及 组织移植。
本发明的另一目的在于提供配制如上所述的生物材料的方法。
本发明提供了一种从鱼鳞配制生物材料的方法， 该方法包括步骤： 将鱼鳞去细胞化， 再将其磨碎至一定量的颗粒， 其中这些颗粒包含海 绵态基质以及粉末的混合物。
本发明的配制生物材料的方法还可包括： 挤压所述海绵态基质以及粉 末， 以在 200°C以下的温度形成片状或块状或通过特定模具得到特定形状。
本发明的配制生物材料的方法中， 在磨碎鱼鳞之前， 还可包括将鱼鳞 脱水的步骤。 所述脱水步骤可包括将鱼鳞脱水直到含水量少于 50%。 在所 述将鱼鳞脱水步骤之前， 还可包括将鱼鳞洗净的步骤。 其中， 将鱼鳞去细 胞化的步骤可以是在所述将鱼鳞洗净步骤之前。 将鱼鳞去细胞化的步骤也 可以是在将鱼鳞脱水步骤之前。
本发明的配制生物材料的方法还可包括将海绵态基质以及粉末从颗粒 中分离的步骤。
本发明中的原料鱼鳞可以是直径平均尺寸小于 20cm的鱼鳞，例如大尺 寸（10-20cm) 的鱼鳞， 也可以是较小尺寸的鱼鳞。 本发明的配制生物材料 的方法中， 重点是保留鱼鳞生物材料的天然键结与立体结构， 对于鱼鳞的 磨碎程度没有特别限定， 一般是将鱼鳞磨碎至颗粒直径平均尺寸小于 10，000μπι。 如果所用鱼鳞原料的直径平均尺寸小于 10，000μπι， 仍需要进行 适当磨碎， 将其磨碎至更小的颗粒尺寸， 例如， 可将其磨碎至颗粒直径平 均尺寸小于 10μπι。
本发明还提供了另一种从鱼鳞配制生物材料的方法， 该方法包括步骤: 将鱼鳞去细胞化， 再将其进行挤压过程以形成片状或块状或通过特定 模具得到特定形状的材料。
本发明的配制生物材料的方法中，所述挤压过程是在 200°C以下的温度 进行。 其中， 在进行鱼鳞挤压过程之前， 还可包括洗净鱼鳞的步骤。 在洗 净步骤结束后， 且在进行鱼鳞挤压过程之前， 还可包括将鱼鳞脱水的步骤。 所述脱水步骤包括将鱼鳞脱水直到含水量少于 50%。 其中， 将鱼鳞去细胞 化的步骤可以是在将鱼鳞洗净步骤之前。 将鱼鳞去细胞化的步骤也可以是 在将鱼鳞脱水步骤之前。 在进行鱼鳞挤压过程之前， 还可包括将鱼鳞浸泡 于水中的步骤。
根据本发明的一具体的由鱼鳞配制生物材料的方法， 所述的配制过程 包括将去细胞化的鱼鳞脱水直到含水量少于 50%， 以及磨碎该脱水过后的 鱼鳞直到磨碎的颗粒直径平均尺寸小于 10，000μπι， 其中每一磨碎的颗粒皆 包含海绵态基质以及粉末的混合物。 在本发明的一实施例中， 是将鱼鳞脱 水到含水量少于 25%， 且磨碎的颗粒直径平均尺寸小于 5000μπι。
另一方面，本发明由鱼鳞配制生物材料的过程包括在 200 °C以下的温度 进行处理。
本发明还提供了利用所述方法由鱼鳞配制得到的生物材料。
利用本发明的方法配制得到的生物材料， 保留了鱼鳞生物材料的天然键 结与立体结构， 该生物材料具有较佳的机械支撑力， 可以用以修复组织以及 组织移植， 且于组织修复以及移植过程中人畜共通疾病传染机率大幅减低。
为使本发明的优点及精神能更进一步的被揭示， 现配合图式作一详细 说明如后。 附图说明
图 1A为本发明的配制生物材料的方法的第一流程示意图；
图 1B为本发明的配制生物材料的方法的第二流程示意图；
图 1C 为本发明第一实施例以及第二实施例配制生物材料的方法的第 一流程示意图；
图 1D 为本发明第一实施例以及第二实施例配制生物材料的方法的第 二流程示意图；
图 1E为本发明第三实施例配制生物材料的方法的第一流程示意图； 图 1F为本发明第三实施例配制生物材料的方法的第二流程示意图； 图 2A为本发明的生物材料培养 3T3 (纤维细胞)细胞五天的 SEM图； 图 2B为本发明的生物材料培养成骨细胞五天的 SEM图； 图 3A为本发明的生物材料培养 3T3 (纤维细胞)细胞五天的共焦显微镜 图；
图 3B为本发明的生物材料培养成骨细胞五天的共焦显微镜图； 图 4为本发明的生物材料培养成骨细胞五天的 H&E染色图。 具体实施方式
请参照图 1A， 其为本发明的配制生物材料的方法的第一流程示意图， 本发明的生物材料配制过程包括：将鱼鳞 9去细胞化 (步骤 S10)且脱水 (步骤 S12)直到鱼鳞 9含水量少于 50%; 这些脱水磨碎过后的鱼鳞 9颗粒直径平 均尺寸小于 10，000μπι， 优选的， 是将鱼鳞 9脱水至含水量少于 25%， 且磨 碎至其颗粒直径平均尺寸小于 5000μπι。
该鱼鳞 9是以冷藏或冷冻的方式以提供给生物材料作配制， 本发明选 用直径平均尺寸小于 20cm的鱼鳞 9， 首先， 将鱼鳞 9去细胞化 (步骤 S10)， 去细胞化是利用低渗液（hypotonic solution) 、 清洁剂（detergent) 、 Triton X-100, 十二垸基硫酸钠 （SDS) 、 蛋白酶抑制剂 （protease inhibitor) 、 脱 氧核糖核酸酶 （DNase) 及核糖核酸酶 （RNase) 等试剂去除鱼鳞 9上的细 胞， 目的是避免由鱼鳞 9所配制而成的生物材料会因为上面所附着的细胞 影响组织修复以及移植， 甚至可能引发宿主免疫反应， 进而产生排斥现象； 要进行鱼鳞 9脱水动作 (步骤 S12)之前，须先将鱼鳞 9利用其它洗剂洗净 (步 骤 Sl l)， 这些洗剂可以例如是表面活性剂、 洗洁剂、 温水以及极性溶剂， 极性溶剂如约 60°C的乙醇， 但均不以此为限。 然而， 本发明并无限制任何 特定的洗净步骤， 但皆须将鱼鳞 9 洗净至可以通过 LAL 鉴定 (limulus amebocyte lysate, LAL test),而洗净后的鱼鳞 9在 LAL鉴定中的检验值必须 小于 1000 Eu/ml。
请参照图 1B， 其为本发明的配制生物材料的方法的第二流程示意图， 其步骤大致与第一流程示意图所述的步骤相同， 唯一不同之处在于去细胞 化 (步骤 S10)的步骤是位在洗净 (步骤 S11)步骤之后， 不论是在将鱼鳞 9洗 净 (步骤 S11)前进行去细胞化 (步骤 S10)动作， 或是将鱼鳞 9洗净 (步骤 S11) 后进行去细胞化 (步骤 S10)动作， 二者皆能如序进入以下的步骤。
之后，将鱼鳞 9脱水 (步骤 S12)，脱水方法可以使用压缩空气脱水器（空 气喷器） 、 烤箱、 冷冻干燥或其它传统的脱水方法， 此外， 也可将鱼鳞 9浸 泡于乙醇或其它极性有机溶剂中以干燥该鱼鳞 9， 目的是将鱼鳞 9脱水直到 含水量少于 50%， 最佳状态是含水量少于 25%; 接着将这些脱水后的鱼鳞 9 磨碎至颗粒直径平均尺寸小于 10，000μπι， 颗粒最佳状态是直径约 5000μπι， 而磨碎过后颗粒包含海绵态基质 14以及粉末 15的混合物， 因此， 本发明使 用过筛器例如筛网对该磨碎过后的颗粒进行过筛动作， 进而分离海绵态基质 与粉末； 该过筛步骤中， 可使用震动工具以加强过筛效果， 藉由过筛器所过 筛出的颗粒以粉末的形式提供生物材料使用， 而过筛后所剩余的颗粒则是以 基质的形式提供生物材料使用； 因此， 磨碎鱼鳞 9的设备并没有限定为特定 的研磨器，只要能使磨碎后的颗粒平均尺寸不超过 ιο，οοομηι，最佳状态是直 径小于 5000μπι， 研磨机或是任何减小鱼鳞 9尺寸的设备皆可。
配制过程中还可包括挤压 (步骤 S16)、 过筛、 全部或部分干燥以及消毒 的步骤， 以产生无菌的生物材料， 在最佳实施例中， 这些步骤可于不论是 否加热的条件下皆可进行。 该生物材料可用于与不同种的连接修复组织或 是活化与非活化的组成物结合。
此外， 可将脱水后的鱼鳞 9通过加压处理以产生一片状或块状或通过 特定模具得到特定形状的生物材料， 其中该加压处理可包括一温度改变 (可 为加温或是低温甚至冷冻)过程， 但不可高于 200°C以上的温度。 在洗净步 骤 (步骤 SI 1)之后，将鱼鳞 9置于 200 °C以下的温度进行一挤压过程 (步骤 S16) 以配制片状或块状或通过特定模具得到特定形状形式 17的生物材料， 而脱 水后的鱼鳞 9易于进行挤压动作。 在进行挤压过程 (步骤 S 16)之前， 可选择 性地外加一将鱼鳞 9浸泡于水中的步骤 (步骤 S18)， 但该步骤需于洗净步骤 (步骤 Sl l)之后进行； 此外， 本发明还有另一个实施例， 该生物材料不论是 基质或是粉末形式皆可通过挤压过程 (步骤 S16)， 以将该生物材料转换为片 状或块状或通过特定模具得到特定形状形式 17， 而该挤压过程是于 200°C 以下的温度进行。 然而， 本发明的加热处理并非如上所述限定于挤压过程， 任何熟知该项技术的人员也可能采用其它加热处理方式， 如任何形式的热 挤压、 热重压、 模制的步骤， 以配制该片状或块状或通过特定模具得到特 定形状的生物材料。
本发明的生物材料包含组织修复因子且可产生组织修复材料， 该组织 修复材料是用于修复不同组织伤害以及组织缺陷部位； 举例而言， 本发明 的生物材料可用于调制注射剂于邻近骨骼缺陷部位、 软骨修复部位、 齿槽 修复部位， 或其它软组织缺陷部位； 另一实施例是将该生物材料制作为外 科手术于邻近骨骼缺陷部位、 软骨修复部位， 或其它组织缺陷部位移植或 植入用的覆盖材料， 因此， 本发明可用于由鱼鳞取得具有组织修复材料的 外科手术移植的连接组织， 特别是用于连接组织缺陷部位。
综上所述， 本发明是关于一种由鱼鳞 9所配制的生物材料， 且该生物 材料是以粉末、 基质或是片状或块状或通过特定模具得到特定形状形式存 在的材料， 可应用于不同组织修复以及移植方面。 这些由鱼鳞 9取得的材 料亦指出不论是粉末、 基质或是片状或块状或通过特定模具得到特定形状 的生物材料皆包含组织修复因子， 且甚至有可能产生不同的定则 （不同形 状可能产生不同修复方式， 例如修复骨骼与修复皮肤则有所不同） 。
本发明将于以下详细叙述说明技术内容， 但均并不以此为限。 第一实施例： 基质形式
请参照图 1C，其为本发明第一实施例配制生物材料的第一流程示意图， 该过程是起始于将鱼鳞进行去细胞化的步骤 (步骤 S20)， 接着将鱼鳞进行洗 净动作 (步骤 S19)， 而洗净后的鱼鳞在 LAL 鉴定中的检验值必须小于 lOOOEu/ml; 请参照图 1D， 其为本发明第一实施例配制生物材料的第二流 程示意图， 该过程是起始于将鱼鳞进行洗净动作 (步骤 S19)， 而洗净后的鱼 鳞在 LAL鉴定中的检验值必须小于 1000 Eu/ml, 接着将鱼鳞进行去细胞化 的步骤 (步骤 S20)。
不论是在将鱼鳞洗净 (步骤 S19)前进行去细胞化 (步骤 S20)动作， 或是 将鱼鳞洗净 (步骤 S19)后进行去细胞化 (步骤 S20)动作， 二者皆能如序进入 以下的步骤。
之后进入到步骤 S21，鱼鳞将被脱水直到含水量少于 50%，接着是步骤 S22, 脱水的鱼鳞将被磨碎直到其颗粒直径平均尺寸小于 ΙΟΟΟΟμπι, 这些颗 粒包含海绵态基质以及粉末的混合物； 步骤 S23是进行过筛步骤， 利用过 筛器将基质由混合物中分离出来。
因此， 经过过筛步骤后得到基质， 该基质包含由 HAP、 TCP以及胶原 蛋白所组成的纤维组织。 第二实施例： 粉末形式
请再次参照图 1C以及图 1D， 其分别为本发明第二实施例配制生物材 料的第一流程图以及本发明第二实施例配制生物材料的第二流程图， 配制 粉末形式的生物材料的过程相似于配制基质形式的生物材料的过程， 如上 所述， 磨碎后的颗粒包含海绵态基质以及粉末的混合物； 步骤 S23是进行 过筛步骤， 配制粉末形式的生物材料的不同之处在于， 其是利用过筛器将 粉末由混合物中分离出来， 因此粉末形式具有一个确切的机械结构 （这里 所提及的机械结构指的是因为本发明并无将生物材料经过酵素或酶处理， 所以生物材料原本的机械结构被完整保存， 也就是不只胶原蛋白与羟基磷 灰石等成分被保留下来， 甚至连该材料天然的立体结构与交链都被完整保 留） ， 进而使其不同于基质形式， 而粉末颗粒的直径尺寸是小于 5000μιη。 第三实施例： 片状或块状或通过特定模具得到特定形状形式
请参照图 1Ε， 其为本发明第三实施例配制生物材料的第一流程示意图， 目的是利用鱼鳞配制片状或块状或通过特定模具得到特定形状的生物材料。 该过程是起始于将鱼鳞去细胞化步骤 (步骤 S30)， 之后将鱼鳞进行洗净动作 (步骤 S29)， 洗净后的鱼鳞在 LAL鉴定中的检验值必须小于 1000 Eu/ml。 请 参照图 1F，其为本发明第三实施例配制生物材料的第二流程示意图， 目的是 利用鱼鳞配制片状或块状或通过特定模具得到特定形状的生物材料， 该过程 是起始于将鱼鳞进行洗净动作 (步骤 S29)， 洗净后的鱼鳞在 LAL鉴定中的检 验值必须小于 1000 Eu/ml， 之后进行将鱼鳞去细胞化步骤 (步骤 S30)。
不论是在将鱼鳞洗净 (步骤 S29)前进行去细胞化 (步骤 S30)动作， 或是 将鱼鳞洗净 (步骤 S29)后进行去细胞化 (步骤 S30)动作， 二者皆能如序进入 以下的步骤。
之后进入到步骤 S31， 在步骤 S31 中， 鱼鳞将被脱水直到含水量少于 25%, 接着是步骤 S32， 脱水的鱼鳞将被磨碎直到其颗粒直径平均尺寸小于 5000μπι, 该些颗粒包含海绵态基质以及粉末的混合物； 步骤 S33是进行过 筛步骤， 利用过筛器将混合物分离为基质以及粉末； 步骤 S34是将该基质 以及粉末进行挤压。 可将基质或粉末分别进行挤压， 也可将基质与粉末混 合后进行挤压。 根据所配制生物材料的使用用途的不同， 例如修复骨骼用 的生物材料或是修复皮肤用的生物材料， 可将基质与粉末按不同的所需用 量比例进行混合。
配制片状或块状或通过特定模具得到特定形状的生物材料有二种方 法， 方法一是在洗净步骤 (步骤 S29)之后， 直接进行后续的动作； 方法二如 图 1E以及图 1F所示， 在挤压步骤之前， 先将鱼鳞进行洗净以及脱水步骤。
因此， 不论是脱水过后的鱼鳞， 未脱水但已洗净后的鱼鳞， 或是由鱼 鳞所取得的基质形式以及粉末形式的生物材料， 皆进行挤压步骤以产生片 状或块状或通过特定模具得到特定形状的生物材料； 利用完整的鱼鳞或是 由鱼鳞取得不同类型的生物材料， 先将其于一低温下以大于 100g/2.5cm3的 压力进行挤压， 而最佳压力为 lkg/2.5cm3， 再将其送至一小于 200 °C高温下 的压模进行挤压 （该步骤的重点是挤压动作， 挤压温度仅是示例性说明， 可以在低温（例如 -18°C到 4°C ) 下挤压， 或是高温 （例如约 30-60°C ) 下挤 压，最好应不超过 200°C， 因温度不同具体的挤压力与挤压时间会有所不同， 以得到片状或块状或通过特定模具得到特定形状的生物材料） ； 在进行挤压 动作之前， 该生物材料的交联作用可藉由加热或加入适当浓度的化学交联剂 (例如可以是戊二醛 （glutaaldehyde) 、 EDC或是其它通常所用的化学交联 剂）达成； 其中， 在生物材料中， 交联剂会与胺群或其它反应物产生反应。
本发明中的去细胞化的过程中， 除了洗去了部分的水溶性蛋白质与葡 萄糖胺聚合醣外， 原来的胶原蛋白与弹性蛋白纤维与大部分的葡萄糖胺聚 合醣仍然保存于其天然细胞外间质的结构内， 因此去细胞化的生物材料能 够提供细胞迁入的自然结构， 进而具有相当好的生物兼容性。
图 2A是利用本发明的生物材料 20培养 3T3 (纤维细胞 22)细胞五天的 SEM图， 图 2B是利用本发明的生物材料 20培养成骨细胞 24细胞 5天的 SEM图， 这些图显示不论是纤维细胞 22或是成骨细胞 24利用本发明的生 物材料 20皆能够生长得相当完整。
图 3A是利用本发明的生物材料 20培养 3T3 (纤维细胞)细胞五天的共 焦显微镜 (confocol microscope)图， 图 3B是利用本发明的生物材料 20培养 成骨细胞五天的共焦显微镜 (confocol microscope)图， 这些图显示不论是纤 维细胞或是成骨细胞利用本发明的生物材料 20皆能够生长得相当完整。
图 4是利用本发明的生物材料 20培养成骨细胞 24五天的 H&E染色图， 这些图显示成骨细胞 24利用本发明的生物材料 20皆能够生长得相当完整。
以上所述是利用一较佳实施例及不同实施例以详细说明本发明， 其并 非用以限制本发明的实施范围， 并且熟悉该技术的人员都能明了， 适当做 些微的修改仍不脱离本发明的精神及范围。

Claims

权利要求书
1.一种从鱼鳞配制生物材料的方法， 该方法包括步骤：
将鱼鳞去细胞化， 再将其磨碎成颗粒， 其中这些颗粒包含海绵态基质 以及粉末的混合物。
2.如权利要求 1所述的配制生物材料的方法， 该方法还包括： 挤压所 述海绵态基质以及粉末，以在 200 °C以下的温度形成片状或块状或通过特定 模具得到特定形状。
3.如权利要求 1所述的配制生物材料的方法，其中，在磨碎鱼鳞之前， 还包括将鱼鳞脱水的步骤。
4.如权利要求 3所述的配制生物材料的方法， 其中， 所述脱水步骤包 括将鱼鳞脱水直到含水量少于 50%。
5.如权利要求 3所述的配制生物材料的方法， 其中， 在将鱼鳞脱水步 骤之前， 还包括将鱼鳞洗净的步骤。
6.如权利要求 5所述的配制生物材料的方法， 其中， 将鱼鳞去细胞化 的步骤是在将鱼鳞洗净步骤之前。
7.如权利要求 3所述的配制生物材料的方法， 其中， 将鱼鳞去细胞化 的步骤是在将鱼鳞脱水步骤之前。
8.如权利要求 1所述的配制生物材料的方法， 该方法还包括将海绵态 基质以及粉末从颗粒中分离的步骤。
9.如权利要求 1所述的配制生物材料的方法， 其中是将鱼鳞磨碎至颗 粒直径平均尺寸小于 10，000μπι。
10.如权利要求 1所述的配制生物材料的方法，其中，所述鱼鳞直径平 均尺寸小于 20cm。
11.一种从鱼鳞配制生物材料的方法， 该方法包括步骤： 将鱼鳞去细胞化， 再将其进行挤压过程以形成片状或块状或通过特 模具得到特定形状的材料。
12.如权利要求 11所述的配制生物材料的方法， 其中， 所述挤压过禾王 是在 200 °C以下的温度进行。
13.如权利要求 11所述的配制生物材料的方法， 其中， 在进行鱼鳞挤 压过程之前， 还包括洗净鱼鳞的步骤。
14.如权利要求 13所述的配制生物材料的方法， 其中， 在洗净步骤结 束后， 且在进行鱼鳞挤压过程之前， 还包括将鱼鳞脱水的步骤。
15.如权利要求 14所述的配制生物材料的方法， 其中， 所述脱水步骤 包括将鱼鳞脱水直到含水量少于 50%。
16.如权利要求 14所述的配制生物材料的方法， 其中， 将鱼鳞去细胞 化的步骤是在将鱼鳞脱水步骤之前。
17.如权利要求 13所述的配制生物材料的方法， 其中， 将鱼鳞去细胞 化的步骤是在将鱼鳞洗净步骤之前。
18.如权利要求 11所述的配制生物材料的方法， 其中， 在进行鱼鳞挤 压过程之前， 还包括将鱼鳞浸泡于水中的步骤。
19.如权利要求 11所述的配制生物材料的方法， 其中， 所述鱼鳞直径 平均尺寸小于 20cm。
PCT/CN2008/070309 2008-01-09 2008-02-15 配制生物材料的方法 Ceased WO2009086743A1 (zh)

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