WO2017054335A1 - 含仿生牙周膜的生物牙根复合体及其制作方法 - Google Patents

含仿生牙周膜的生物牙根复合体及其制作方法 Download PDF

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WO2017054335A1
WO2017054335A1 PCT/CN2015/098312 CN2015098312W WO2017054335A1 WO 2017054335 A1 WO2017054335 A1 WO 2017054335A1 CN 2015098312 W CN2015098312 W CN 2015098312W WO 2017054335 A1 WO2017054335 A1 WO 2017054335A1
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periodontal
collagen
root
stem cells
bio
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PCT/CN2015/098312
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English (en)
French (fr)
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王松灵
胡磊
韩子韵
高振华
王秀梅
仇志烨
王硕
崔福斋
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首都医科大学
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Publication of WO2017054335A1 publication Critical patent/WO2017054335A1/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
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges

Definitions

  • the present invention relates to the field of biomedical materials, and in particular to a biodental complex comprising a bionic periodontal membrane and a method of fabricating the same.
  • Tissue engineering method The seed cells are combined with a pre-designed bio-stent with a complete tooth morphology to form a regenerated tooth similar to the normal tooth structure, shape and function.
  • the root is the main support and load-bearing part of the entire tooth, during the chewing process. Play an irreplaceable role.
  • the dentin of the root has a high density and a certain pore structure. About 70% of the composition is inorganic mineral, 20% is organic, 10% is water, and the microstructure is inorganic minerals arranged in the form of nanoparticles in the collagen molecule. Mineralized collagen structure between the surface and the molecular surface. Root regeneration can avoid the technical problems of controlling tooth shape and tooth eruption relative to whole tooth regeneration, and can solve the defect that the implanted denture lacks biological activity and metal foreign matter.
  • the regenerated root has a periodontal membrane and a dentin structure, and can also be repaired in morphology and function, so it has unique advantages in dental tissue engineering research.
  • the regenerated root can be fitted with an artificial crown, which is comparable in shape and function to the natural crown, thus creating a tooth with a complete structure and function.
  • bioengineered tooth root having a normal root physiological anatomy and function in the tooth missing site, and four conditions must be met to achieve this process: , suitable scaffold material; 2, good seed cells; 3, reliable stent / cell complex construction method; 4, suitable for micro-environment of biological root regeneration.
  • the synthetic bio-root support material is mainly calcium phosphate bioceramic material, such as tris/hydroxyapatite composite ceramic.
  • This calcium phosphate bioceramic has a large disadvantage: 1. Calcium phosphate bioceramic is only a porous or dense structure of inorganic ceramic material, does not have the bionic structure and chemical composition of the root, which is not conducive to the reconstruction of the biological root; Calcium phosphate bioceramics are more brittle and are not conducive to the maintenance of intraoperative and postoperative stent integrity. 3. Calcium phosphate bioceramics degrade slowly, which is not conducive to reconstruction of bio-roots within short sputum (3-6 months).
  • a periodontal (periodontium) tissue is present on the outer circumference of the natural root and on the inner side of the alveolar bone.
  • the periodontal ligament tissue is also called periodontal ligament, which is composed of cells (fibroblasts, osteoblasts, osteoblasts, osteoclasts, etc.), extracellular matrix, collagen fibers, nerves, blood vessels. It is an essential component of the biological roots. It has many functions such as supporting teeth, feeling, nutrition and renewing the cementum and alveolar bone. Therefore, the construction of biological roots should also include the construction of periodontal ligaments.
  • the cell sheet avoids the conventional cell digestion process, thus retaining important cell surface proteins and extracellular matrix.
  • the cell membrane is wrapped outside the bio-root stent and can be used simply.
  • the scaffold material is more conducive to the construction of the biological root.
  • the periodontal membrane is very tightly bound to the cementum and has a certain chemical bond. Simply wrapping the cell membrane outside the stent does not result in a tightly bonded structure similar to the natural root and periodontal membrane. It is even more difficult to form a chemical bond.
  • the present invention provides a biodental complex comprising a biomimetic periodontal membrane and a bioroot support.
  • the material of the bio-root stent is mainly a mineralized collagen with high inorganic content, and has the same microstructure and chemical composition as the natural dentin; the stent has a macroscopic shape similar to the natural root, and has a macro blind hole inside or
  • the through-hole structure can be used to load cells, growth factors, drugs, etc., as well as for cell growth.
  • the biodental complex containing the bionic periodontal ligament is compounded with odontogenic stem cells such as dental pulp stem cells in the bio-root stent; the outer layer is tightly bound with a layer of periodontal cell membrane made of dental stem cells, which is rich in cells and extracellular.
  • odontogenic stem cells such as dental pulp stem cells in the bio-root stent
  • periodontal cell membrane made of dental stem cells, which is rich in cells and extracellular.
  • the main component of natural periodontal ligaments such as matrix and collagen fibers.
  • the present invention also provides a method for preparing a biological root stent and a method for preparing a biological root complex comprising a biomimetic periodontal membrane.
  • a biodental complex comprising a bionic periodontal membrane.
  • the biodental complex containing the bionic periodontal membrane is composed of a bio-root stent, a odontogenic stem cell and a periodontal cell membrane.
  • the bio-root stent component comprises mineralized collagen, wherein the bio-root stent is grown with a dental stem cell, and the bio-root stent is tightly bound to a periodontal cell membrane made of a dental stem cell.
  • the periodontal cell membrane is also called the bionic periodontal membrane.
  • the odontogenic stem cells are dental follicle stem cells, dental pulp stem cells, apical papillary stem cells, deciduous deciduous stem cells or gingival stem cells.
  • the dental pulp stem cells or the apical papillary stem cells are inoculated in the bio-root stent, and the periodontal ligament is used in the periodontal cell membrane.
  • the biological root canal is also referred to as a stent
  • the periodontal cell membrane is also called a periodontal membrane, a cell membrane and a membrane.
  • the material of the scaffold is mineralized collagen, and the component comprises collagen and apatite, wherein the apatite is arranged in the form of weakly crystallized nanoparticles between the collagen molecules and the molecular surface, collagen.
  • /Apatite 5/5 ⁇ 2/8 ⁇ );
  • the biomimetic periodontal membrane is a membrane of the periodontal ligament stem cells rich in cells, extracellular matrix and collagen fibers, with a thickness of 0.7 mm, composed of 2-3 layers of cells.
  • the microstructure between the stent and the bionic periodontal membrane is microscopically embedded and chemically bonded through the extracellular matrix.
  • the collagen in the scaffold material is animal-derived type I collagen, preferably type I collagen derived from bovine Achilles tendon.
  • the main phase of the apatite component in the material of the stent is hydroxyapatite.
  • the material of the stent may also contain a bioabsorbable medical polymer, including polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL),
  • the bioabsorbable medical polymer is contained in the scaffold material in an amount of not more than 50% by weight. Accordingly, the content of mineralized collagen in the scaffold material is 50 wt% - 100 wt ⁇ 3 ⁇ 4.
  • the material of the stent can also be made of bioabsorbable medical polymer, including polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL).
  • PLA polylactic acid
  • PGA polyglycolic acid
  • PLGA lactic acid-glycolic acid copolymer
  • PCL polycaprolactone
  • the stent has a microscopic pore structure with a porosity of 70% to 95% and a pore diameter of 50 to 500 ⁇ .
  • the shape of the bracket is a columnar body; the height of the columnar body is 9 ⁇ 12 mm; the outer side surface of the columnar body is circular or approximately circular by a plane perpendicular to the axial direction of the columnar body Shape, the circumference is 11 ⁇ 14 mm; the upper bottom surface of the columnar body is a plane or a convex surface, if it is a convex surface, the height of the protrusion is not higher than 1 mm, and the height is included in the total height of the columnar body; the lower bottom surface of the columnar body is Plane or convex surface, if convex, the height of the protrusion is not higher than 1 mm, and the height is included in the total height of the column.
  • the inside of the bracket may have a through hole in the axial direction of the columnar body, or a blind hole in the bottom of the columnar body, and the length of the blind hole in the axial direction of the column body is not less than 2/3 of the height of the columnar body; 0 ⁇ 6.0 ⁇
  • the inner side of the blind hole or the through hole is perpendicular to the plane of the axial direction of the shape of the circular or nearly circular shape, the circumference is 3. 0 ⁇ 6.0 mm.
  • the membrane is composed of 2-3 layers of cells, wherein the moisture accounts for 50-80 wt%, and the moisture is removed. Among other substances, collagen fibers account for 60 to 75 wt%.
  • a bio-root stent is provided, the features of which are consistent with the bio-root stent in the biodental complex of the first aspect comprising a biomimetic periodontal membrane.
  • a method for preparing a biological root stent comprises the following steps:
  • Step Sl-1 dissolving collagen in any one of hydrochloric acid, nitric acid or acetic acid to prepare an acid solution of collagen
  • collagen concentration is 5.0x10 - 5 ⁇ 5.0x10 - 3 g / mL;
  • Step Sl-2 continue to stir the solution obtained in step S1-1, slowly add a solution containing calcium ions, the amount of calcium ions added per gram of collagen corresponding to the addition of calcium ions 0.01 ⁇ 0.16 mol;
  • Step S1-4 continuously stirring the solution obtained in the step S1-3, slowly adding the NaOH solution to the pH of the mixed system
  • Step Sl-5 the mixing system obtained in step S1-4 is allowed to stand for 24 ⁇ 120 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated by centrifugation to obtain mineralized collagen jelly;
  • Step Sl-6 detecting the content of the solid material in the mineralized collagen jelly obtained in step Sl-5, diluting or concentrating the jelly, so that the content of the solid matter therein reaches 0.72 ⁇ 0.9 g / mL;
  • Step Sl-7 taking a certain amount of the mineralized collagen jelly obtained in step Sl-6, filling the mold, performing sufficient freeze-drying to obtain a mineralized collagen scaffold, and performing cutting and cutting;
  • Step S1-8 preparing a concentration of 0.005 ⁇ 0.25 wt% of glutaraldehyde in ethanol as a cross-linking agent, the mineralized collagen scaffold obtained in step S1-7 is immersed in the cross-linking agent solution 24 ⁇ 48 small ⁇ , cross-linking;
  • Step Sl-9 the mineralized collagen scaffold is taken out from the cross-linking agent solution, placed in a chromatography column, and washed with flowing pure water for 48-72 hours to remove residual cross-linking agent;
  • Step S10-10 the mineralized collagen scaffold obtained in step S1-9 is vacuum dried or freeze-dried;
  • Step S1-1 the post-treatment of the mineralized collagen scaffold obtained in step S1-10, Wash and sterilize to obtain a mineralized collagen-based bio-root stent.
  • post-processing of the bracket includes but is not limited to: machining through holes or blind holes, machining surface grooves/grains/patterns, edge chamfering, trimming. Sealed after sterilization.
  • a method for fabricating a biodental complex comprising a bionic periodontal membrane is provided, the method comprising the steps of:
  • the step S2 specifically includes:
  • Step S2-l selecting allogeneic dental pulp stem cells cultured under the conditions of production quality management regulations, 1x10
  • pulp cells After the pulp cells are digested, they are mixed with the medium by air blowing, and inoculated on the biological root support material by dropping and vacuum adsorption;
  • Step S2-2 the scaffold material obtained in step S2-1 is allowed to stand in the incubator for 4 hours, and then placed in a rotary three-dimensional incubator for further cultivation, the rotation speed is 2-5 times/min, every three days. Change the medium and incubate for 5 days.
  • the step S3 specifically includes:
  • Step S3-l selecting autologous or allogeneic periodontal ligament stem cells cultured in accordance with production quality management specifications
  • MEM medium changing every three days
  • Step S3-2 after 10-14 days of culture, wrinkles appear on the edge cells of the culture dish, and the periodontal cell membrane is completely removed by a relatively blunt blade or a cell scraper, and the periodontal cell membrane is not dried during the process;
  • Step S3-3 the periodontal cell membrane obtained in step S3-2 is completely expanded, folded in half, and the liquid is blotted dry, and the biological root-support/cell complex obtained in step S2 is placed on the periodontal cell membrane.
  • the bio-root stent/cell complex is rolled up to the other side, and the tissue is loosely fitted to form a biodental complex.
  • a method for preparing a biological root canal comprising a bionic periodontal membrane is provided.
  • the preparation method combines the in vitro biomimetic mineralization technology and the cell membrane technology, and mainly includes the following steps: [0050] Step S1-1, dissolving the collagen in any one of hydrochloric acid, nitric acid or acetic acid to prepare collagen Acid solution , wherein the collagen concentration is 5.0x10 - 5 ⁇ 5.0x10 - 3 g / mL;
  • Step Sl-2 continuously stirring the solution obtained in step Sl-1, slowly adding a solution containing calcium ions, the amount of calcium ions added is 0.1 ⁇ 0.16 mol per kg of collagen;
  • Step Sl-4 continuously stirring the solution obtained in the step S1-3, slowly adding the NaOH solution to the pH of the mixed system.
  • Step Sl-5 the mixing system obtained in step S1-4 is allowed to stand for 24 to 120 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated to obtain a mineralized collagen jelly;
  • Step Sl-6 the jelly obtained in the step S1-5 is freeze-dried, and after grinding, a mineralized collagen powder is obtained.
  • a method of making a biological root canal/cell complex comprising a bionic periodontal membrane is provided.
  • the preparation process of the bio-root stent/cell complex containing the bionic periodontal membrane comprises the following steps:
  • Step S2-l selecting allogeneic dental pulp stem cells cultured in accordance with the production quality management (GMP) condition
  • 1x10 7 dental pulp stem cells were digested, mixed with medium and mixed, and seeded on the biological root scaffold material by dropping and vacuum adsorption.
  • Step S2-2 the scaffold material obtained in step S2-1 is allowed to stand in the incubator for 4 hours, and then placed in a rotary three-dimensional incubator for further cultivation, the rotation speed is 2-5 times/min, every three days. Change liquid, culture for 5 days
  • Step S3-1 selecting autologous or allogeneic periodontal ligament stem cells cultured in accordance with the production quality management (GMP) condition, and inoculating the vigorously growing second or third generation periodontal ligament stem cells in a 10 cm culture dish, cultivating Ingredients are ⁇ - ⁇ medium (containing 15% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin)
  • Step S3-2 After 10-14 days of culture, the cells on the edge of the culture dish are wrinkled, and the cell membrane is completely removed by a relatively blunt blade or a cell scraper, and the cell membrane is not dried during the process.
  • Step S3-3 the periodontal membrane obtained in step S3-2 is completely spread, folded in half, and the liquid is blotted dry, and the stent/cell complex obtained in step S2-2 is placed on the periodontal membrane. On one side, roll up the wrapped stent/cell complex to the other side and tighten it properly.
  • the allogeneic stem cells cultured under the conditions of production quality management may also be the following odontogenic stem cells, such as dental follicle stem cells, dental pulp stem cells, apical papillary stem cells, deciduous deciduous stem cells, and gingival stem cells.
  • a tissue engineering construction method of a biological tooth root comprising a bionic periodontal membrane.
  • the construction method mainly comprises the following steps:
  • Step Sl-1 dissolving the collagen in any one of hydrochloric acid, nitric acid or acetic acid to prepare an acid solution of collagen
  • collagen concentration is 5.0x10 - 5 ⁇ 5.0x10 - 3 g / mL;
  • Step Sl-2 continuously stirring the solution obtained in step S1-1, slowly adding a solution containing calcium ions, the amount of calcium ions added is 0.1 ⁇ 0.16 mol per kg of collagen;
  • Step S1-4 continuously stirring the solution obtained in the step S1-3, slowly adding the NaOH solution to the pH of the mixed system
  • Step Sl-5 the mixing system obtained in step S1-4 is allowed to stand for 24 ⁇ 120 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated to obtain mineralized collagen jelly;
  • Step Sl-6 detecting the content of the solid matter in the mineralized collagen jelly obtained in step S1-5, diluting or concentrating the jelly, so that the content of the solid matter therein reaches 0.72 ⁇ 0.9 g / mL;
  • Step S1-5 measuring a certain amount of the mineralized collagen jelly obtained in step S1-6 is filled into the mold, and is fully freeze-dried to obtain a mineralized collagen scaffold, and cutting and cutting;
  • Step S8-8 preparation The concentration is 0.005 ⁇
  • the 0.25 wt% ethanol solution of glutaraldehyde is used as a crosslinking agent, and the mineralized collagen scaffold obtained in step S1-7 is immersed in the cross-linking agent solution for 24 to 48 hours to carry out crosslinking;
  • Step Sl-9 the mineralized collagen scaffold is taken out from the cross-linking agent solution, placed in a chromatography column, and washed with flowing pure water for 48-72 hours to remove residual cross-linking agent;
  • Step S10-10 the mineralized collagen scaffold obtained in step S1-9 is vacuum dried or freeze-dried;
  • Step S1-1 the post-treatment of the mineralized collagen scaffold obtained in step S1-10, Cleaning and sterilizing to obtain a mineralized collagen-based bio-root stent, wherein post-treatment of the stent includes, but is not limited to, processing through-holes or blind holes, machined surface grooves/grains/patterns, edge chamfering, trimming. Sealed after sterilization.
  • the stent has a microscopic pore structure with a porosity of 70% to 95% and a pore diameter of 50 to 500 ⁇ .
  • the shape of the bracket is a columnar body; the height of the columnar body is 9 ⁇ 12 mm; the outer side of the columnar body is circular or nearly circular in shape perpendicular to the plane of the axial direction of the columnar body, The length may be 11 ⁇ 14 mm; the inside may have a through hole in the axial direction of the columnar body, or a blind hole in the bottom of the columnar body, the length of the blind hole along the axial direction of the column body is not less than 2/3 of the height of the column body; The side surface of the blind hole or the through hole of the columnar body is circular or nearly circular in shape perpendicular to the plane perpendicular to the axial direction of the column body, and has a circumference of 3.0 to 6.0 mm.
  • Step S2-l selecting a heterogeneous dental pulp stem cell cultured in accordance with the production quality management (GMP) condition
  • 1x107 dental pulp stem cells were digested, mixed with medium, and mixed with the biological root scaffold material obtained in step S1-11 by dropping and vacuum adsorption.
  • the dental pulp stem cells cultured under the conditions of production quality management may also be the following odontogenic stem cells, such as dental follicle stem cells, periodontal ligament stem cells, apical papilla stem cells, deciduous deciduous stem cells, and gingiva stem cell.
  • odontogenic stem cells such as dental follicle stem cells, periodontal ligament stem cells, apical papilla stem cells, deciduous deciduous stem cells, and gingiva stem cell.
  • step S2-2 the scaffold material obtained in step S2-1 is allowed to stand in the incubator for 4 hours, and then placed in a rotary three-dimensional incubator for further cultivation, the rotation speed is 2-5 times/min, and is changed every three days. Liquid, cultured for 5 days.
  • Step S3-1 selecting autologous or allogeneic periodontal ligament stem cells cultured in accordance with the production quality management (GMP) condition, and inoculating the vigorously growing second or third generation periodontal ligament stem cells in a 10 cm culture dish, cultivating Ingredients are ⁇ - ⁇ medium (containing 15% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin)
  • Step S3-2 After 10-14 days of culture, the cells on the edge of the culture dish are wrinkled, and the periodontal membrane is completely removed by a relatively blunt blade or a cell scraper, and the periodontal membrane is not dried during the process.
  • Step S3-3 the periodontal membrane obtained in step S3-2 is completely expanded, folded in half, and the liquid is blotted dry, and the stent/cell complex obtained in step S2-2 is placed on the periodontal membrane. On one side, roll up the wrapped stent/cell complex onto the other side and tighten as appropriate to form a stent/cell/membrane complex.
  • Step S4-l 3 months after tooth extraction in the mandibular anterior region of the mini-pigs, bio-root implantation is performed.
  • Anesthesia Suspension new sputum + ketamine 15 mg / kg intramuscular injection anesthesia. After skin preparation, use 2% iodophor for disinfection of the head and face and mouth, and spread the towel. Partial infiltration anesthesia with atecaine.
  • (2) The mandibular anterior region is designed to cut the alveolar crest, cut to the bone surface, and turn up the periosteal flap. See the mandibular anterior teeth in the edentulous area. The alveolar bone is flat, the width is sufficient, the york-garde drill is positioned.
  • the stage is reamed, the hole is prepared, and the stent/cell/membrane complex is implanted at the alveolar crest.
  • the implant has a torque of about 35 Ncm.
  • the submerged implant can absorb the suture and tightly suture the wound.
  • Step S4-2 After the implantation operation, the small pigs were intramuscularly injected (antibiotic) with penicillin 800,000 u/day, and fed with penicillin for three consecutive days. Wound healing was observed at 1 week, 2 weeks, and 4 weeks after surgery.
  • Step S4-3 6 months after the implantation of the biological root, the biological root regeneration is successful, and the superstructure is repaired.
  • Step S4-5 on the wearing gold core post, the position is smooth, the edge is close, the glass ion bonding, the full crown tooth preparation, the silicone rubber take the impression mold, inject the plaster model, and make the porcelain full crown.
  • Step S4-6 in the edentulous area, waxing teeth, silicone rubber embossing mold, filling the dentition area of the female mold with self-setting resin
  • Step S4-7 1 week later, the Linyi crown was removed from the small pig mouth, and the porcelain crown was tried, and the position was smooth, the edges were close, and the glass ions were bonded.
  • Steps S4-8 respectively, after wearing the whole crown for 3 or 6 months, the tissue was taken for histological and clinical evaluation, and it was found that the periodontal membrane-like tissue was surrounded by the root of the tooth, and the sharpy fiber was obliquely inserted into the alveolar from the root surface. In the bone, the periodontal tissue is healthy.
  • the present invention prepares an animal-derived type I collagen and hydroxyapatite into a root-like biological root-supporting stent, and then attaches the dental pulp stem cells thereto, and the outer layer is wrapped with a periodontal cell membrane to form a biological root-supporting stent/ The cell/membrane complex is implanted into the jaw bone of the animal to regenerate the biological root.
  • the biodental scaffold containing the mineralized collagen of the present invention has suitable mechanical properties and biodegradability, and can be combined with periodontal
  • the cell membrane is tightly bound, and the biodental root regenerated in the jaw of the animal is not only similar in structure and composition to the natural root, but also contains the periodontal ligament structure, can function after the crown, and can be functional later. Remodeled.
  • FIG. 1 is a flow chart of a tissue engineering construction of a bio-rooted stent containing a bionic periodontal membrane according to the present invention
  • 2A-2D are a composite method of a dental pulp stem cell and a stent of the present invention.
  • 5A-5D are transmission electron micrographs of a bionic periodontal patch of the present invention.
  • 6A-6F are the results of gene and protein expression detection of the bionic periodontal membrane of the present invention.
  • 7A-7C are the histological comparisons of the regenerated periodontal tissue with the normal tissue and the implant periodontal tissue of the present invention.
  • the present invention is directed to providing a material and a scaffold having a root biomimetic structure and chemical composition, having suitable mechanical properties and biodegradability, mixing the material with cells for rotational three-dimensional culture to ensure nutrient supply, and A tightly bound biomimetic periodontal ligament tissue was constructed outside the scaffold to successfully construct the histology of the biodental root.
  • FIG. 1 is a flow chart showing the construction of a biological root tissue containing a biomimetic periodontal membrane according to the present invention.
  • the manufacturing process of the biodental complex containing the bionic periodontal membrane of the present invention comprises the following steps:
  • the dental stem cells are inoculated on the bio-root stent, and the bio-root stent/cell complex is obtained by compound culture in a three-dimensional rotating environment.
  • Step S1-1 the collagen is dissolved in 0.5% acetic acid solution, formulated into an acid solution of collagen, wherein the collagen concentration is 5.0x10 - 4 g / mL;
  • Step S1-2 continuously stirring the solution obtained in the step S1-1, slowly adding a solution containing calcium ions, the amount of calcium ions added is 0.05 mol per kg of collagen, and some of the calcium ions are free in the solution. In, failed to be adsorbed on collagen fibers;
  • Step Sl-4 continuously stirring the solution obtained in step S1-3, slowly adding NaOH solution to the pH of the mixed system
  • Step Sl-5 the mixed system obtained in step S1-4 is allowed to stand for 48 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated by centrifugation to obtain a mineralized collagen jelly;
  • Step Sl-6 detecting the content of the solid matter in the mineralized collagen jelly obtained in step S1-5, diluting or concentrating the jelly, so that the content of the solid matter therein reaches 0.76 g / mL;
  • Step S1-7 taking a certain amount of the mineralized collagen jelly obtained in the step S1-6 into the mold, performing sufficient freeze-drying to obtain a mineralized collagen scaffold, and cutting and cutting into a diameter of 4.1 mm and a height of 10.8 mm.
  • Step S1-8 preparing a concentration of 0.01 wt% of glutaraldehyde in ethanol as a crosslinking agent, immersing the mineralized collagen scaffold obtained in step S1-7 in a crosslinking agent solution for 48 hours , cross-linking;
  • Step Sl-9 the mineralized collagen scaffold is taken out from the cross-linking agent solution, placed in a chromatography column, and washed with flowing pure water for 48 hours to remove residual cross-linking agent;
  • Step Sl-10 the mineralized collagen scaffold obtained in step S1-9 is sufficiently freeze-dried
  • Step Sl-1 l blind hole processing of the mineralized collagen scaffold obtained in step Sl-10, the blind hole depth is 7 mm
  • the porosity of the stent was determined to be 90.23%.
  • biodental scaffold/cell complex containing the biomimetic periodontal membrane further includes the following periodontal cell membrane Tablet preparation steps:
  • Step S3-1 selecting autologous or allogeneic periodontal ligament stem cells cultured in accordance with the production quality management (GMP) condition, and inoculating the vigorously growing second or third generation periodontal ligament stem cells in a 10 cm culture dish, cultivating Ingredients are ⁇ - ⁇ medium (containing 15% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 20. (Vg/ml vitamin C), every three days Change the liquid.
  • GMP production quality management
  • Step S3-2 After 10-14 days of culture, the cells on the edge of the culture dish are wrinkled, and the cell membrane is completely removed by a relatively blunt blade or a cell scraper, and the cell membrane is not dried during the process.
  • Step S1-1 the collagen is dissolved in 0.5% acetic acid solution, formulated into an acid solution of collagen, wherein the collagen concentration is 5.0 ⁇ 10 4 g/mL;
  • Step S1-2 continuously stirring the solution obtained in the step S1-1, slowly adding a solution containing calcium ions, the amount of calcium ions added is 0.05 mol per kg of collagen, and some of the calcium ions are free in the solution. In, failed to be adsorbed on collagen fibers;
  • Step Sl-4 continuously stirring the solution obtained in the step S1-3, slowly adding the NaOH solution to the pH of the mixed system.
  • Step S1-5 the mixed system obtained in the step S1-4 is allowed to stand for 48 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated by centrifugation to obtain a mineralized collagen jelly;
  • Step Sl-6 detecting the content of the solid matter in the mineralized collagen jelly obtained in step S1-5, diluting or concentrating the jelly, so that the content of the solid matter therein reaches 0.72 ⁇ 0.9 g / mL;
  • Step Sl-7 taking a certain amount of the mineralized collagen jelly obtained in step S1-6 into the mold, performing sufficient freeze-drying to obtain a mineralized collagen scaffold, and performing cutting and cutting;
  • Step S1-8 preparing a solution of glutaraldehyde in a concentration of 0.005 to 0.25 wt ⁇ 3 ⁇ 4 as a crosslinking agent, soaking the mineralized collagen scaffold obtained in step S1-7 in a crosslinking agent solution 24 to 48 Xiaoyan, cross-linking;
  • Step S1-1 removing the mineralized collagen scaffold from the cross-linking agent solution, placing it in a chromatography column, and flowing pure Wash water for 48 ⁇ 72 hours to remove residual crosslinker;
  • Step S10-10 the mineralized collagen scaffold obtained in step S3-4 is vacuum dried or freeze-dried;
  • Steps S1-1l the post-treatment of the mineralized collagen scaffold obtained in step S1-10, Cleaning and sterilizing to obtain a mineralized collagen-based bio-root stent, wherein post-treatment of the stent includes, but is not limited to, processing through-holes or blind holes, machined surface grooves/grains/patterns, edge chamfering, trimming. Sealed after sterilization.
  • Step S2-l selecting allogeneic dental pulp stem cells cultured in accordance with the Good Manufacturing Practice (GMP)
  • 1 ⁇ 10 7 dental pulp stem cells were digested, mixed with a medium, and inoculated on the biological root scaffold material obtained in step S3-6 by dropping and vacuum adsorption.
  • the dental pulp stem cells cultured under the conditions of production quality management may also be odontogenic stem cells, such as dental follicle stem cells, periodontal ligament stem cells, apical papilla stem cells, deciduous deciduous stem cells, and gingiva. stem cell.
  • odontogenic stem cells such as dental follicle stem cells, periodontal ligament stem cells, apical papilla stem cells, deciduous deciduous stem cells, and gingiva. stem cell.
  • Step S2-2 the scaffold material obtained in step S2-1 is allowed to stand in the incubator for 4 hours, and then placed in a rotary three-dimensional incubator for further cultivation, the rotation speed is 2-5 times/min, every three days. Change liquid, culture for 5 days
  • Step S3-1 selecting autologous or allogeneic periodontal ligament stem cells cultured in accordance with the production quality management (GMP) condition, and inoculating the vigorously growing second or third generation periodontal ligament stem cells in a 10 cm culture dish, cultivating Ingredients are ⁇ - ⁇ medium (containing 15% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin)
  • Step S3-2 After 10-14 days of culture, wrinkles appear on the cells at the edge of the culture dish, and the cell membrane is completely removed by a relatively blunt blade or a cell scraper, and the cell membrane is not dried during the process.
  • Step S3-3 the periodontal membrane obtained in step S3-2 is completely expanded, folded in half, and the liquid is blotted dry, and the stent/cell complex obtained in step S2-2 is placed on the periodontal membrane. On one side, roll up the wrapped stent/cell complex onto the other side and tighten as appropriate to form a stent/cell/membrane complex.
  • the step S4 can be performed to perform the bio-root implantation and the crown observation is performed. Histological and clinical efficacy observations. Specifically include:
  • Step S4-l 3 months after tooth extraction in the mandibular anterior region of the mini-pigs, bio-root implantation is performed.
  • the mandibular anterior region is designed to cut the alveolar crest, cut to the bone surface, and turn up the periosteal flap. See the mandibular anterior teeth in the edentulous area.
  • the alveolar bone is flat, the width is sufficient, the york-garde drill is positioned.
  • the stage is reamed, the hole is prepared, and the stent/cell/membrane complex is implanted at the alveolar crest.
  • the implant has a torque of about 35 Ncm.
  • the submerged implant can absorb the suture and tightly suture the wound.
  • Step S4-2 After the implantation operation, the small pigs were intramuscularly injected (antibiotics) with penicillin 800,000 u/day, and fed with penicillin for three consecutive days. Wound healing was observed at 1 week, 2 weeks, and 4 weeks after surgery.
  • Step S4-3 6 months after the implantation of the biological root, the biological root regeneration is successful, and the superstructure is repaired.
  • Step S4-4 1 week later, it can be seen that the shape of the gums recovered well, the silicone rubber was used to test the internal teeth of the roots, the zinc oxide was temporarily sealed, and the plaster model was poured to make the metal post core.
  • Step S4-5 on the wearing gold core post, the position is smooth, the edge is close, the glass ion bonding, the full crown tooth preparation, the silicone rubber take the impression mold, inject the plaster model, and make the porcelain full crown.
  • Step S4-6 in the edentulous area, waxing teeth, silicone rubber embossing mold, filling the dentition area of the female mold with self-setting resin
  • Step S4-7 1 week later, the Linyi crown was removed from the small pig's mouth, and the porcelain crown was tried, and the seat was smooth, the edges were close, and the glass ions were bonded.
  • Steps S4-8 respectively, after wearing the whole crown for 3 or 6 months, the histology and clinical efficacy evaluation were performed, and it was found that the periodontal membrane-like tissue was surrounded by the root of the tooth, and the sharpy fiber was obliquely inserted into the alveolar from the root surface. In the bone, the periodontal tissue is healthy.
  • a mineralized collagen-based bio-root stent material having a HA content of 50 wt% was prepared.
  • Step S1-1 the collagen is dissolved in 0.5% acetic acid solution, formulated into an acid solution of collagen, wherein the collagen concentration is 5.0 ⁇ 10 3 g/mL;
  • Step S1-2 continuously stirring the solution obtained in the step S1-1, slowly adding a solution containing calcium ions, calcium ions
  • the amount of addition is 0.01 mol of calcium ion per gram of collagen, and some of the calcium ions are free in the solution and are not adsorbed on the collagen fibers;
  • Step Sl-4 continuously stirring the solution obtained in the step S1-3, slowly adding the NaOH solution to the pH of the mixed system.
  • Step S1-5 the mixing system obtained in step S1-4 is allowed to stand for 24 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated by centrifugation to obtain a mineralized collagen jelly;
  • Step Sl-6 detecting the content of the solid substance in the mineralized collagen jelly obtained in step S1-5, diluting or concentrating the jelly, so that the content of the solid matter therein reaches 0.9 g / mL;
  • Step S1-7 taking a certain amount of the mineralized collagen jelly obtained in the step S1-6 into the mold, performing sufficient freeze-drying to obtain a mineralized collagen scaffold, and cutting and cutting into a diameter of 4.1 mm and a height of 10.8 mm.
  • Step S1-8 preparing a 0.25 wt% ethanol solution of glutaraldehyde as a crosslinking agent, immersing the mineralized collagen scaffold obtained in step S1-7 in a crosslinking agent solution for 24 hours , cross-linking;
  • Step Sl-9 the mineralized collagen scaffold is taken out from the cross-linking agent solution, placed in a chromatography column, and washed with flowing pure water for 72 hours to remove residual cross-linking agent;
  • Step Sl-10 the mineralized collagen scaffold obtained in step S1-9 is sufficiently freeze-dried
  • Step Sl-1 blind hole processing of the mineralized collagen scaffold obtained in step Sl-10, the blind hole depth is 7 mm
  • the stent was tested to have a porosity of 95%.
  • a mineralized collagen-based bio-root stent material having a HA content of 80% by weight was prepared.
  • Step S1-1 the collagen is dissolved in 0.5% acetic acid solution, formulated into an acid solution of collagen, wherein the collagen concentration is 5.0 x 10 5 g / mL;
  • Step S1-2 continuously stirring the solution obtained in the step S1-1, slowly adding a solution containing calcium ions, the amount of calcium ions added is 0.16 mol of calcium ions per gram of collagen, and some of the calcium ions are free in the solution. In, failed to be adsorbed on collagen fibers;
  • Step Sl-5 the mixed system obtained in the step S1-4 is allowed to stand for 24 hours, the supernatant is removed, the impurity ions are washed away by centrifugation, and concentrated by centrifugation to obtain a mineralized collagen jelly;
  • Step Sl-6 detecting the content of the solid matter in the mineralized collagen jelly obtained in step S1-5, diluting or concentrating the jelly, so that the content of the solid matter therein reaches 0.72 g / mL;
  • Step S1-5 measuring a certain amount of the mineralized collagen jelly obtained in step S1-6 is filled into the mold, and is sufficiently freeze-dried to obtain a mineralized collagen scaffold, and cut and cut into a diameter of 4.1 mm and a height of 10.8 mm.
  • Step S1-8 preparing a solution of glutaraldehyde in a concentration of 0.005 wt ⁇ 3 ⁇ 4 as a crosslinking agent, immersing the mineralized collagen scaffold obtained in step S1-7 in a cross-linking agent solution for 24 hours ⁇ , cross-linking;
  • Step Sl-9 the mineralized collagen scaffold is taken out from the cross-linking agent solution, placed in a chromatography column, and washed with flowing pure water for 72 hours to remove residual cross-linking agent;
  • Step Sl-10 the mineralized collagen scaffold obtained in step S1-9 is sufficiently freeze-dried
  • Step Sl-1 the blinded hole processing of the mineralized collagen scaffold obtained in step Sl-10, the blind hole depth is 7 mm
  • the porosity of the stent was determined to be 85.5%.
  • FIG. 2A-2D are a method for culturing a dental pulp stem cell and a stent according to the present invention.
  • Fig. 2A is a general observation photograph of the scaffold material after inoculation of cells;
  • Fig. 2B is a rotary three-dimensional culture system;
  • Fig. 2C is an electron microscope observation after 2 days of inoculation, in which a small amount of cells are initially adhered to the scaffold material (600x)
  • Fig. 2D shows an electron microscope observation after 5 days of culture, in which the cells are connected to each other in a piece and arranged closely (1000x).
  • FIG. 3 is a general observation photograph of a bionic periodontal membrane of the present invention.
  • 2 Vg/ml of ascorbic acid is added to the medium component, it can promote rapid cell proliferation, secrete a large amount of extracellular matrix collagen component, connect all the cells together, and after 10-13 days of growth, the whole cell membrane can be removed. Completely uncovered.
  • Periodontal stem cell membrane visible on the slice It consists of 2-3 layers of cells, which are rich in extracellular matrix and tightly connected.
  • 5A-5D are transmission electron micrographs of a bionic periodontal patch of the present invention. Transmission electron microscopy showed that the periodontal ligament membrane maintained a tight junction between cells and had normal physiological activities of the cells.
  • Figure 5A shows the intact cell morphology.
  • Figure 5B shows that the obtained periodontal ligament stem cell membrane maintains a close junction between cells.
  • Fig. 5C shows that the cell growth and differentiation are very good, a large number of microfilaments are present in the cytoplasm, and exocytosis vesicles are visible along the surface of the cell membrane.
  • Figure 5D shows that the matrix and collagen fibers (mainly type 2 collagen) are visible around the cells.
  • FIGS. 6A-6F are the results of gene and protein expression detection of the bionic periodontal membrane of the present invention. The results showed that the extracellular matrix of the periodontal ligament cell membrane was significantly higher than that of the enzyme-digested cells.
  • Figures 6A, 6B, and 6 C are real-time PCR comparisons of collagen I (COLI), fibronectin, and integrin pl, respectively.
  • Figures 6D, 6E, and 6F show the results of immunofluorescence staining of the above proteins, respectively, as confirmed by protein levels.
  • 7A-7C are the histological comparisons of the regenerated periodontal tissue with the normal tissue and the implant periodontal tissue of the present invention.
  • Six months after replantation histological observation showed that the morphology of the regenerated bio-root tissue was different from that of the new bone tissue.
  • the newly formed hard tissue area is surrounded by fibrous connective tissue.
  • the fiber travels similarly to the normal periodontal ligament, with a sharpy fibrous-like structure inserted into the newly formed hard tissue area.
  • FIG. 7A shows the normal root structure 6 months after the biological root implant
  • Figure 7C shows the implant grinding plate.
  • PDL periodontal ligament
  • D dentin
  • C cementum
  • B bone
  • IM implant.

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Abstract

含仿生牙周膜的生物牙根复合体、生物牙根支架及其制备方法，所述含仿生牙周膜的生物牙根复合体由生物牙根支架、牙源性干细胞和牙周细胞膜片复合而成，所述生物牙根支架的成分包括矿化胶原，支架中生长有牙源性干细胞，支架外部紧密结合有牙源性干细胞制成的牙周细胞膜片。

Description

含仿生牙周膜的生物牙根复合体及其制作方法 技术领域
[0001] 本发明涉及生物医用材料领域， 具体涉及一种含仿生牙周膜的生物牙根复合体 及其制作方法。
背景技术
[0002] 各种原因造成的牙齿缺失一直困扰人类的身心健康， 缺失牙齿的修复方法伴随 着人类的进步也由简单粗糙变得复杂精细， 尽管如此， 目前常用的修复方法诸 如活动义齿、 固定义齿、 种植体等都是机械性的修复， 存在着各种缺陷， 缺乏 真正生理意义上的修复， 不能满足人类的需要。 为此， 如何利用人工方法再造 出具有生物学活性的牙齿以修复缺牙， 实现牙再生 （tooth regeneration) 是目前 国内外学者着力研究的热点课题之一。
[0003] 从近年牙齿、 牙根再生研究的发展脉络不难看出， 当今口腔再生研究基本遵循 如下规律： 即从经典的牙齿发育学研究技术发展到胚层重组实验 （发育期牙胚 组织或细胞的重组体） ， 并逐步过渡到成体干细胞的研究， 期望利用牙齿 /牙根 / 牙周发育的自然规律来实现牙齿 /牙根 /牙周的再生。 目前， 全牙再生 （the whole-tooth regeneration) 主要包括两禾中方式：
[0004] 1、 细胞培养方法： 按照发育生物学的基本原理， 将牙间充质细胞与牙上皮细 胞体外重组培养， 使诱导形成新牙的过程重复牙齿发育的组织形态学过程；
[0005] 2、 组织工程学方法： 将种子细胞与预先设计好的具有完整牙形态的生物支架 复合， 以期形成与正常牙齿结构、 外形和功能相似的再生牙。
[0006] 然而， 上述两种全牙再生方法在发展和应用过程中均遇到瓶颈。 对于细胞培养 方法而言， 同其他器官再生一样， 关于牙齿发生发育的机理尚未完全解析， 而 且啮齿类动物模型得到的牙齿发育信号网络可能很难为人类牙齿再生提供借鉴 ； 对于组织工程学方法而言， 全牙再生涉及到了牙冠的再生以及萌出等一系列 问题， 这些因素均大大阻碍了牙组织工程的研究进程， 因此成功仍需吋曰。
[0007] 从解剖学的角度分析， 牙根是整个牙齿主要的支持和承重部分， 在咀嚼过程中 发挥着不可替代的作用。 牙根的牙本质具有较高的密度和一定的孔隙结构， 成 分上约 70%为无机矿物， 20%为有机物， 10%为水分， 微观结构为无机矿物以纳 米微粒形式有序排列在胶原分子之间与分子表面的矿化胶原结构。 牙根再生 （ro ot regeneration) 相对于全牙再生可以避免控制牙齿外形和牙齿萌出的技术难题 ， 而且能解决种植的义齿缺乏生物活性和存在金属异物的缺陷。 此外， 再生的 牙根因具有牙周膜及牙本质结构， 还可实现形态及功能的修复， 故而在牙齿组 织工程研究中具有独特的优势。 另外， 再生的牙根可以安装人造牙冠， 而人造 牙冠在外形与功能上都可与天然牙冠相媲美， 从而构建出具有完整结构和功能 的牙齿。
[0008] 要实现以上构想， 需要在牙齿缺失部位构建出具有正常牙根生理解剖结构及其 功能的生物牙根 （bioengineered tooth root, bio-root) ， 而实现这一过程则必须 满足四个条件： 1、 合适的支架材料； 2、 良好的种子细胞； 3、 可靠的支架 /细胞 复合体构建方法； 4、 适宜生物牙根再生的微环境。
[0009] 现有技术中对于这种实现生物牙根再生的支架材料来源有两种： 动物源性材料
(即经过处理加工的同种异体或异种牙根） 和人工合成材料。 动物源性材料具 有免疫原性， 临床使用中存在较大的排异、 感染风险。 人工合成的生物牙根支 架材料主要是磷酸钙生物陶瓷材料， 如磷酸三 /羟基磷灰石复合陶瓷等。 然而 ， 这种磷酸钙生物陶瓷存在较大不足： 1、 磷酸钙生物陶瓷仅为多孔或致密结构 的无机陶瓷材料， 并不具有牙根的仿生结构和化学组成， 不利于生物牙根的重 建； 2、 磷酸钙生物陶瓷脆性较大， 不利于术中及术后支架完整性的保持； 3、 磷酸钙生物陶瓷的降解较慢， 不利于在短吋间 （3 ~ 6个月） 内重建生物牙根。
[0010] 另一方面， 在天然牙根的外周、 牙槽骨的内侧有一层牙周膜 （periodontium) 组织。 该牙周膜组织也被称为牙周韧带 （periodontal ligament) ， 由细胞 （成纤 维细胞、 成骨细胞、 成牙骨质细胞、 破骨细胞等） 、 细胞外基质、 胶原纤维、 神经、 血管等组成， 具有支持牙齿、 感觉、 营养和更新牙骨质和牙槽骨等多方 面的功能， 是生物牙根必不可少的组成部分。 因此， 生物牙根的构建也应包含 牙周膜的构建。 有研究表明， 通过化学药物刺激体外培养的牙周膜干细胞 （peri odontal ligament stem cell, PDLSCs) ， 可以使细胞分泌大量细胞外基质而形成 细胞膜片 （cell sheet) ， 避免了常规的细胞消化过程， 从而保留了重要的细胞表 面蛋白以及细胞自分泌的外基质 （extracellular matrix) ， 将该细胞膜片包裹在生 物牙根支架外部， 能够较单纯使用支架材料更有利于生物牙根的构建。 然而， 在天然牙根周围， 牙周膜与牙骨质的结合非常紧密， 并具有一定的化学键合， 简单地将细胞膜片包裹在支架外部并不能获得与天然牙根和牙周膜类似的紧密 结合结构， 更无法形成化学键结合。
[0011] 此外， 现有的生物医用材料、 支架和组织工程产品尚不能很好地满足上述生物 牙根构建的各方面要求。
技术问题
[0012] 针对上述现有技术的不足， 本发明提供了一种含仿生牙周膜的生物牙根复合体 以及生物牙根支架。 该生物牙根支架的材料主要为一种无机成分含量较高的矿 化胶原， 具有与天然牙本质一致的微观结构和化学组成； 支架具有与天然牙根 近似的宏观外形， 内部具有宏观的盲孔或者通孔结构， 可用于装载细胞、 生长 因子、 药物等， 以及供细胞长入。 含仿生牙周膜的生物牙根复合体则在生物牙 根支架内部复合有牙源性干细胞如牙髓干细胞； 外部紧密结合有一层牙源性干 细胞制成的牙周细胞膜片， 富含细胞、 细胞外基质、 胶原纤维等天然牙周膜的 主要成分。
[0013] 本发明还提供了生物牙根支架的制备方法以及含仿生牙周膜的生物牙根复合体 的制作方法。
问题的解决方案
技术解决方案
[0014] 本发明的第一方面， 提供了一种含仿生牙周膜的生物牙根复合体。 该含仿生牙 周膜的生物牙根复合体由生物牙根支架、 牙源性干细胞和牙周细胞膜片复合而 成。 所述生物牙根支架成分包括矿化胶原， 所述生物牙根支架中生长有牙源性 干细胞， 所述生物牙根支架外部紧密结合有牙源性干细胞制成的牙周细胞膜片 。 其中牙周细胞膜片又称为仿生牙周膜。 牙源性干细胞为牙囊干细胞、 牙髓干 细胞、 根尖牙乳头干细胞、 脱落乳牙干细胞或牙龈干细胞。 优选地， 在生物牙 根支架中接种牙髓干细胞或根尖牙乳头干细胞， 牙周细胞膜片则使用牙周膜干 细胞。 本发明中生物牙根支架又简称为支架， 牙周细胞膜片又被称为牙周膜片 、 细胞膜片和膜片。
[0015] 除仿生牙周膜部分， 支架的材料为矿化胶原， 成分包含胶原和磷灰石， 其中磷 灰石以弱结晶的纳米微粒形式有序排列在胶原分子之间与分子表面， 胶原 /磷灰 石= 5/5 ~ 2/8 ^ )； 仿生牙周膜为富含细胞、 细胞外基质、 胶原纤维的牙周膜 干细胞细胞膜片， 厚度为 0.7mm， 由 2-3层细胞构成； 支架与仿生牙周膜之间具 有微观结构镶嵌， 并通过细胞外基质形成化学键合。
[0016] 所述支架材料中的胶原为动物源性的 I型胶原， 优选为牛跟腱来源的 I型胶原。
[0017] 所述支架的材料中磷灰石成分的主要物相为羟基磷灰石。
[0018] 所述支架的材料还可以含有生物可吸收医用高分子， 包括聚乳酸 （PLA) 、 聚 羟基乙酸 （PGA) 、 乳酸-羟基乙酸共聚物 （PLGA) 、 聚己内酯 （PCL) ， 该生 物可吸收医用高分子在支架材料中的含量为不高于 50 wt%。 相应地， 支架材料 中矿化胶原的含量为 50 wt%-100 wt<¾。
[0019] 所述支架的材料还可以由生物可吸收医用高分子， 包括聚乳酸 （PLA) 、 聚羟 基乙酸 （PGA) 、 乳酸-羟基乙酸共聚物 （PLGA) 、 聚己内酯 （PCL) 制作成的 3维纳米结构支架。
[0020] 所述支架在微观上具有一定的孔隙结构， 孔隙率为 70% ~ 95%， 孔径为 50 ~ 500 μηι。
[0021] 在宏观上： 该支架的外形为柱状体； 柱状体的高度为 9 ~ 12 mm; 柱状体的外 侧面被垂直于柱状体轴向的平面所截得的形状为圆形或者近似圆形， 周长为 11 ~ 14 mm; 柱状体的上底面为平面或者凸面， 若为凸面， 凸起高度不高于 l mm， 且该高度计入柱状体的总高度； 柱状体的下底面为平面或者凸面， 若为凸面， 凸起高度不高于 1 mm, 且该高度计入柱状体的总高度。
[0022] 所述支架内部可以具有沿柱状体轴向方向的通孔， 或者幵口朝柱状体底面的盲 孔， 盲孔沿柱状体轴向的长度不小于柱状体高度的 2/3; 柱状体内部盲孔或通孔 的侧面被垂直于柱状体轴向的平面所截得的形状为圆形或者近似圆形， 周长为 3. 0〜 6.0 mm。
[0023] 所述仿生牙周膜中， 膜片由 2-3层细胞构成， 其中水分占 50 ~ 80 wt%， 除水分 以外的物质组成中， 胶原纤维占 60 ~ 75 wt%。
[0024] 本发明的第二方面， 提供了一种生物牙根支架， 其特征与前述第一方面含仿生 牙周膜的生物牙根复合体中的生物牙根支架一致。
[0025] 本发明的第三方面， 提供了一种生物牙根支架的制备方法， 其特征在于， 所述 制备方法包括以下步骤：
[0026] 步骤 Sl-1、 将胶原溶于盐酸、 硝酸或醋酸中的任何一种， 配制成胶原的酸溶液
， 其中胶原浓度为 5.0x10 - 5 ~ 5.0x10 -3 g/mL;
[0027] 步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.01 ~ 0.16 mol;
[0028] 步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = 1/1 ~ 2/1； [0029] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH
= 6 - 8 , 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现 白色悬浊液；
[0030] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 24 ~ 120小吋， 抽去上清液， 用离 心的方法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0031] 步骤 Sl-6、 检测步骤 Sl-5获得的矿化胶原胶冻中固体物质的含量， 对胶冻进行 稀释或者浓缩， 使其中固体物质的含量达到 0.72 ~ 0.9 g/mL;
[0032] 步骤 Sl-7、 量取一定量步骤 Sl-6获得的矿化胶原胶冻填入模具中， 进行充分冷 冻干燥， 获得矿化胶原支架， 并进行切割剪裁；
[0033] 步骤 Sl-8、 配制浓度为 0.005 ~ 0.25 wt%的戊二醛的乙醇溶液作为交联剂， 将步 骤 S1-7获得的矿化胶原支架浸泡于交联剂溶液中 24 ~ 48小吋， 进行交联；
[0034] 步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以流动的纯 水洗涤 48 ~ 72小吋， 以除去残留的交联剂；
[0035] 步骤 Sl-10、 将步骤 S1-9获得的矿化胶原支架进行真空干燥或者冷冻干燥； [0036] 步骤 Sl-l l、 对步骤 S1-10获得的矿化胶原支架进行后处理、 清洗和灭菌， 获得 矿化胶原基生物牙根支架。 其中， 对支架的后处理包括但不限于： 加工通孔或 者盲孔、 加工表面沟槽 /纹路 /图案、 边缘倒角、 修剪。 灭菌后封存。 [0037] 本发明的第四方面， 提供了一种含仿生牙周膜的生物牙根复合体的制作方法， 所述制作方法包括以下步骤：
[0038] Sl、 采用第三方面所述的生物牙根支架的制备方法制作生物牙根支架；
[0039] S2、 在所述生物牙根支架上接种牙源性干细胞， 并在三维旋转环境中复合培养 得到生物牙根支架 /细胞复合体；
[0040] S3、 使用牙源性干细胞制作牙周细胞膜片， 并将牙周细胞膜片包裹在所述生物 牙根支架 /细胞复合体上得到含仿生牙周膜的生物牙根复合体。
[0041] 在根据本发明优选实施例所述的含仿生牙周膜的生物牙根复合体的制作方法中
， 所述步骤 S2具体包括：
[0042] 步骤 S2-l、 选用符合生产质量管理规范条件下培养的异体牙髓干细胞， 将 1x10
7牙髓干细胞消化后用培养基吹打混匀， 通过滴加和负压吸附的方式接种于生物 牙根支架材料上；
[0043] 步骤 S2-2、 将步骤 S2-1获得的支架材料在培养箱中静置 4小吋， 然后放入旋转 式三维培养器内继续培养， 转速 2-5次 /分钟， 每三天换液， 培养 5天。
[0044] 在根据本发明优选实施例所述的含仿生牙周膜的生物牙根复合体的制作方法中
， 所述步骤 S3具体包括：
[0045] 步骤 S3-l、 选用符合生产质量管理规范条件下培养的自体或异体牙周膜干细胞
， 将生长旺盛的第二或第三代牙周膜干细胞接种在 10cm培养皿， 培养成分为
MEM培养基， 每三天换液；
[0046] 步骤 S3-2、 培养 10-14天后， 培养皿边缘细胞出现皱褶， 用较钝刀片或细胞刮子 将牙周细胞膜片整体揭下来， 过程中牙周细胞膜片不要干燥；
[0047] 步骤 S3-3、 将步骤 S3-2获得的牙周细胞膜片完整展幵， 对折， 并将液体吸干， 将步骤 S2获得的生物牙根支架 /细胞复合体放置于牙周细胞膜片的一侧， 向另一 侧卷起包裹生物牙根支架 /细胞复合体， 松紧合适， 形成生物牙根复合体。
[0048] 此外， 本发明的第五方面， 还提供了含仿生牙周膜的生物牙根支架的制备方法
[0049] 该制备方法综合了体外仿生矿化技术和细胞膜片技术， 主要包括以下步骤： [0050] 步骤 Sl-1、 将胶原溶于盐酸、 硝酸或醋酸中的任何一种， 配制成胶原的酸溶液 ， 其中胶原浓度为 5.0x10 - 5 ~ 5.0x10 -3 g/mL;
[0051] 步骤 Sl-2、 持续搅拌步骤 Sl-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.01 ~ 0.16 mol;
[0052] 步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = 1/1 ~ 2/1；
[0053] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH
= 6 - 8 , 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现 白色悬浊液；
[0054] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 24 ~ 120小吋， 抽去上清液， 用离 心的方法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0055] 步骤 Sl-6、 将步骤 S1-5获得的胶冻进行冷冻干燥， 研磨后获得矿化胶原粉体。
[0056] 本发明的第六方面， 提供了含仿生牙周膜的生物牙根支架 /细胞复合体制作方 法。
[0057] 含仿生牙周膜的生物牙根支架 /细胞复合体制备过程包括以下步骤：
[0058] 步骤 S2-l、 选用符合生产质量管理规范 （GMP) 条件下培养的异体牙髓干细胞
， 将 1x10 7牙髓干细胞消化后用培养基吹打混匀， 通过滴加和负压吸附的方式接 种于生物牙根支架材料上。
[0059] 步骤 S2-2、 将步骤 S2-1获得的支架材料在培养箱中静置 4小吋， 然后放入旋转 式三维培养器内继续培养， 转速 2-5次 /分钟， 每三天换液， 培养 5天
[0060] 步骤 S3-l、 选用符合生产质量管理规范 （GMP) 条件下培养的自体或异体牙周 膜干细胞， 将生长旺盛的第二或第三代牙周膜干细胞接种在 10cm培养皿， 培养 成分为 α-ΜΕΜ培养基 （含 15%胎牛血清， 2 mmol/L谷氨酰胺， 100 U/ ml青霉素
， 100 g/ ml链霉素， 20.(Vg/ml维生素 C) ， 每三天换液。
[0061] 步骤 S3-2、 培养 10-14天后， 培养皿边缘细胞出现皱褶， 用较钝刀片或细胞刮子 将细胞膜片整体揭下来， 过程中细胞膜片不要干燥。
[0062] 步骤 S3-3、 将步骤 S3-2获得的牙周膜片完整展幵， 对折， 并将液体吸干， 将步 骤 S2-2获得的支架 /细胞复合体放置于牙周膜片的一侧， 向另一侧卷起包裹支架 / 细胞复合体， 松紧合适。 [0063] 所述符合生产质量管理规范 （GMP) 条件下培养的异体干细胞还可以为以下牙 源性干细胞， 如牙囊干细胞、 牙髓干细胞、 根尖牙乳头干细胞、 脱落乳牙干细 胞、 牙龈干细胞。
[0064] 本发明的第七方面， 提供了含仿生牙周膜的生物牙根的组织工程学构建方法。
[0065] 该构建方法主要包括以下步骤：
[0066] 步骤 Sl-1、 将胶原溶于盐酸、 硝酸或醋酸中的任何一种， 配制成胶原的酸溶液
， 其中胶原浓度为 5.0x10 - 5 ~ 5.0x10 -3 g/mL;
[0067] 步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.01 ~ 0.16 mol;
[0068] 步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = 1/1 ~ 2/1； [0069] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH
= 6 - 8 , 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现 白色悬浊液；
[0070] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 24 ~ 120小吋， 抽去上清液， 用离 心的方法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0071] 步骤 Sl-6、 检测步骤 S1-5获得的矿化胶原胶冻中固体物质的含量， 对胶冻进行 稀释或者浓缩， 使其中固体物质的含量达到 0.72 ~ 0.9 g/mL;
[0072] 步骤 Sl-7、 量取一定量步骤 S1-6获得的矿化胶原胶冻填入模具中， 进行充分冷 冻干燥， 获得矿化胶原支架， 并进行切割剪裁； 步骤 Sl-8、 配制浓度为 0.005 ~
0.25 wt%的戊二醛的乙醇溶液作为交联剂， 将步骤 S1-7获得的矿化胶原支架浸泡 于交联剂溶液中 24 ~ 48小吋， 进行交联；
[0073] 步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以流动的纯 水洗涤 48 ~ 72小吋， 以除去残留的交联剂；
[0074] 步骤 Sl-10、 将步骤 S1-9获得的矿化胶原支架进行真空干燥或者冷冻干燥； [0075] 步骤 Sl-l l、 对步骤 S1-10获得的矿化胶原支架进行后处理、 清洗和灭菌， 获得 矿化胶原基生物牙根支架， 其中， 对支架的后处理包括但不限于： 加工通孔或 者盲孔、 加工表面沟槽 /纹路 /图案、 边缘倒角、 修剪。 灭菌后封存。 [0076] 支架在微观上具有一定的孔隙结构， 孔隙率为 70% ~ 95%， 孔径为 50 ~ 500 μηι
。 在宏观上： 该支架的外形为柱状体； 柱状体的高度为 9 ~ 12 mm; 柱状体的外 侧面被垂直于柱状体轴向的平面所截得的形状为圆形或者近似圆形， 周长为 11 ~ 14 mm; 内部可以具有沿柱状体轴向方向的通孔， 或者幵口朝柱状体底面的盲孔 ， 盲孔沿柱状体轴向的长度不小于柱状体高度的 2/3; 柱状体内部盲孔或通孔的 侧面被垂直于柱状体轴向的平面所截得的形状为圆形或者近似圆形， 周长为 3.0 〜 6.0 mm。
[0077] 步骤 S2-l、 选用符合生产质量管理规范 （GMP) 条件下培养的异体牙髓干细胞
， 将 1x107牙髓干细胞消化后用培养基吹打混匀， 通过滴加和负压吸附的方式接 种于步骤 S1-11获得的生物牙根支架材料上。
[0078] 所述符合生产质量管理规范 （GMP) 条件下培养的牙髓干细胞还可以为以下牙 源性干细胞， 如牙囊干细胞、 牙周膜干细胞、 根尖牙乳头干细胞、 脱落乳牙干 细胞、 牙龈干细胞。
[0079] S2-2、 将步骤 S2-1获得的支架材料在培养箱中静置 4小吋， 然后放入旋转式三 维培养器内继续培养， 转速 2-5次 /分钟， 每三天换液， 培养 5天。
[0080] 步骤 S3-l、 选用符合生产质量管理规范 （GMP) 条件下培养的自体或异体牙周 膜干细胞， 将生长旺盛的第二或第三代牙周膜干细胞接种在 10cm培养皿， 培养 成分为 α-ΜΕΜ培养基 （含 15%胎牛血清， 2 mmol/L谷氨酰胺， 100 U/ ml青霉素
， 100 g/ ml链霉素， 20.(Vg/ml维生素 C) ， 每三天换液。
[0081] 步骤 S3-2、 培养 10-14天后， 培养皿边缘细胞出现皱褶， 用较钝刀片或细胞刮子 将牙周膜片整体揭下来， 过程中牙周膜片不要干燥。
[0082] 步骤 S3-3、 将步骤 S3-2获得的牙周膜片完整展幵， 对折， 并将液体吸干， 将步 骤 S2-2获得的支架 /细胞复合体放置于牙周膜片的一侧， 向另一侧卷起包裹支架 / 细胞复合体， 松紧合适， 形成支架 /细胞 /膜片复合体。
[0083] 步骤 S4-l、 小型猪下颌前牙区牙齿拔除后 3个月后， 行生物牙根植入术。
[0084] 手术过程：
[0085] (1) 麻醉： 速眠新 Π+氯胺酮 15mg/kg肌肉注射麻醉。 备皮后使用 2%碘伏进行 头面部及口腔内消毒， 铺巾。 阿替卡因局部浸润麻醉。 [0086] (2) 下颌前牙区设计牙槽嵴顶切口， 切幵至骨面， 翻起粘骨膜瓣， 见下颌骨 前牙缺牙区牙槽骨平整， 宽度充足， 先锋钻定位， 逐级扩孔， 备洞， 牙槽嵴顶 处植入支架 /细胞 /膜片复合体， 植入吋扭矩约 35Ncm， 采用潜入式植入， 可吸收 缝线严密缝合伤口。
[0087] 步骤 S4-2、 种植手术后， 小型猪当天肌肉注射 （抗生素） 青霉素 80万 u/只， 连 续三天给予添加青霉素饲料喂养。 分别于术后 1周、 2周、 4周观察伤口愈合情况
， 有无感染。
[0088] 步骤 S4-3、 生物牙根植入术后 6个月， 生物牙根再生成功， 行上部结构修复。
沿原手术切口切幵， 翻瓣， 暴露生物牙根， 修整牙龈形态， 将生物牙根暴露口 腔内， 严密缝合伤口。
[0089] 步骤 S4-4、 1周后， 可见牙龈外形恢复良好， 硅橡胶采生物牙根内部印模， 氧 化锌暂封， 灌注石膏模型， 制作金属桩核。
[0090] 步骤 S4-5、 就戴金素桩核， 就位顺利， 边缘密合， 玻璃离子粘接， 全冠牙体预 备， 硅橡胶取印模， 灌注石膏模型， 制作烤瓷全冠。
[0091] 步骤 S4-6、 在缺牙区排蜡牙， 硅橡胶翻印模， 阴模内缺牙区部位加注自凝树脂
， 复位印模， 待自凝树脂凝固后， 取下硅橡胶印模， 调磨、 修整自凝树脂牙冠
， 抛光、 消毒， 玻璃离子粘接。
[0092] 步骤 S4-7、 1周后于小型猪口内卸下临吋冠， 试戴烤瓷牙冠， 就位顺利， 边缘 密合， 玻璃离子粘接。
[0093] 步骤 S4-8、 分别在全冠就戴 3、 6个月后取组织进行组织学及临床疗效评价， 发 现生物牙根周围有牙周膜样组织， sharpy纤维从牙根表面斜插入牙槽骨内， 牙周 组织健康。
发明的有益效果
有益效果
[0094] 本发明将动物源性的 I型胶原与羟基磷灰石制备成牙根样生物牙根支架， 再将 牙髓干细胞附着在其上， 并且外侧包裹有牙周细胞膜片， 形成生物牙根支架 /细 胞 /膜片复合体， 再将复合体植入动物颌骨内进行生物牙根的再生， 本发明含有 矿化胶原的生物牙根支架具有合适的机械性能和生物降解特性， 且能够与牙周 细胞膜片紧密结合， 同吋植入动物颌骨内进行再生出的生物牙根不仅在结构和 成分上与天然牙根相似， 而且含有牙周膜结构， 带冠后能行使功能， 并能后期 进行功能性改建。
对附图的简要说明
附图说明
[0095] 图 1为本发明的含仿生牙周膜的生物牙根支架组织工程构建流程图；
[0096] 图 2A-2D为本发明的牙髓干细胞与支架复合方法；
[0097] 图 3为本发明的仿生牙周膜片的大体观察照片；
[0098] 图 4为本发明的仿生牙周膜片的组织学染色切片；
[0099] 图 5A-5D为本发明的仿生牙周膜片的透射电镜照片；
[0100] 图 6A-6F为本发明的仿生牙周膜片的基因和蛋白表达检测结果；
[0101] 图 7A-7C为本发明的再生牙周组织与正常组织及种植体牙周组织的组织学情况 对比。
本发明的实施方式
[0102] 为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及实施例
， 对本发明进行进一步详细说明。
[0103] 本项发明旨在提供一种具有牙根仿生结构和化学组成、 具备合适机械性能和生 物降解特性的材料和支架， 将材料与细胞混合进行旋转式三维培养以保证其营 养供应， 并且在支架外部构建一层紧密结合的仿生牙周膜组织， 在临床上实现 生物牙根的组织学成功构建。
[0104] 图 1为本发明的含仿生牙周膜的生物牙根组织工程学构建流程图。 如图所示， 本发明中含仿生牙周膜的生物牙根复合体的制作过程包括以下步骤：
[0105] Sl、 制作含有矿化胶原成分的生物牙根支架；
[0106] S2、 在生物牙根支架上接种牙源性干细胞， 并在三维旋转环境中复合培养得到 生物牙根支架 /细胞复合体。
[0107] S3、 使用牙源性干细胞制作牙周细胞膜片， 并将牙周细胞膜片包裹在所述生物 牙根支架 /细胞复合体上得到含仿生牙周膜的生物牙根复合体。 [0108] 实施例 1
[0109] 一、 制备 HA含量为 70wt%的矿化胶原基生物牙根支架材料。
[0110] 步骤 Sl-1、 将胶原溶于 0.5\^%的醋酸溶液中， 配制成胶原的酸溶液， 其中胶原 浓度为 5.0x10 - 4 g/mL;
[0111] 步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.05 mol, 其中部分钙离子游离在溶液里， 未能被吸附于胶原蛋白纤维上；
[0112] 步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = 5/3;
[0113] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH
= 7， 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现白色 悬浊液；
[0114] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 48小吋， 抽去上清液， 用离心的方 法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0115] 步骤 Sl-6、 检测步骤 S1-5获得的矿化胶原胶冻中固体物质的含量， 对胶冻进行 稀释或者浓缩， 使其中固体物质的含量达到 0.76 g/mL;
[0116] 步骤 Sl-7、 量取一定量步骤 S1-6获得的矿化胶原胶冻填入模具中， 进行充分冷 冻干燥， 获得矿化胶原支架， 并切割剪裁成直径 4.1 mm、 高 10.8 mm的圆柱； [0117] 步骤 Sl-8、 配制浓度为 0.01 wt%的戊二醛的乙醇溶液作为交联剂， 将步骤 S1-7 获得的矿化胶原支架浸泡于交联剂溶液中 48小吋， 进行交联；
[0118] 步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以流动的纯 水洗涤 48小吋， 以除去残留的交联剂；
[0119] 步骤 Sl-10、 将步骤 S1-9获得的矿化胶原支架进行充分冷冻干燥；
[0120] 步骤 Sl-l l、 对步骤 Sl-10获得的矿化胶原支架进行盲孔加工， 盲孔深度为 7 mm
， 直径为 4.71 mm， 并进行清洗和灭菌， 获得矿化胶原基生物牙根支架。
[0121] 经检测， 该支架孔隙率为 90.23%。
[0122] 二、 含仿生牙周膜的生物牙根支架 /细胞 /膜片复合体制备过程：
[0123] 含仿生牙周膜的生物牙根支架 /细胞复合体制备过程中还包括以下牙周细胞膜 片的制备步骤：
[0124] 步骤 S3-l、 选用符合生产质量管理规范 （GMP) 条件下培养的自体或异体牙周 膜干细胞， 将生长旺盛的第二或第三代牙周膜干细胞接种在 10cm培养皿， 培养 成分为 α-ΜΕΜ培养基 （含 15%胎牛血清， 2 mmol/L谷氨酰胺， 100 U/ ml青霉素 ， 100 g/ ml链霉素， 20.(Vg/ml维生素 C) ， 每三天换液。
[0125] 步骤 S3-2、 培养 10-14天后， 培养皿边缘细胞出现皱褶， 用较钝刀片或细胞刮子 将细胞膜片整体揭下来， 过程中细胞膜片不要干燥。
[0126] 三、 含仿生牙周膜的生物牙根的组织工程学构建， 即含仿生牙周膜的生物牙根 复合体的制作方法如下：
[0127] 步骤 Sl-1、 将胶原溶于 0.5\^%的醋酸溶液中， 配制成胶原的酸溶液， 其中胶原 浓度为 5.0x10 4 g/mL;
[0128] 步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.05 mol, 其中部分钙离子游离在溶液里， 未能被吸附于胶原蛋白纤维上；
[0129] 步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = 5/3;
[0130] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH
= 7， 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现白色 悬浊液；
[0131] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 48小吋， 抽去上清液， 用离心的方 法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0132] 步骤 Sl-6、 检测步骤 S1-5获得的矿化胶原胶冻中固体物质的含量， 对胶冻进行 稀释或者浓缩， 使其中固体物质的含量达到 0.72 ~ 0.9 g/mL;
[0133] 步骤 Sl-7、 量取一定量步骤 S1-6获得的矿化胶原胶冻填入模具中， 进行充分冷 冻干燥， 获得矿化胶原支架， 并进行切割剪裁；
[0134] 步骤 Sl-8、 配制浓度为 0.005~0.25 wt<¾的戊二醛的乙醇溶液作为交联剂， 将步 骤 S1-7获得的矿化胶原支架浸泡于交联剂溶液中 24 ~ 48小吋， 进行交联；
[0135] 步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以流动的纯 水洗涤 48 ~ 72小吋， 以除去残留的交联剂；
[0136] 步骤 Sl-10、 将步骤 S3-4获得的矿化胶原支架进行真空干燥或者冷冻干燥； [0137] 步骤 Sl-l l、 对步骤 S1-10获得的矿化胶原支架进行后处理、 清洗和灭菌， 获得 矿化胶原基生物牙根支架， 其中， 对支架的后处理包括但不限于： 加工通孔或 者盲孔、 加工表面沟槽 /纹路 /图案、 边缘倒角、 修剪。 灭菌后封存。
[0138] 步骤 S2-l、 选用符合生产质量管理规范 （GMP) 条件下培养的异体牙髓干细胞
， 将 1x10 7牙髓干细胞消化后用培养基吹打混匀， 通过滴加和负压吸附的方式接 种于步骤 S3-6获得的生物牙根支架材料上。
[0139] 所述符合生产质量管理规范 （GMP) 条件下培养的牙髓干细胞还可以为以下牙 源性干细胞， 如牙囊干细胞、 牙周膜干细胞、 根尖牙乳头干细胞、 脱落乳牙干 细胞、 牙龈干细胞。
[0140] 步骤 S2-2、 将步骤 S2-1获得的支架材料在培养箱中静置 4小吋， 然后放入旋转 式三维培养器内继续培养， 转速 2-5次 /分钟， 每三天换液， 培养 5天
[0141] 步骤 S3-l、 选用符合生产质量管理规范 （GMP) 条件下培养的自体或异体牙周 膜干细胞， 将生长旺盛的第二或第三代牙周膜干细胞接种在 10cm培养皿， 培养 成分为 α-ΜΕΜ培养基 （含 15%胎牛血清， 2 mmol/L谷氨酰胺， 100 U/ ml青霉素
， 100 g/ ml链霉素， 20.(Vg/ml维生素 C) ， 每三天换液。
[0142] 步骤 S3-2、 培养 10-14天后， 培养皿边缘细胞出现皱褶， 用较钝刀片或细胞刮子 将细胞膜片整体揭下来， 过程中细胞膜片不要干燥。
[0143] 步骤 S3-3、 将步骤 S3-2获得的牙周膜片完整展幵， 对折， 并将液体吸干， 将步 骤 S2-2获得的支架 /细胞复合体放置于牙周膜片的一侧， 向另一侧卷起包裹支架 / 细胞复合体， 松紧合适， 形成支架 /细胞 /膜片复合体。
[0144] 在制得含有仿生牙周膜的生物牙根复合体， 即上述步骤 S3-3所得的支架 /细胞 / 膜片复合体后， 可以执行步骤 S4行生物牙根植入术并制作牙冠观察组织学及临 床疗效观察。 具体包括：
[0145] 步骤 S4-l、 小型猪下颌前牙区牙齿拔除后 3个月后， 行生物牙根植入术。
[0146] 手术过程：
[0147] (1) 麻醉： 速眠新 Π+氯胺酮 15mg/kg肌肉注射麻醉。 备皮后使用 2%碘伏进行 头面部及口腔内消毒， 铺巾。 阿替卡因局部浸润麻醉。
[0148] (2) 下颌前牙区设计牙槽嵴顶切口， 切幵至骨面， 翻起粘骨膜瓣， 见下颌骨 前牙缺牙区牙槽骨平整， 宽度充足， 先锋钻定位， 逐级扩孔， 备洞， 牙槽嵴顶 处植入支架 /细胞 /膜片复合体， 植入吋扭矩约 35Ncm， 采用潜入式植入， 可吸收 缝线严密缝合伤口。
[0149] 步骤 S4-2、 种植手术后， 小型猪当天肌肉注射 （抗生素） 青霉素 80万 u/只， 连 续三天给予添加青霉素饲料喂养。 分别于术后 1周、 2周、 4周观察伤口愈合情况
， 有无感染。
[0150] 步骤 S4-3、 生物牙根植入术后 6个月， 生物牙根再生成功， 行上部结构修复。
沿原手术切口切幵， 翻瓣， 暴露生物牙根， 修整牙龈形态， 将生物牙根暴露口 腔内， 严密缝合伤口。
[0151] 步骤 S4-4、 1周后， 可见牙龈外形恢复良好， 硅橡胶采生物牙根内部印模， 氧 化锌暂封， 灌注石膏模型， 制作金属桩核。
[0152] 步骤 S4-5、 就戴金素桩核， 就位顺利， 边缘密合， 玻璃离子粘接， 全冠牙体预 备， 硅橡胶取印模， 灌注石膏模型， 制作烤瓷全冠。
[0153] 步骤 S4-6、 在缺牙区排蜡牙， 硅橡胶翻印模， 阴模内缺牙区部位加注自凝树脂
， 复位印模， 待自凝树脂凝固后， 取下硅橡胶印模， 调磨、 修整自凝树脂牙冠
， 抛光、 消毒， 玻璃离子粘接。
[0154] 步骤 S4-7、 1周后于小型猪口内卸下临吋冠， 试戴烤瓷牙冠， 就位顺利， 边缘 密合， 玻璃离子粘接。
[0155] 步骤 S4-8、 分别在全冠就戴 3、 6个月后取组织进行组织学及临床疗效评价， 发 现生物牙根周围有牙周膜样组织， sharpy纤维从牙根表面斜插入牙槽骨内， 牙周 组织健康。
[0156] 实施例 2
[0157] 制备 HA含量为 50 wt%的矿化胶原基生物牙根支架材料。
[0158] 步骤 Sl-1、 将胶原溶于 0.5\^%的醋酸溶液中， 配制成胶原的酸溶液， 其中胶原 浓度为 5.0x10 3 g/mL;
[0159] 步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.01 mol, 其中部分钙离子游离在溶液里， 未能被吸附于胶原蛋白纤维上；
[0160] 步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = l/1 ;
[0161] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH
= 7， 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现白色 悬浊液；
[0162] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 24小吋， 抽去上清液， 用离心的方 法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0163] 步骤 Sl-6、 检测步骤 S1-5获得的矿化胶原胶冻中固体物质的含量， 对胶冻进行 稀释或者浓缩， 使其中固体物质的含量达到 0.9 g/mL;
[0164] 步骤 Sl-7、 量取一定量步骤 S1-6获得的矿化胶原胶冻填入模具中， 进行充分冷 冻干燥， 获得矿化胶原支架， 并切割剪裁成直径 4.1 mm、 高 10.8 mm的圆柱； [0165] 步骤 Sl-8、 配制浓度为 0.25wt%的戊二醛的乙醇溶液作为交联剂， 将步骤 S1-7 获得的矿化胶原支架浸泡于交联剂溶液中 24小吋， 进行交联；
[0166] 步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以流动的纯 水洗涤 72小吋， 以除去残留的交联剂；
[0167] 步骤 Sl-10、 将步骤 S1-9获得的矿化胶原支架进行充分冷冻干燥；
[0168] 步骤 Sl-l l、 对步骤 Sl-10获得的矿化胶原支架进行盲孔加工， 盲孔深度为 7 mm
， 直径为 4.71 mm， 并进行清洗和灭菌， 获得矿化胶原基生物牙根支架。
[0169] 经检测， 该支架孔隙率为 95%。
[0170] 实施例 3
[0171] 制备 HA含量为 80wt%的矿化胶原基生物牙根支架材料。
[0172] 步骤 Sl-1、 将胶原溶于 0.5\^%的醋酸溶液中， 配制成胶原的酸溶液， 其中胶原 浓度为 5.0x10 5 g/mL;
[0173] 步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液， 钙离子 的加入量为每克胶原对应加入钙离子 0.16mol， 其中部分钙离子游离在溶液里， 未能被吸附于胶原蛋白纤维上； [0174] 步骤 Sl-3、 持续搅拌步骤 Sl-2获得的溶液， 缓慢滴加含磷酸根离子的溶液， 磷 酸根离子的加入量与步骤 S1-2中钙离子加入量的摩尔比为 Ca/P = 2/1 ;
[0175] 步骤 Sl-4、 持续搅拌步骤 S1-3获得的溶液， 缓慢滴加 NaOH溶液至混合体系 pH = 7， 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH = 7日寸， 混合体系出现白色 悬浊液；
[0176] 步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 24小吋， 抽去上清液， 用离心的方 法洗去杂质离子， 离心浓缩得到矿化胶原胶冻；
[0177] 步骤 Sl-6、 检测步骤 S1-5获得的矿化胶原胶冻中固体物质的含量， 对胶冻进行 稀释或者浓缩， 使其中固体物质的含量达到 0.72 g/mL;
[0178] 步骤 Sl-7、 量取一定量步骤 S1-6获得的矿化胶原胶冻填入模具中， 进行充分冷 冻干燥， 获得矿化胶原支架， 并切割剪裁成直径 4.1 mm、 高 10.8 mm的圆柱； [0179] 步骤 Sl-8、 配制浓度为 0.005wt<¾的戊二醛的乙醇溶液作为交联剂， 将步骤 S1-7 获得的矿化胶原支架浸泡于交联剂溶液中 24小吋， 进行交联；
[0180] 步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以流动的纯 水洗涤 72小吋， 以除去残留的交联剂；
[0181] 步骤 Sl-10、 将步骤 S1-9获得的矿化胶原支架进行充分冷冻干燥；
[0182] 步骤 Sl-l l、 对步骤 Sl-10获得的矿化胶原支架进行盲孔加工， 盲孔深度为 7 mm
， 直径为 4.71 mm， 并进行清洗和灭菌， 获得矿化胶原基生物牙根支架。
[0183] 经检测， 该支架孔隙率为 85.5%。
[0184] 以下为本发明实施例 1的实验结果分析。
[0185] 图 2A-图 2D为本发明的牙髓干细胞与支架复合培养方法。 其中图 2A为接种细胞 后的支架材料大体观察照片； 图 2B为旋转式三维培养系统； 图 2C为接种 2天后电 镜观察图， 图中可见有少量的细胞幵始黏附在支架材料上 (600x)； 图 2D为培养 5 天后电镜观察图， 图中可见细胞相互连接成一片， 排列紧密 (1000x)。
[0186] 图 3为本发明的仿生牙周膜片的大体观察照片。 当培养基成分中加入 2(Vg/ml的 抗坏血酸后， 可以促进细胞快速增殖， 并分泌大量细胞外基质胶原成分， 将所 有细胞连接在一起， 当生长到 10-13天后， 可以将整个细胞膜片完整揭下来。
[0187] 图 4为本发明的仿生牙周膜片的组织学染色切片。 切片上可见牙周干细胞膜片 由 2-3层细胞构成， 细胞之间细胞外基质丰富， 连接紧密。
[0188] 图 5A-5D为本发明的仿生牙周膜片的透射电镜照片。 透射电镜显示， 牙周膜细 胞膜片保持了细胞间的紧密连接， 且具有细胞正常的生理活动。 其中图 5A显示 为完整的细胞形态。 图 5B表明获得的牙周膜干细胞膜片保持了细胞间的紧密连 接。 图 5C示细胞生长分化很好， 胞浆内大量微丝， 沿细胞膜表面可见胞吐小泡 。 图 5D中表明细胞周围可见基质和胶原纤维 (主要为 2型胶原)。
[0189] 图 6A-6F为本发明的仿生牙周膜片的基因和蛋白表达检测结果。 检测结果表明 牙周膜细胞膜片与酶消化下来的细胞相比， 细胞外基质明显增高。 图 6A、 6B、 6 C分别为 1型胶原 (collagen I， COLI)， 纤维连接蛋白（ fibronectin )及整联蛋白 β1( integrin pl)的 real-time PCR对比结果。 图 6D、 6E、 6F分别为以上蛋白的免疫荧 光化学染色结果， 也从蛋白水平证实了这一点。
[0190] 图 7A-7C为本发明的再生牙周组织与正常组织及种植体牙周组织的组织学情况 对比。 回植后 6个月， 组织学观察显示再生的生物牙根组织形态与新生的骨组织 不同， 其散在分布着少量细胞， 推测可能是成牙本质细胞或成牙骨质细胞， 中 央为均匀、 粉染的基质。 新形成的硬组织区域外为纤维结缔组织包绕， 纤维走 行与正常牙周膜类似， 有穿通 （sharpy) 纤维样结构插入新形成的硬组织区。 种 植体磨片染色结果显示形成单纯的骨结合 7， 并没有正常的牙周膜结构。 其中图 7A为生物牙根植入后 6个月， 图 7B为正常的牙根结构， 图 7C为种植体磨片。 PDL ： 牙周膜； D: 牙本质； C: 牙骨质； B: 骨； IM: 种植体。
[0191] 本发明是根据特定实施例进行描述的， 但本领域的技术人员应明白在不脱离本 发明范围吋， 可进行各种变化和等同替换。 此外， 为适应本发明技术的特定场 合或材料， 可对本发明进行诸多修改而不脱离其保护范围。 因此， 本发明并不 限于在此公幵的特定实施例， 而包括所有落入到权利要求保护范围的实施例。

Claims

权利要求书
一种含仿生牙周膜的生物牙根复合体， 其特征在于， 由生物牙根支架 、 牙源性干细胞和牙周细胞膜片复合而成， 所述生物牙根支架成分包 括矿化胶原， 所述生物牙根支架中生长有牙源性干细胞， 所述生物牙 根支架外部紧密结合有牙源性干细胞制成的牙周细胞膜片。
根据权利要求 1所述的含仿生牙周膜的生物牙根复合体， 其特征在于 ， 所述生物牙根支架的矿化胶原中胶原为动物源性的 I型胶原， 磷灰 石的主要物相为羟基磷灰石， 其中磷灰石以弱结晶的纳米微粒形式有 序排列在胶原分子之间与分子表面， 胶原 /磷灰石 = 5/5 ~ 2/8 (w/w)。 根据权利要求 1所述的含仿生牙周膜的生物牙根复合体， 其特征在于 ， 所述生物牙根支架内部具有供牙源性干细胞生长贴附的孔隙结构， 孔隙率为 70% ~ 95%， 孔径为 50 ~ 500 μηι。
根据权利要求 1所述的含仿生牙周膜的生物牙根复合体， 其特征在于
， 所述牙周细胞膜片由 2-3层牙源性干细胞构成， 所述牙周细胞膜片 富含细胞、 细胞外基质以及胶原纤维， 其中水分占 50 ~ 80 wt%， 除 水分以外的物质组成中， 胶原纤维占 60 ~ 75 wt%。
一种生物牙根支架， 其特征在于， 所述生物牙根支架的成分包括矿化 胶原， 所述矿化胶原中胶原为动物源性的 I型胶原， 磷灰石的主要物 相为羟基磷灰石， 其中磷灰石以弱结晶的纳米微粒形式有序排列在胶 原分子之间与分子表面， 胶原 /磷灰石 = 5/5 ~ 2/8 (w/w)=
一种生物牙根支架的制备方法， 其特征在于， 所述制备方法包括以下 步骤：
步骤 Sl-1、 将胶原溶于盐酸、 硝酸或醋酸中的任何一种， 配制成胶原 的酸溶液， 其中胶原浓度为 5.0x10 5 ~ 5.0x10 3 g/mL;
步骤 Sl-2、 持续搅拌步骤 S1-1获得的溶液， 缓慢滴加含钙离子的溶液
， 钙离子的加入量为每克胶原对应加入钙离子 0.01 ~ 0.16 mol;
步骤 Sl-3、 持续搅拌步骤 S1-2获得的溶液， 缓慢滴加含磷酸根离子的 /P = 1/1 ~ 2/1；
步骤 Sl-4、 持续搅拌步骤 Sl-3获得的溶液， 缓慢滴加 NaOH溶液至混 合体系 pH = 6 ~ 8， 当 pH = 5 ~ 6日寸， 混合体系幵始出现沉淀， 当 pH =
7吋， 混合体系出现白色悬浊液；
步骤 Sl-5、 将步骤 S1-4获得的混合体系静置 24 ~ 120小吋， 抽去上清 液， 用离心的方法洗去杂质离子， 离心浓缩得到矿化胶原胶冻； 步骤 Sl-6、 检测步骤 S1-5获得的矿化胶原胶冻中固体物质的含量， 对 胶冻进行稀释或者浓缩， 使其中固体物质的含量达到 0.72 ~ 0.9 g/mL 步骤 Sl-7、 量取一定量步骤 S1-6获得的矿化胶原胶冻填入模具中， 进 行充分冷冻干燥， 获得矿化胶原支架， 并进行切割剪裁；
步骤 Sl-8、 配制浓度为 0.005 ~ 0.25 <¾的戊二醛的乙醇溶液作为交联 剂， 将步骤 S1-7获得的矿化胶原支架浸泡于交联剂溶液中 24 ~ 48小吋 ， 进行交联；
步骤 Sl-9、 将矿化胶原支架从交联剂溶液中取出， 置于层析柱中， 以 流动的纯水洗涤 48 ~ 72小吋， 以除去残留的交联剂；
步骤 Sl-10、 将步骤 S1-9获得的矿化胶原支架进行真空干燥或者冷冻 干燥；
步骤 Sl-l l、 对步骤 S1-10获得的矿化胶原支架进行后处理、 清洗和灭 菌， 获得矿化胶原基生物牙根支架。
[权利要求 7] 含仿生牙周膜的生物牙根复合体的制作方法， 其特征在于， 所述制 作方法包括以下步骤：
51、 采用权利要求 6所述的生物牙根支架的制备方法制作生物牙根支 架；
52、 在所述生物牙根支架上接种牙源性干细胞， 并在三维旋转环境中 复合培养得到生物牙根支架 /细胞复合体；
53、 使用牙源性干细胞制作牙周细胞膜片， 并将牙周细胞膜片包裹在 所述生物牙根支架 /细胞复合体上得到含仿生牙周膜的生物牙根复合 体。
[权利要求 8] 根据权利要求 7所述的含仿生牙周膜的生物牙根复合体的制作方法， 其特征在于， 所述步骤 S2具体包括：
步骤 S2-l、 选用符合生产质量管理规范条件下培养的异体牙髓干细胞 ， 将 1x10 7牙髓干细胞消化后用培养基吹打混匀， 通过滴加和负压吸 附的方式接种于生物牙根支架材料上；
步骤 S2-2、 将步骤 S2-1获得的支架材料在培养箱中静置 4小吋， 然后 放入旋转式三维培养器内继续培养， 转速 2-5次 /分钟， 每三天换液， 培养 5天。
[权利要求 9] 根据权利要求 7所述的含仿生牙周膜的生物牙根复合体的制作方法， 其特征在于， 所述步骤 S3具体包括：
步骤 S3-l、 选用符合生产质量管理规范条件下培养的自体或异体牙周 膜干细胞， 将生长旺盛的第二或第三代牙周膜干细胞接种在 10cm培 养皿， 培养成分为 α-ΜΕΜ培养基， 每三天换液； 步骤 S3-2、 培养 10-14天后， 培养皿边缘细胞出现皱褶， 用较钝刀片 或细胞刮子将牙周细胞膜片整体揭下来， 过程中牙周细胞膜片不要干 燥；
步骤 S3-3、 将步骤 S3-2获得的牙周细胞膜片完整展幵， 对折， 并将液 体吸干， 将步骤 S2获得的生物牙根支架 /细胞复合体放置于牙周细胞 膜片的一侧， 向另一侧卷起包裹生物牙根支架 /细胞复合体， 松紧合 适， 形成生物牙根复合体。
[权利要求 10] 根据权利要求 7所述的含仿生牙周膜的生物牙根复合体的制作方法， 其特征在于， 所述牙源性干细胞为牙囊干细胞、 牙髓干细胞、 根尖牙 乳头干细胞、 脱落乳牙干细胞、 牙龈干细胞或牙周膜干细胞。
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