WO2022135487A1 - 一种用于下鼻甲重建的组织工程骨移植物 - Google Patents

一种用于下鼻甲重建的组织工程骨移植物 Download PDF

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WO2022135487A1
WO2022135487A1 PCT/CN2021/140551 CN2021140551W WO2022135487A1 WO 2022135487 A1 WO2022135487 A1 WO 2022135487A1 CN 2021140551 W CN2021140551 W CN 2021140551W WO 2022135487 A1 WO2022135487 A1 WO 2022135487A1
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bone
cells
tissue
bone graft
bmsc
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PCT/CN2021/140551
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English (en)
French (fr)
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周广东
石润杰
刘豫
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上海软馨生物科技有限公司
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Priority to US18/258,630 priority Critical patent/US20240108786A1/en
Priority to CN202180087453.0A priority patent/CN117858733A/zh
Publication of WO2022135487A1 publication Critical patent/WO2022135487A1/zh

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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
    • A61L27/3604—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
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • 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
    • A61L27/3695—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 characterised by the function or physical properties of the final product, where no specific conditions are defined to achieve this
    • 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
    • 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
    • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor 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
    • A61L27/36—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
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L27/36—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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • 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
    • 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
    • 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 invention belongs to the field of biomedical tissue engineering, in particular to a tissue engineering bone graft for inferior turbinate reconstruction.
  • ENS Empty nose syndrome
  • ENS is an iatrogenic complication caused by excessive turbinate resection. It is manifested as secondary nasal mucosal atrophy and a series of accompanying symptoms, including dryness in the nasal cavity and pharynx, inability to concentrate, fatigue, irritability, anxiety, depression, etc. About 20% of patients with inferior turbinate resection will develop ENS. Due to the poor treatment effect, patients and their families have hostile feelings towards the surgeon and the hospital, and even violent conflicts occur, which brings certain unstable factors to the society.
  • the present invention provides a tissue engineering bone graft for inferior turbinate reconstruction.
  • a tissue engineered bone graft comprising:
  • BMSC human bone marrow stromal stem cell
  • the decalcified bone matrix carrier has a degree of decalcification of 95-85%;
  • the thickness of the demineralized bone matrix carrier is 3-8 mm.
  • the decalcification degree of the decalcified bone matrix carrier is 92%-86%.
  • the thickness of the demineralized bone matrix carrier is 4.5-5.5 mm.
  • the BMSCs are autologous cells.
  • the BMSCs are derived from cancellous bone.
  • the cancellous bone includes: ilium, sternum, and rib.
  • the graft is a solid cell-material composite
  • the concentration of BMSC in the composite material is 1 ⁇ 10 7 cells/cm 3 -1 ⁇ 10 8 cells/cm 3 , preferably 2 ⁇ 10 7 cells/cm 3 to 7 ⁇ 10 7 cells/cm 3 .
  • the content of BMSC in the composite material is 1 ⁇ 10 7 cells/g-1 ⁇ 10 8 cells/g, preferably 2 ⁇ 10 7 cells/g-7 ⁇ 10 7 cells/g.
  • the shape of the tissue-engineered bone graft conforms to the shape of the inferior turbinate defect in the human body that needs to be transplanted.
  • a method for preparing a bone graft as described in the first aspect of the present invention comprising the steps of:
  • the BMSC cell is derived from autologous bone marrow;
  • BMSCs are cultured by external expansion in a culture liquid containing basic fibroblast growth factor (bFGF);
  • bFGF basic fibroblast growth factor
  • BMSC-decalcified bone complex (3) Inoculate BMSC cells on demineralized bone matrix carrier, induce chondrogenic culture in vitro, and form tissue engineered bone (BMSC-decalcified bone complex).
  • the in vitro culture medium is a low-sugar medium.
  • step (2) the BMSCs are expanded and cultured to passages 2-5.
  • the concentration of bFGF in the in vitro culture medium is 0-10 ng/mL; preferably 2-5 ng/mL.
  • the expanded BMSC cells are long spindle-shaped, with small cell volume and strong proliferation activity.
  • the seeding concentration of the BMSCs is 1 ⁇ 10 7 cells/g-1 ⁇ 10 8 cells/g; preferably 2 ⁇ 10 7 cells/g-7 ⁇ 10 7 cells/g; more preferably, 3.5 ⁇ 10 7 cells/g to 5 ⁇ 10 7 cells/g.
  • step (3) the in vitro chondrogenesis is induced and cultured for 0.5-8 weeks; preferably, 0.5-4 weeks.
  • a use of the bone graft according to the first aspect of the present invention is provided for preparing a medicament for repairing the defect of the inferior turbinate.
  • the drug is a material containing living cells.
  • the defect site of the inferior turbinate is selected from the bottom of the inferior turbinate, the surrounding tissue of the inferior turbinate, and the lateral wall of the nasal cavity.
  • the tissue engineered bone graft is also used to increase the volume of the inferior turbinate, reduce the volume of the nasal cavity, and improve the ventilation function of the nasal cavity.
  • a method of repairing a defect site of the inferior turbinate by administering the bone graft according to the first aspect of the present invention to a subject in need.
  • Figure 1 shows the effect of fibroblast growth factor (bFGF) in the medium on the growth of BMSC cells; among them, "+bFGF” in Figure A means the culture with bFGF added, and “-bFGF” means the culture without bFGF;
  • Figure B In the line graph of , the ordinate is the OD (optical density) value of the CCK-8 detection result, and the abscissa is the culture days (d).
  • Figure 2 shows a graph of demineralized bone samples.
  • Figure 3 shows the tissue engineered bone formed after 4 weeks of in vitro osteogenic induction of BMSC-composite demineralized bone.
  • Figure 4 shows the schematic diagram of the formation of cartilage-like grafts by inoculating BMSCs with polyglycolic acid/polylactic acid scaffolds in vitro for 8 weeks, wherein, picture A is the cartilage-like graft, and pictures B, D, and F are the histology of the graft, respectively , safranin-O, and type II collagen immunohistochemical staining (scale bar is 1 mm), C, E, and G are the magnifications of the black box content in B, D, and F, respectively (scale bar is 100 ⁇ m).
  • Figure 5 shows a schematic diagram of tissue engineering cartilage formed by seeding BMSCs with polyglycolic acid/polylactic acid scaffolds and implanted in a subcutaneous environment for 12 weeks.
  • picture A is the tissue-engineered bone formed by the cartilage-like graft of BMSC-polyglycolic acid/polylactic acid tissue under the skin of nude mice
  • picture B is the histological staining of picture A (scale bar is 100 ⁇ m).
  • Figure 6 shows the tissue engineered bone tissue formed by BMSC-decalcified bone complex transplanted into the subcutaneous environment of nude mice.
  • Figure 7 shows histological staining of BMSC-decalcified bone complexes.
  • Figure 8 shows the preoperative and postoperative MRI images of BMSC-demineralized bone complex implanted into the lateral wall of the nasal cavity in a patient with empty nose, equivalent to the inferior turbinate site.
  • the present inventor has developed for the first time a tissue engineered bone based on a specific material demineralized bone matrix and compounded with BMSC cells.
  • the tissue engineered bone of the present invention is particularly suitable for repairing inferior turbinate defects.
  • the demineralized bone matrix in the present invention has specific hardness and thickness: a suitable thickness is beneficial to support the inferior turbinate part and facilitate the loading of BMSC cells; the specific hardness is convenient for trimming, while providing the required strength for the inferior turbinate support.
  • the BMSCs loaded on the material are beneficial for nasal mucosa repair.
  • the inventors constructed a special tissue-engineered bone graft material by optimizing the thickness and degree of decalcification of the decalcified bone carrier material and the in vitro culture conditions of BMSCs, which facilitates the reconstruction of the inferior turbinate.
  • the present invention has been completed on this basis.
  • the invention is based on minimally invasively taking a small amount of BMSC cells, in vitro culture, high-density inoculation on demineralized bone materials of a specific size and thickness, and obtaining a biologically active BMSC cell-demineralized bone material complex by culturing, Relying on the osteogenesis of autologous tissue cells and the degradation and absorption of biomaterials, the new inferior turbinate is finally formed.
  • tissue-engineered cartilage/bone graft of the present invention As used herein, “inferior turbinate reconstructed tissue-engineered bone graft", “tissue-engineered cartilage/bone graft of the present invention” and “cartilage/bone graft of the present invention” of the present invention may be used interchangeably, all refer to The tissue engineered bone graft for inferior turbinate repair mentioned in the first aspect of the present invention.
  • demineralized bone-gelatin composite scaffolds polyglycolic acid/polylactic acid (PGA/PLA), polycaprolactone (PCL), or polycaprolactone composite hydroxyapatite can be used as scaffolds for tissue engineering bone .
  • PGA/PLA polyglycolic acid/polylactic acid
  • PCL polycaprolactone
  • polycaprolactone composite hydroxyapatite can be used as scaffolds for tissue engineering bone .
  • induction refers to the process of providing a special biochemical environment to transform a cell population such as stem cells with multi-directional differentiation ability into another cell population with different functional properties.
  • seeding refers to the process of uniformly distributing cells on a three-dimensional scaffold material.
  • autologous transplantation refers to the process of removing a desired biological living material (eg, bone marrow stromal stem cells) from an individual and re-administering it to the same individual.
  • a desired biological living material eg, bone marrow stromal stem cells
  • the selected material of the tissue engineering carrier is demineralized bone matrix.
  • Demineralized bone matrix is a bone graft material that is decalcified from allogeneic or xenogeneic bone to reduce immunogenicity. Different degrees of decalcification correspond to different mechanical strengths. It has good biological properties, osteoinductivity, osteoconductivity and biodegradability, promotes new bone formation and bone tissue mineralization, thereby accelerating bone healing, and can be effectively repaired alone or in combination with autologous bone, other biomaterials, and growth factors It is an ideal scaffold material for bone tissue engineering.
  • the length of the demineralized bone material is 10-40 mm, preferably 34.5-35.5 mm; the width is 5-15 mm, preferably 9.5-10.5 mm.
  • the length, width and height of the demineralized bone material are respectively 35 mm, 10 mm and 5 mm.
  • the size of the tissue engineered bone graft of the present invention can be customized according to the conditions of different patients. For example, it can be trimmed according to the position of the inferior turbinate defect in different patients, which has met the actual requirements.
  • the demineralized bone material in the tissue engineering bone graft of the present invention needs to have a certain thickness. If the thickness is too large, it is not conducive to the inoculation of stem cells and the full penetration of the cell suspension, and the cultured cell-material composite will appear hollow, which will affect the repair effect after implantation; The liquid is lost, and the cells cannot be effectively loaded, reducing the seeding efficiency.
  • the thickness of the decalcified bone material of the present invention is 1-8 mm; more preferably, the thickness is 2-5 mm.
  • the source of bone tissue that can be used in the tissue engineering carrier of the present invention is not particularly limited, and can be allogeneic bone tissue derived from humans, or xenogeneic bone tissue derived from animals (such as pigs, cattle, sheep, dogs, etc.). Preferred are xenogeneic bone tissues derived from pigs and bovines.
  • the degree of decalcification when the degree of decalcification is small, the toughness of the material is low, and when the material is trimmed, the material is more prone to fragmentation, which increases the difficulty of operation and prolongs the degradation time of the material in the body; but the degree of decalcification is too high.
  • the strength of the material is insufficient, and it is difficult to meet the strength required for repairing the inferior turbinate, which affects the prognosis of the patient.
  • the decalcified bone matrix carrier of the decalcified bone material of the present invention has a decalcification degree of 95-85%, preferably 92%-86%. That is, the calcium content of the demineralized bone material in the present invention should be controlled at about 5-15%, preferably 8-14%.
  • the bone tissue was immersed in liquid nitrogen for 5 min, and then placed in 75% ethanol solution for degreasing treatment; then added to 0.5M HCL solution for decalcification treatment, the HCL solution was replaced every two hours for a total of 3 times; deionized After washing with water for 3 times, 0.05% trypsin solution was added and placed in a constant temperature shaker at 37°C for digestion for 2 h for decellularization treatment; finally, demineralized bone matrix was prepared by freeze-drying, and stored in a dry box for future use.
  • Measured using plasma emission spectrometry Take 0.5g of freeze-dried demineralized bone, fully grind it into demineralized bone matrix powder, put it into a 100ml volumetric flask, add 5ml of concentrated HNO 3 , digest it in a microwave at 190°C for 18 minutes, and dilute to 100ml; take 1.5ml of the solution and add plasma An emission spectrometer (ICP) was used for detection, and the value was read; repeated three times, and the average value was taken.
  • ICP emission spectrometer
  • Bone marrow stromal cells are a kind of tissue stem cells with multi-directional differentiation potential. tissue cells. The cell population is abundant, easy to obtain, and has strong proliferation ability.
  • BMSCs are directly expanded with osteogenic induction liquid, and after the cells reach a certain number, they are seeded on demineralized bone or other tissue engineering scaffold materials, and continue to be cultured with osteogenic induction liquid for 1-3 weeks.
  • BMSCs are expanded in a low-sugar medium containing bFGF.
  • bFGF can significantly increase the proliferation activity of BMSCs, which is beneficial to maintain their stem cell properties, thereby saving the number of BMSCs needed and improving osteogenic activity.
  • a high concentration of BMSC is inoculated into the demineralized bone material, and the size of the demineralized bone is determined according to the degree of atrophy of the patient's inferior turbinate to form a tissue engineered bone (BMSC-decalcified bone complex).
  • the concentration of BMSCs used for seeding in the present invention is usually 1 ⁇ 10 7 cells/g-8 ⁇ 10 7 cells/g, preferably 2 ⁇ 10 7 cells/g-5 ⁇ 10 7 cells/g.
  • concentration of seed cells is adjusted with the culture medium, and then mixed with the tissue engineering carrier of the present invention.
  • the ratio of the culture medium to the solid material is not particularly limited when mixing, but the maximum amount of the culture medium that the carrier of the present invention can adsorb shall prevail.
  • various other cells, growth factors, and various transgenic components can also be added or compounded, so as to maintain cell phenotype, promote cell growth or matrix synthesis ability, etc., or promote tissue growth, blood vessel, nerve growth, etc. Enter and so on.
  • the formed bone graft can be directly implanted into the bone defect and other parts of the body to repair the bone tissue defect or fill the bone tissue.
  • the cell seeding concentration of the hBMSC-demineralized bone complex in the present invention is usually about 1 ⁇ 10 7 cells/g to 8 ⁇ 10 7 cells/g or higher.
  • the material is demineralized bone material or other solid material, solid or liquid composite material.
  • the cell concentration is adjusted with the culture medium, and then mixed with the decalcified bone material.
  • the ratio of the culture medium to the decalcified bone material is not particularly limited, but the maximum amount of the culture medium that can be adsorbed by the decalcified bone material shall prevail.
  • the stent material is a special three-dimensional shape, such as the following turbinate, the calculation is performed according to the actual volume.
  • the tissue engineered cartilage graft of the present invention is easy to manufacture.
  • the BMSCs are first expanded in a low-glucose medium containing bFGF, and after the cells reach a certain number, they are seeded on demineralized bone or other tissue engineering scaffold materials, and then the BMSCs are used in Example 6 of the patent (ZL201110268830.9).
  • the disclosed in vitro chondrogenesis induction solution is cultured for 1-8 weeks to form chondrogenic grafts.
  • the demineralized bone matrix material of the present invention can effectively reconstruct the inferior turbinate.
  • the stem cells required by the present invention are obtained from autologous materials, have no immunogenicity, and are highly safe.
  • the BMSC microenvironment is beneficial to the mucosal repair of the nasal cavity.
  • the present invention only needs a small amount of stem cells, and the material collection process is a routine operation, which does not damage normal tissues.
  • the size of the graft can be prepared according to the shape of the tissue defect to achieve precise repair.
  • the in vitro culture method is simple and easy to learn, easy to popularize, and easy to form industrialized products.
  • the tissue-engineered cartilage/bone graft of the present invention has good plasticity and certain mechanical strength, is easy to be processed into a desired shape and has a supporting function, and meets the requirements of the specific position of the inferior turbinate.
  • the culture medium was DMEM (delbecco's modified eagle medium), 10 nmol/L of sodium glycerol ⁇ -phosphate, 0.1 ⁇ mol/L of dexamethasone, 50 ⁇ mol/L of L- ⁇ -ascorbic acid phosphate, 300 mg/L of L-glutamine, 1.25 ( OH) 2 VD 3 10nmol/L and 10% fetal bovine serum (hyclone, USA), all unspecified reagents were Sigma Company, USA.
  • DMEM delbecco's modified eagle medium
  • 10 nmol/L of sodium glycerol ⁇ -phosphate 10 nmol/L of sodium glycerol ⁇ -phosphate
  • 0.1 ⁇ mol/L of dexamethasone 50 ⁇ mol/L of L- ⁇ -ascorbic acid phosphate
  • 300 mg/L of L-glutamine 300 mg/L of L-glutamine
  • Expansion medium for BMSCs low sugar medium containing bFGF
  • the medium used to expand BMSCs contains 10 g of low-sugar DMEM medium, 300 mg of L-glutamine, 50 mg of vitamin C, and 3.7 g of sodium bicarbonate per liter of liquid. Preferably, 2-5 ng/mL of basic fibroblast growth factor (bFGF) is added.
  • bFGF basic fibroblast growth factor
  • the high glucose DMEM medium contains 10% FBS, ⁇ -glycerol phosphate 10 mM, vitamin D 310 nM, and dexamethasone 0.1 ⁇ M.
  • the medium was changed 48 hours after primary cell inoculation, and after the cells reached 80% to 90% confluence, digested with 0.25% trypsin, subcultured at 2 ⁇ 10 3 cells/cm 2 , and cultured in a 37°C, 5% CO 2 incubator By passage 3, cells were harvested and counted.
  • BMSCs were expanded in low-glucose medium containing bFGF.
  • the medium used to expand BMSCs contained 10 g of low-sugar DMEM medium, 300 mg of L-glutamine, 50 mg of vitamin C, and 3.7 g of sodium bicarbonate per liter of liquid. 0ng, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL basic fibroblast growth factor (bFGF) was added to the medium.
  • BMSCs were cultured in the above-mentioned medium, respectively.
  • BMSCs cultured with bFGF can maintain a long spindle shape and have smaller cell volume;
  • the BMSCs expanded in the medium were more spread in morphology.
  • Figure 1B shows that BMSCs cultured with bFGF can still have good proliferation activity on the ninth day of culture; BMSCs cultured without bFGF continued to decrease after the fifth day of culture.
  • Example 3 In vitro culture of BMSC composite tissue engineering scaffolds
  • Method 1 BMSCs were directly expanded with the osteogenic induction solution. After the cells reached a certain number, they were inoculated into demineralized bone or other tissue engineering scaffold materials, and cultured with the above osteogenic induction solution for 1-3 weeks.
  • Method 2 BMSCs were first expanded in low-glucose medium containing bFGF, and when the cells reached a certain number, they were seeded on demineralized bone or other tissue engineering scaffolds, and then cultured in osteogenic induction solution for 1-3 weeks.
  • Method 3 First, expand BMSCs in a low-glucose medium containing bFGF. After the cells reach a certain number, they are seeded on demineralized bone or other tissue engineering scaffold materials. The chondrogenic induction solution was cultured for 0.5-8 weeks to form cartilage-like grafts (ie, tissue engineered cartilage).
  • method 1 provides an osteogenic environment through the osteogenic induction solution from the cell expansion stage, which may be more conducive to subsequent bone regeneration;
  • Method 2 expands cells by bFGF-containing low-glucose medium, which can obtain a higher number of cells than the first method;
  • Method 3 is on the basis of method 2, using chondrogenesis-inducing solution to culture the cell-material complex for a longer period of time, the regenerated tissue in vitro is closer to cartilage, and the surrounding environment is better after implantation in the inferior turbinate defect. Tolerance is expected to improve the chance of survival (the inferior turbinate defect site is in the submucosal microenvironment, and the blood supply is not very rich, which is not conducive to the long-term survival of general tissue engineered bone, but tissue engineered cartilage is more tolerant to the environment of ischemic supply) .
  • the above-mentioned tissue engineered cartilage implanted under the nasal mucosa will continue to develop into bone tissue (matching the type of tissue defect) due to the terminal ossification of internal BMSCs. .
  • Method 3 is better than 1 and better than 2.
  • Demineralized bone was selected as the scaffold material for tissue engineering bone. Through the decalcification treatment, the calcium content of the demineralized bone material is controlled to be about 8-10%. The size of demineralized bone material needs to be customized according to the situation of different patients. Generally, the length, width and height of the suitable demineralized bone matrix carrier material are respectively 35mm, 10mm and 5mm, and the mass of the material is 4-5g. A diagram of the demineralized bone sample is shown in Figure 2.
  • BMSCs were inoculated into demineralized bone material at a concentration of 3.5 ⁇ 10 7 cells/g, and cultured for 0.5-8 weeks.
  • the size of demineralized bone was determined according to the degree of inferior turbinate atrophy of the patient, and a BMSC-decalcified bone complex was formed. bone.
  • the BMSC-decalcified bone complexes formed after 4 weeks of in vitro osteogenic induction are shown in Figure 3.
  • BMSCs were expanded in low-glucose medium containing bFGF. After the cells reached a certain number, they were inoculated on polyglycolic acid/polylactic acid scaffolds, and cultured in chondrogenic induction solution for 0.5, 1, 2, 3, 4, 6, 8, and 12 weeks. Afterwards, cartilage-like grafts can be formed in vitro. The experimental results are shown in Figure 4.
  • Fig. 4A After 8 weeks of in vitro culture, the construct developed a porcelain-white cartilage-like appearance (Fig. 4A), histological results showed a typical cartilage lacuna structure (Fig. 4B, Fig. 4C), and expressed abundant cartilage-specific matrix, including glycosamine Glycans (Fig. 4D, Fig. 4E, red) and collagen type II (Fig. 4F, Fig. 4G, brown).
  • the cartilage-like grafts of the BMSC-polyglycolic acid/polylactic acid scaffold prepared in Example 3 were implanted subcutaneously in nude mice, respectively.
  • a tissue engineered bone sample developed after 12 weeks is shown in Figure 5. The sample is removed and tested.
  • FIG. 5A The cartilage-like graft is tissue-engineered cartilage in vitro.
  • BMSCs undergo terminal ossification after implantation in vivo, and finally form tissue-engineered bone (hard bone).
  • Figure 5B shows the histological staining of 5A, showing a typical bone-like tissue structure.
  • Figure 5A is a general view: the tissue engineered cartilage regenerated in vitro, after implanting in the subcutaneous non-cartilage regeneration microenvironment, can develop into bone-like tissue;
  • Figure 5B is HE staining: histological staining results show a typical bone-like structure.
  • tissue engineered bone formed after 4 weeks of in vitro osteogenic induction of BMSC-composite demineralized bone (Fig. 3) was implanted subcutaneously in nude mice.
  • tissue engineered bone tissue was formed after 4-8 weeks of development.
  • a tissue engineered bone tissue sample formed after 6 weeks of development is shown in FIG. 6 .
  • the tissue-engineered cartilage regenerated in vitro is implanted in a subcutaneous environment (a non-cartilage-regenerative microenvironment), and the tissue-engineered cartilage undergoes heterotopic ossification to form tissue-engineered bone.
  • the inferior turbinate defect site belongs to the non-cartilage regeneration microenvironment, in which the BMSC-decalcified bone complex induced by chondrogenesis can undergo heterotopic ossification to form tissue engineered bone.
  • FIG. 7 Histological staining of the BMSC-decalcified bone complex samples shown in FIG. 6 is shown in FIG. 7 .
  • the results showed that the BMSC-demineralized bone composite samples had typical bone-like structures.
  • Figure 7 has a bone tissue-specific trabecular structure compared to Figure 5B, which is more in line with the tissue structure of natural bone tissue.
  • the final ossification of the constructed product cultured in the chondrogenic induction solution for 0.5-8 weeks was relatively sufficient, and hard bone with suitable hardness could be formed.
  • the constructed product still had the characteristics of cartilage tissue.
  • tissue engineered bone through osteogenic induction.
  • Culture in vitro for about 1-3 weeks.
  • the nasal mucosa is incised under a nasal endoscope, and the nasal mucosa and bone are separated to form an implantation cavity.
  • the tissue engineered bone is repaired in vitro into a suitable shape and then implanted.
  • the inner or outer lateral wall of the inferior turbinate of the volunteers corresponds to the inferior turbinate.
  • the graft is bone-like, so it is convenient for transplantation; on the other hand, the graft of the present invention finally develops into mature bone tissue after transplantation, and the final shape and hardness are closer to the natural inferior turbinate.
  • Cells have multiple differentiation potentials and can differentiate into different tissues in a specific differentiation environment. Therefore, it can be directed to differentiate into bone tissue in the submucosal environment near the bone surface to achieve turbinate regeneration; in the part close to the mucosa, it can be directed to differentiate into mucosal tissue, and tissue repair is performed by cell replacement.
  • Stem cells have a certain paracrine effect, which can secrete VEGF to promote angiogenesis, secrete IL-6 to regulate immune balance and inhibit inflammation, and secrete SDF to inhibit apoptosis in surrounding tissues, etc., all of which are beneficial to the repair of nasal mucosa. Function.
  • Stem cells have certain immunomodulatory effects. It can prevent the inappropriate activation of T lymphocytes, inhibit the proliferation of T cells, and inhibit the differentiation of T cells to Th1 and Th17. In addition, T cells can be immunosuppressed by the production of IDO catabolites. At the same time, stem cells can transform dendritic cells into a tolerant phenotype, and HLA-G5 and B7-H4 produced by stem cells can differentiate effector T cells into Treg (regulatory T cells), maintain T cell quiescence and regulate T cells. Cell subtype balance.
  • Stem cells can also inhibit the activation and function of B cells, blocking these cells in the G0/G1 phase of the cell cycle by paracrine means, thereby inhibiting the proliferation of B cells; it can also induce mature protein-1 ( Blimp-1) mRNA expression to inhibit the differentiation of B cells to plasma cells. Therefore, stem cells can generate an immune tolerance microenvironment in the process of tissue repair, thereby interrupting the immune response and reducing the immune inflammatory response in the local microenvironment, which is beneficial to the repair of mucosal tissue.
  • Blimp-1 mature protein-1
  • stem cells have significant advantages in mucosal regeneration. Neither simple prosthesis implantation nor cell-free implantation can achieve the immunomodulatory effect produced by the stem cell microenvironment, thereby reducing the regeneration efficiency of the nasal septum mucosa after implantation and affecting the prognosis of patients.
  • autologous bone is an autologous source and has no immunogenicity, but its source is limited, which will cause secondary damage to patients, which is unacceptable for some patients;
  • the mineralized mature natural bone tissue has very little viable cells, so it is difficult to survive in the mucosal environment in which the turbinate is transplanted (lack of osteoblasts and blood supply), while the viable cells of tissue engineered bone
  • the content is much higher than that of natural bone, and the porosity is high when it is just implanted in the body. Before the completion of vascularization, it can obtain nutrients through body fluid penetration, and it can survive stably after vascularization is established.
  • the survival rate of autologous tissue-engineered bone implanted in vivo is much higher than that of autologous bone grafting.
  • cell-free implants represented by bioceramics such as hydroxyapatite can improve turbinate status, ventilation and physiological structure, they have no obvious effect on nasal mucosa repair.
  • the nasal environment is susceptible to infection and artificial materials lack biological activity, there are many risks such as material exposure, prolapse, infection and rejection.
  • the bone graft of BMSC-polyglycolic acid/polylactic acid will degrade after transplantation to produce acidic degradation products, which will interfere with the regeneration of the inferior turbinate.
  • the BMSC-decalcified bone bone graft of the present invention has a certain degree of flexibility, and is easy to be trimmed into a graft of suitable size and shape.
  • the graft of the present invention has excellent mechanical strength properties, and does not produce acidic degradation products after transplantation, and by providing a microenvironment favorable for bone regeneration, the graft of the present invention is more likely to achieve bone regeneration and bone regeneration in the submucosal environment.
  • the mucosa regenerates and thus contributes to the formation of inferior turbinates with desirable properties (eg hardness, etc.).
  • the tissue-engineered bone graft for inferior turbinate reconstruction of the present invention has significant repairing effect, and has the characteristics of high safety, good compatibility, strong plasticity, and excellent repairing effect.

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Abstract

本发明提供了一种用于下鼻甲重建的组织工程骨移植物。具体地，本发明提供了一种组织工程化骨移植物，其含有在脱钙骨基质上生长的骨髓基质干细胞(BMSC)；所述组织工程化骨移植物用于下鼻甲重建。本发明的组织工程骨移植物能够有效重建下鼻甲，并且无免疫原性、安全性高。

Description

一种用于下鼻甲重建的组织工程骨移植物 技术领域
本发明属于生物医学组织工程领域，具体涉及一种用于下鼻甲重建的组织工程骨移植物。
背景技术
空鼻综合征(empty nose syndrome,ENS)是鼻甲过分切除导致的一种医源性并发症。表现为继发性鼻腔黏膜萎缩及一系列伴发症状，包括鼻腔咽部干燥感，注意力无法集中，疲劳、烦躁、焦虑、抑郁等。大约20％的下鼻甲切除术患者会发展出ENS。由于治疗效果不佳，患者及其家属对手术医生、医院存在着敌对情绪,甚至发生激烈矛盾，给社会带来一定的不安定因素。
为了治疗ENS，学者们尝试采用了各种填充材料，如硅胶，自体或异体骨、软骨，人工真皮、羟基磷灰石或其复合物。虽然上述方法能达到一定的治疗效果，但存在排异、过敏、组织相容性等问题。
综上所述，本领域迫切需要开发一种用于下鼻甲重建的组织工程骨移植物。
发明内容
本发明提供了一种用于下鼻甲重建的组织工程骨移植物。
在本发明的第一方面，提供了一种组织工程化骨移植物，它包括：
(a)脱钙骨基质载体；
(b)人骨髓基质干细胞(BMSC)细胞；其中，
所述脱钙骨基质载体的脱钙程度为95-85％；和/或
所述脱钙骨基质载体的厚度为3-8mm。
在另一优选例中，所述脱钙骨基质载体的脱钙程度为92％-86％。
在另一优选例中，所述脱钙骨基质载体的厚度为4.5-5.5mm。
在另一优选例中，所述的BMSC为自体细胞。
在另一优选例中，所述的BMSC来源于松质骨。
在另一优选例中，所述的松质骨包括：髂骨、胸骨、肋骨。
在另一优选例中，所述移植物为固态细胞材料复合物，且BMSC在复合材料 中的浓度为1×10 7个细胞/cm 3-1×10 8个细胞/cm 3，优选2×10 7细胞/cm 3-7×10 7细胞/cm 3。BMSC在复合材料中的含量为1×10 7个细胞/g-1×10 8个细胞/g，优选2×10 7细胞/g-7×10 7细胞/g。
在另一优选例中，所述组织工程化骨移植物形状与人体需要移植的下鼻甲缺损部位形状相符。
在本发明的第二方面，提供了一种制备如本发明第一方面所述骨移植物的方法，包括步骤：
(1)提供一自体BMSC细胞，所述BMSC细胞来自自体骨髓；
(2)通过含有碱性成纤维生长因子(bFGF)的培养液体外扩增培养BMSC细胞；
(3)接种BMSC细胞于脱钙骨基质载体，体外成软骨诱导培养，形成组织工程化骨(BMSC-脱钙骨复合物)。
在另一优选例中，步骤(2)中，所述体外培养液为低糖培养基。
在另一优选例中，步骤(2)中，所述扩增培养BMSC至第2-5代。
在另一优选例中，步骤(2)中，所述体外培养液中bFGF的浓度为0-10ng/mL；优选2-5ng/mL。
在另一优选例中，步骤(2)中，所述扩增的BMSC细胞为长梭形，细胞体积小，增殖活性强。
在另一优选例中，步骤(3)中，所述BMSC的接种浓度为1×10 7细胞/g-1×10 8细胞/g；优选为2×10 7细胞/g-7×10 7细胞/g；更优选地，为3.5×10 7细胞/g-5×10 7细胞/g。
在另一优选例中，步骤(3)中，所述体外成软骨诱导培养0.5-8周；优选0.5-4周。
在本发明的第三方面，提供了一种如本发明第一方面所述骨移植物的用途，用于制备一种修复下鼻甲缺损部位的药物。
在另一优选例中，所述的药物为包含活细胞的材料。
在另一优选例中，所述下鼻甲缺损部位选自下鼻甲基底、下鼻甲周边组织、鼻腔外侧壁。
在另一优选例中，所述组织工程化骨移植物还用于增大下鼻甲体积、缩小鼻 腔容积，改善鼻腔通气功能。
在本发明的第四方面，提供了一种修复下鼻甲缺损部位的方法，将如本发明第一方面所述的骨移植物施用给有需要的受试者。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了培养基中有无成纤维生长因子(bFGF)对BMSC细胞生长的影响；其中，A图中“+bFGF”为添加bFGF培养，“-bFGF”为未添加bFGF培养；B图中的折线图中，纵坐标为CCK-8检测结果的OD(optical density)值，横坐标为培养天数(d)。
图2显示了脱钙骨样品图。
图3显示了BMSC-复合脱钙骨体外成骨诱导4周后形成的组织工程骨。
图4显示了BMSC接种聚羟基乙酸/聚乳酸支架材料体外培养8周形成软骨样移植物的示意图，其中，A图为软骨样移植物，B、D、F图分别为该移植物的组织学、蕃红-O、和II型胶原免疫组化染色(比例尺为1mm)，C、E、G图分别为B、D、F图中黑框内容的放大(比例尺为100μm)。
图5显示了BMSC接种聚羟基乙酸/聚乳酸支架材料形成的组织工程软骨植入皮下环境12周后取材的示意图。其中，A图为BMSC-聚羟基乙酸/聚乳酸组织的软骨样移植物在裸鼠皮下继续发育形成的组织工程骨，B图为A图的组织学染色(比例尺为100μm)。
图6显示了BMSC-脱钙骨复合物移植到裸鼠皮下环境中形成的组织工程骨组织。
图7显示了BMSC-脱钙骨复合物的组织学染色。
图8显示了BMSC-脱钙骨复合物植入空鼻症患者鼻腔外侧壁相当于下鼻甲部位术前术后MRI影像。
具体实施方式
本发明人通过广泛而深入的研究，首次开发了一种基于特定材质的脱钙骨基质，复合BMSC细胞构建的组织工程骨，本发明的组织工程骨特别适用于修复下鼻甲缺损。本发明中的脱钙骨基质具有特定的硬度、厚度：合适的厚度有利于支撑下鼻甲部位，便于BMSC细胞负载；特定的硬度便于修剪，同时提供下鼻甲支撑时所需强度。此外，材料上负载的BMSC有利于鼻腔粘膜修复。具体地，本发明人通过优化脱钙骨载体材料的厚度和脱钙程度、BMSC体外培养条件，构建了一种特殊的组织工程骨移植物材料，便于下鼻甲重建。在此基础上完成了本发明。
本发明基于微创取少量的BMSC细胞，通过体外培养，高密度接种于特定规模大小和厚度的脱钙骨材料后，通过培养得到一种有生物活性的BMSC细胞-脱钙骨材料复合物，依靠自体组织细胞的成骨作用及生物材料的降解吸收，最终形成新生的下鼻甲骨。
术语
如本文所用，本发明的“下鼻甲重建的组织工程骨移植物”、“本发明的组织工程化软骨/骨移植物”、“本发明的软骨/骨移植物”可以互换使用，皆指本发明第一方面述及的用于下鼻甲修复的组织工程骨移植物。
一般地，可选用脱钙骨-明胶复合支架，聚羟基乙酸/聚乳酸(PGA/PLA)，聚己内酯(PCL)，或者聚己内酯复合羟基磷灰石作为组织工程骨的支架材料。
术语“诱导”指提供特殊的生化环境，将具有多向分化能力的干细胞等细胞群体转变为另一种功能特性不同的细胞群体的过程。
术语“接种”指将细胞均匀分布于三维支架材料上的过程。
术语“自体移植”指将所需生物活体材料(如骨髓基质干细胞)从某个体中取出并再施用于同一个体的过程。
在本发明的一个优选例中，选用的组织工程载体的材料为脱钙骨基质。
脱钙骨基质
脱钙骨基质(DBM)是由同种异体骨或异种骨经脱钙处理，能降低免疫原性的骨移植材料。脱钙程度不同对应的机械强度也不同。具有良好的生物学特性、骨诱导性和骨传导性和生物降解性，促进新骨形成及骨组织矿化，进而加速骨愈合，可以单独或与自体骨、其它生物材料、生长因子联合有效修复骨损 伤，是比较理想的骨组织工程支架材料。
在另一优选例中，所述脱钙骨材料的长度为10-40mm，优选为34.5-35.5mm；宽度为5-15mm，优选为9.5-10.5mm。
在本发明的一个实施例中，脱钙骨材料的长、宽、高分别为35mm、10mm、5mm。
应理解，本发明的组织工程化骨移植物大小可以根据不同患者的情况来定制。例如，可以根据不同患者下鼻甲缺损的部位进行修剪，已满足实际要求。
为了在下鼻甲这一特定位置起支撑作用，本发明的组织工程骨移植物中的脱钙骨材料需要一定厚度。厚度过大，不利于干细胞接种和细胞悬液的充分渗透，并且培养的细胞材料复合体会出现中空现象，影响植入后的修复效果；厚度过小，无法满足力学强度要求，导致接种时细胞悬液流失，进而无法有效的负载细胞，降低接种效率。
本发明人优化了脱钙骨材料的厚度，在一优选例中，本发明的脱钙骨材料厚度为1-8mm；更佳地，厚度为2-5mm。
脱钙程度
可用于本发明组织工程载体的骨组织来源没有特别限制，可以是来源于人的同种异体骨组织，也可以是来源于动物(如猪、牛、羊、狗等)的异种骨组织。优选的是来源于猪、牛的异种骨组织。
在同样的处理条件下，脱钙程度较小时材料韧性较低，在修剪材料时会导致材料较易发生碎裂现象，增加操作难度，同时会延长材料在体内的降解时间；但脱钙程度过大时，材料强度不足，难以符合修复下鼻甲所需的强度，影响患者预后。
在本发明的一个实施例中，本发明的脱钙骨材料所述脱钙骨基质载体的脱钙程度为95-85％，优选为92％-86％。即，本发明中的脱钙骨材料的含钙量应控制在5-15％左右，优选为8-14％。
脱钙的方法
将骨组织浸入液氮中5min，随后置于75％乙醇溶液中进行脱脂处理；随后加入0.5M的HCL溶液中进行脱钙处理，HCL溶液每两小时更换一次，共更换3次；使用去离子水洗涤3次后加入0.05％胰酶溶液置于37℃恒温摇床消化2h进行脱 细胞处理；最后经冷冻干燥制成脱钙骨基质，干燥箱储存备用。
脱钙程度的测定
使用等离子发射光谱分析法测量。取0.5g冻干脱钙骨，充分研磨制成脱钙骨基质粉末粉末，置入100ml容量瓶，加入5ml浓HNO 3，微波190℃消解18分钟，定容至100ml；取溶液1.5ml加入等离子发射光谱仪(ICP)进行检测，读取数值；重复三次，取平均值。
骨髓基质干细胞
骨髓基质干细胞(bone marrow stromal cell，BMSC)是一类具有多向分化潜能的组织干细胞,在体内外适当的诱导环境下可以分化为骨、软骨、脂肪、肌肉、神经、肌腱及韧带等多种组织细胞。该细胞群来源充足,取材方便,增殖能力强。
在本发明的一个实施例中，直接以成骨诱导液扩增BMSC，待细胞达到一定数量之后，接种于脱钙骨或其他组织工程支架材料，继续以成骨诱导液培养1-3周。
在另一优选例中，以含bFGF的低糖培养基扩增BMSC。bFGF可显著提高BMSC增殖活性，有利于维持其干细胞特性，进而节省所需BMSC的数量并且提高成骨活性。
在另一优选例中，以高浓度的BMSC接种于脱钙骨材料，依患者下鼻甲萎缩程度决定脱钙骨大小，形成组织工程化骨(BMSC-脱钙骨复合物)。
本发明用于接种的BMSC浓度通常为1×10 7细胞/g-8×10 7细胞/g，较佳地为2×10 7细胞/g-5×10 7细胞/g。通常，以培养液调整种子细胞浓度，然后与本发明的组织工程载体混合，其中混合时培养液与固体性材料的比例没有特别限制，但是以本发明载体能够吸附的培养液最大量为准。
在本发明的移植物中，还可添加或复合其他各种细胞、生长因子、各种转基因成分，从而保持细胞表型、促进细胞生长或基质合成能力等，或者促进组织生长、血管、神经长入等。
形成的骨移植物，可直接植入体内骨缺损处等部位，修复骨组织缺损或填充骨组织。
hBMSC-脱钙骨复合物
本发明中hBMSC-脱钙骨复合物的细胞接种浓度通常约为1×10 7细胞/g-8×10 7细胞/g或更高。材料为脱钙骨材料或者其他固体性材料，固、液体复合材料。以培养液调整细胞浓度，然后与脱钙骨材料混合，其中混合时培养液与脱钙骨材料的比例没有特别限制，但是以该脱钙骨材料所能够吸附培养液的最大量为准。当支架材料为特殊三维形状时，如下鼻甲时，按实际体积的大小来进行计算。
制备方法
本发明的组织工程化软骨移植物制作简便。
在一个具体实施例中，先以含bFGF的低糖培养基扩增BMSC，待细胞达到一定数量之后，接种于脱钙骨或其他组织工程支架材料，再以专利(ZL201110268830.9)中实施例6所公开的体外成软骨诱导液培养1-8周，形成软骨样移植物。
本发明的主要优点包括
(1)本发明的脱钙骨基质材料能够有效重建下鼻甲。
(2)本发明所需干细胞取材于自体，无免疫原性，安全性高。
(3)BMSC微环境有利于鼻腔的黏膜修复。
(4)本发明仅需微量干细胞，取材过程为常规操作，不损伤正常组织。
(5)可按照组织缺损形状制备移植物大小，以达到精确修复。
(6)体外培养方法简便易学，便于推广，易形成产业化产品。
(7)本发明的组织工程化软骨/骨移植物具有较好的可塑性及一定的机械强度，容易加工成所需的形状并有支撑作用，满足下鼻甲特定位置的要求。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
成骨条件培养液
培养液为DMEM(delbecco's modified eagle medium)，加入β-磷酸甘油钠10nmol/L，地塞米松0.1μmol/L，L-α-磷酸抗坏血酸50μmol/L，L-谷氨酰胺300mg/L，1.25(OH) 2VD 3 10nmol/L和10％胎牛血清(hyclone，USA)，未注明试剂均为Sigma公司，USA。
体外成软骨诱导液
如专利(ZL 201110268830.9)中实施例6所公开的体外成软骨诱导液。
BMSC的扩增培养基(含bFGF的低糖培养基)：
用于扩增BMSC的培养基每升液体含低糖DMEM培养基10g，L-谷氨酰胺300mg，维生素C 50mg，碳酸氢钠3.7g。优选地，加入2-5ng/mL的碱性成纤维生长因子(bFGF)。
成骨诱导液
高糖DMEM培养液中含10％FBS，β-磷酸甘油10mM，维生素D310nM，地塞米松0.1μM。
实施例1：BMSC的获取和培养
从患者髂前上棘经穿刺取骨髓3～5ml，置于PercoII分离液上(密度1.073g/L)行梯度密度离心，骨髓与分离液比例为1:2。2550r/min离心30分钟，吸取中间云雾状细胞层，磷酸缓冲液(PBS)洗1次。1550r/min离心后弃上清获取有核细胞，以2×10 7细胞/cm 2接种培养皿，进行体外细胞扩增，以成骨条件培养液予成骨诱导。
原代细胞接种后48小时换液，细胞达80％～90％融合后，采用0.25％胰酶消化，2×10 3细胞/cm 2传代培养，置于37℃，5％CO 2培养箱培养至第3代，收集细胞并计数。
实施例2：BMSC培养条件的优化
以含bFGF的低糖培养基扩增BMSC。用于扩增BMSC的培养基中，每升液体含低糖DMEM培养基10g，L-谷氨酰胺300mg，维生素C 50mg，碳酸氢钠3.7g。培养基中添加0ng、1ng/mL、2ng/mL、5ng/mL、10ng/mL碱性成纤维生长因子(bFGF)。分别以上述培养基培养BMSC。
结果显示，在表明含bFGF的培养基中，bFGF浓度范围为2-5ng/mL左右最佳。
添加(+bFGF)与未添加(-bFGF)bFGF培养的细胞形态结果如图1A所示：添加bFGF培养的BMSC更能维持长梭形，且细胞体积较小；而未添加bFGF培养的普通体系中扩增的BMSC，形态较为铺展。
图1B中显示，添加bFGF培养的BMSC在培养的第九天依然可以有较好的增殖活性；未添加bFGF培养的BMSC在培养的第五天后增殖活性不断降低。
实施例3：BMSC复合组织工程支架材料的体外培养
(1)实验方法：
方法1：直接以成骨诱导液扩增BMSC，待细胞达到一定数量之后，接种于脱钙骨或其他组织工程支架材料，继续以上述成骨诱导液培养1-3周。
方法2：先以含bFGF的低糖培养基扩增BMSC，待细胞达到一定数量之后，接种于脱钙骨或其他组织工程支架材料，再以成骨诱导液培养1-3周。
方法3：先以含bFGF的低糖培养基扩增BMSC，待细胞达到一定数量之后，接种于脱钙骨或其他组织工程支架材料，再以专利(ZL 2011 1 0268830.9)实施例6中优选的体外成软骨诱导液培养0.5-8周，形成软骨样移植物(即组织工程软骨)。
其中，方法1从细胞扩增阶段就通过成骨诱导液提供成骨环境，可能更有利于后续骨再生；
方法2通过含bFGF的低糖培养基扩增细胞，与第一种方法相比可以获得更多的细胞数量；
方法3则在方法2的基础上，采用成软骨诱导液对细胞材料复合物进行为期更长的体外培养，体外再生出来的组织更加接近于软骨，植入下鼻甲缺损部位之后对周围的环境更加耐受，有望提高存活几率(下鼻甲缺损部位处于粘膜下的微环境，血供并不是非常丰富，不利于一般组织工程骨的长久存活，但组织工程软骨对缺血供的环境更加耐受)。上述被植入鼻腔黏骨膜下的组织工程软骨，由于其内部BMSC的终末骨化作用，会进一步通过“软骨内骨化”的方式继续发育成骨组织(与所缺损的组织类型相匹配)。
注：方法3优于1优于2。
(2)BMSC接种于脱钙骨材料：
选用脱钙骨作为组织工程骨的支架材料。通过脱钙处理，控制脱钙骨材料的含钙量在8-10％左右。脱钙骨材料的大小需根据不同患者的情况来定制。通 常较为适宜的脱钙骨基质载体材料的长、宽、高分别为35mm、10mm、5mm，材料的质量为4-5g。脱钙骨样品图如图2所示。
成骨诱导后BMSC以3.5×10 7细胞/g浓度接种于脱钙骨材料，培养0.5-8周，依患者下鼻甲萎缩程度决定脱钙骨大小，形成BMSC-脱钙骨复合物组织工程化骨。体外成骨诱导4周后形成的BMSC-脱钙骨复合物如图3所示。
(3)BMSC接种于其他材料：
以含bFGF的低糖培养基扩增BMSC，待细胞达到一定数量之后，接种于聚羟基乙酸/聚乳酸支架材料，成软骨诱导液培养0.5、1、2、3、4、6、8、12周之后，在体外均可形成软骨样移植物。实验结果如图4所示。
体外培养8周后，构建产物形成了瓷白色软骨样外观(图4A)，组织学结果显示典型的软骨陷窝结构(图4B、图4C)，并且表达了丰富的软骨特异基质，包括糖胺聚糖(图4D、图4E，红色)和II型胶原(图4F、图4G，褐色)。
这表明，用成软骨诱导液培养0.5-12周所得构建产物具有典型的软骨组织特征。
实施例4：动物移植实验
将实施例3制备的BMSC-聚羟基乙酸/聚乳酸支架材料的软骨样移植物(培养时间为0.5、1、2、3、4、6、8、12周)，分别植入裸鼠皮下，一个12周后发育而成的组织工程骨样本如图5所示。取出所述样本，进行检测。
结果如图5A所示。软骨样移植物在体外是组织工程软骨，植入体内之后BMSC发生终末骨化，最终形成了组织工程骨(硬骨)；图5B为5A的组织学染色，可见典型的骨样组织结构。
其中：图5A为大体观：体外再生的组织工程软骨，植入皮下非软骨再生微环境之后，可发育为骨样组织；
图5B为HE染色：组织学染色结果显示了典型的骨样结构。
将BMSC-复合脱钙骨体外成骨诱导4周后形成的组织工程骨(图3)，植入裸鼠皮下。
结果表明，4-8周发育后形成组织工程骨组织。其中，一个6周发育后所形成组织工程骨组织样品示于图6。
体外再生的组织工程软骨植入皮下环境中(非软骨再生微环境)，组织工程软骨发生异位骨化，形成组织工程骨。下鼻甲缺损部位属于非软骨再生微环 境，经成软骨诱导的BMSC-脱钙骨复合物在该环境中可发生异位骨化形成组织工程骨。
图6所示的BMSC-脱钙骨复合物样品的组织学染色如图7所示。结果表明，所述BMSC-脱钙骨复合物样品具有典型的骨样结构。图7较图5B拥有骨组织特异性的骨小梁结构，更加符合天然骨组织的组织结构。
此外，成软骨诱导液培养时间0.5-8周的构建产物的终末骨化较为充分，可形成硬度合适的硬骨。当成软骨诱导液培养时间≥12周时，所构建产物仍具有软骨组织特征，然而作为下鼻甲重建的移植物植入体内后，不易通过软骨内骨化发生终末骨化。
这提示，采用0.5周-4周(或3-30天，或7-28天)的构建产物是优选的，因为一方面移植时为软骨状，便于移植操作；另一方面，移植物在移植后易于通过软骨内骨化发生终末骨化，形成形状和硬度与天然下鼻甲更为接近。
(注：体外成软骨诱导的时间如果超过8周，则会降低移植物在体内发生异位骨化的几率)。
实施例5：下鼻甲重建
根据实施例3中的方法1，将细胞接种于脱钙骨材料，通过成骨诱导形成组织工程骨。体外培养1-3周左右，待细胞与生物材料附着良好后，鼻内镜下切开鼻腔黏膜，分离鼻粘膜和骨质，形成植入腔，将组织工程化骨体外修成合适形状后植入志愿者下鼻甲内或鼻腔外侧壁相当于下鼻甲部位。
结果：图8显示志愿者的症状改善明显，显著影像学数据及主观量表评分均有显著改善。
结果说明由于移植物为骨状，因此便于移植手术；另一方面，本发明的移植物在移植后最终发育为成熟的骨组织，最终的形成形状和硬度与天然下鼻甲更为接近。
讨论：
本发明采用的BMSC细胞复合的生物材料有诸多优点：
(1)细胞拥有多项分化潜能，在特定分化环境中可分化为不同的组织。因此其在鼻黏膜下环境靠近骨面部分可定向分化为骨组织，实现鼻甲再生；在靠近黏膜部位可定向分化为黏膜组织，以细胞替代的方式进行组织修复。
(2)干细胞具有一定的旁分泌(Paracrine)作用，可以分泌VEGF促进血管新生，分泌IL-6调节免疫平衡抑制炎症，分泌SDF抑制周围组织中的细胞凋亡等，这些均有利于修复鼻黏膜功能。
(3)干细胞具有一定的免疫调节作用。可以防止T淋巴细胞的不适当激活，抑制T细胞增殖，抑制T细胞向Th1、Th17分化。此外，可以通过产生IDO分解代谢物来对T细胞进行免疫抑制。同时，干细胞可以将树突状细胞转化为耐受性表型，干细胞产生的HLA-G5和B7-H4可使效应T细胞向Treg(调节性T细胞)分化，维持T细胞静止状态和调节T细胞亚型平衡。干细胞也可抑制B细胞的活化和功能，通过旁分泌的方式将这些细胞阻滞在细胞周期的G0/G1期，从而抑制B细胞的增殖；也可通过下调B淋巴细胞诱导成熟蛋白-1(Blimp-1)的mRNA表达来抑制B细胞向浆细胞的分化。因此，干细胞在组织修复过程中可产生免疫耐受微环境，从而中断免疫反应，减少局部微环境的免疫炎症反应，有利于黏膜组织的修复。
(4)相对于无生物活性的假体植入，或羟基磷灰石这类的无细胞负载植入，干细胞在黏膜再生方面具有其显著优势。单纯假体植入或无细胞负载植入都无法实现干细胞微环境所产生的免疫调节作用，进而降低植入后鼻中隔黏膜再生效率，影响患者预后。
与其他材料对比，如使用自体骨、或使用无细胞移植物羟基磷灰石修复：自体骨为自体来源无免疫原性，但其来源有限，对于患者会造成二次伤害，部分患者难以接受；此外，矿化后的成熟天然骨组织中活细胞含量极少，因此，将其移植到鼻甲所处的黏膜环境中很难存活(缺乏成骨细胞和血供)，而组织工程骨的活细胞含量远高于天然骨，且刚植入体内时孔隙率较高，血管化完成前可通过体液渗透获得营养，血管化建立后即可稳定存活。因此，自体组织工程骨植入体内后(尤其在非成骨环境下)的存活率远高于自体骨移植。以羟基磷灰石等生物陶瓷为代表的无细胞植入物虽可改善鼻甲状态，改善通气量及生理结构，但其对于鼻黏膜修复并无明显效果。此外，由于鼻腔环境易感染，人工材料又缺乏生物活性，因此存在材料外露、脱出、感染和排异等诸多风险。BMSC-聚羟基乙酸/聚乳酸的骨移植物，移植后降解会产生酸性降解产物，会干扰下鼻甲的再生。
与现有技术相比，本发明的BMSC-脱钙骨的骨移植物具有一定柔韧度，便于修剪为适宜大小和形状的移植物。此外，本发明的移植物具有优异的力学强 度特性，并且移植后不会产生酸性降解产物，并且通过提供利于骨再生的微环境使得本发明的移植物更易于在鼻黏膜下环境实现骨再生与黏膜再生，因此有助于形成性能(如硬度等)符合要求的下鼻甲。
因此，本发明用于下鼻甲重建的组织工程骨移植物具有显著的修复效果，具有安全性高、相容性好、可塑性强、修复效果优异等特点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种组织工程化骨移植物，其特征在于，它包括：
    (a)脱钙骨基质载体；
    (b)人骨髓基质干细胞(BMSC)细胞；其中，
    所述脱钙骨基质载体的脱钙程度为95-85％；和/或
    所述脱钙骨基质载体的厚度为3-8mm。
  2. 如权利要求1所述的骨移植物，其特征在于，所述脱钙骨基质载体的脱钙程度为92％-86％。
  3. 如权利要求1所述的骨移植物，其特征在于，所述脱钙骨基质载体的厚度为4.5-5.5mm。
  4. 如权利要求1所述的骨移植物，其特征在于，所述的BMSC为自体细胞。
  5. 如权利要求1所述的骨移植物，其特征在于，所述的BMSC来源于松质骨。
  6. 如权利要求5所述的骨移植物，其特征在于，所述的松质骨包括：髂骨、胸骨、肋骨。
  7. 如权利要求1所述的骨移植物，其特征在于，所述移植物为固态细胞材料复合物，且BMSC在复合材料中的浓度为1×10 7个细胞/cm 3-1×10 8个细胞/cm 3，优选2×10 7细胞/cm 3-7×10 7细胞/cm 3。
  8. 如权利要求1所述的骨移植物，其特征在于，所述组织工程化骨移植物形状与人体需要移植的下鼻甲缺损部位形状相符。
  9. 一种制备如权利要求1所述骨移植物的方法，其特征在于，包括步骤：
    (1)提供一自体BMSC细胞，所述BMSC细胞来自自体骨髓；
    (2)通过含有碱性成纤维生长因子(bFGF)的培养液体外扩增培养BMSC细胞；和
    (3)接种BMSC细胞于脱钙骨基质载体，体外成软骨诱导培养，形成组织工程化骨(BMSC-脱钙骨复合物)。
  10. 如权利要求9所述的方法，其特征在于，步骤(2)中，所述体外培养液中bFGF的浓度为0-10ng/mL；优选2-5ng/mL。
  11. 如权利要求9所述的方法，其特征在于，步骤(3)中，所述BMSC的接种浓度为1×10 7细胞/g-1×10 8细胞/g；优选为2×10 7细胞/g-7×10 7细胞/g；更优选地，为3.5×10 7细胞/g-5×10 7细胞/g。
  12. 如权利要求9所述的方法，其特征在于，步骤(3)中，所述体外成软骨诱导培养0.5-8周；优选0.5-4周。13.一种如权利要求1所述骨移植物的用途，其特征在于，用于制备一种修复下鼻甲缺损部位的药物。
  13. 如权利要求13所述的用途，其特征在于，所述下鼻甲缺损部位选自下鼻甲基底、下鼻甲周边组织、鼻腔外侧壁。
  14. 一种修复下鼻甲缺损部位的方法，其特征在于，将如权利要求1所述的骨移植物施用给有需要的受试者。
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