CN114652894A - Bone repair material and preparation method thereof - Google Patents
Bone repair material and preparation method thereof Download PDFInfo
- Publication number
- CN114652894A CN114652894A CN202210356618.6A CN202210356618A CN114652894A CN 114652894 A CN114652894 A CN 114652894A CN 202210356618 A CN202210356618 A CN 202210356618A CN 114652894 A CN114652894 A CN 114652894A
- Authority
- CN
- China
- Prior art keywords
- microvascular
- bone repair
- repair material
- matrix carrier
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000463 material Substances 0.000 title claims abstract description 30
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 210000000577 adipose tissue Anatomy 0.000 claims abstract description 26
- 239000011159 matrix material Substances 0.000 claims abstract description 20
- 210000001519 tissue Anatomy 0.000 claims abstract description 14
- 239000000560 biocompatible material Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 30
- 102000029816 Collagenase Human genes 0.000 claims description 15
- 108060005980 Collagenase Proteins 0.000 claims description 15
- 229960002424 collagenase Drugs 0.000 claims description 15
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 14
- 239000012634 fragment Substances 0.000 claims description 14
- 239000002953 phosphate buffered saline Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 4
- 108010022355 Fibroins Proteins 0.000 claims description 4
- 108010010803 Gelatin Proteins 0.000 claims description 4
- 238000004132 cross linking Methods 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 235000019322 gelatine Nutrition 0.000 claims description 4
- 235000011852 gelatine desserts Nutrition 0.000 claims description 4
- 229920002674 hyaluronan Polymers 0.000 claims description 4
- 229960003160 hyaluronic acid Drugs 0.000 claims description 4
- GJKGAPPUXSSCFI-UHFFFAOYSA-N 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone Chemical compound CC(C)(O)C(=O)C1=CC=C(OCCO)C=C1 GJKGAPPUXSSCFI-UHFFFAOYSA-N 0.000 claims description 3
- OQORATGQIWQZBQ-UHFFFAOYSA-L dilithium phosphonato-(2,4,6-trimethyl-3-phenylphenyl)methanone Chemical group C1(=CC=CC=C1)C=1C(=C(C(=O)P([O-])([O-])=O)C(=CC=1C)C)C.[Li+].[Li+] OQORATGQIWQZBQ-UHFFFAOYSA-L 0.000 claims 1
- 239000002131 composite material Substances 0.000 description 13
- 238000001914 filtration Methods 0.000 description 13
- 239000000017 hydrogel Substances 0.000 description 11
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 10
- 239000007787 solid Substances 0.000 description 9
- 239000000706 filtrate Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 5
- 210000004088 microvessel Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000011010 flushing procedure Methods 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 3
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 230000001678 irradiating effect Effects 0.000 description 3
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 230000017423 tissue regeneration Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000001365 lymphatic vessel Anatomy 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- RKCKOWJWQLITLM-UHFFFAOYSA-N P(O)(O)=O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C Chemical group P(O)(O)=O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C RKCKOWJWQLITLM-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 235000021231 nutrient uptake Nutrition 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
-
- 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
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/365—Bones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—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
- A61L27/3687—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 use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- 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
- A61L27/52—Hydrogels or hydrocolloids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- 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
Abstract
The invention belongs to the technical field of biomedical materials, and relates to a bone repair material and a preparation method thereof. The bone repair material containing the microvascular tissue is based on the microvascular extracted from the adipose tissue of the bone repair material and a biocompatible material matrix, has an intercommunicated macroporous structure and excellent biocompatibility, is short in preparation period and strong in plasticity, can obtain base materials with different shapes and sizes according to requirements, and is used for simulating a natural tissue structure.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and relates to a bone repair material and a preparation method thereof, in particular to a bone repair material containing microvascular tissue.
Background
With the continuous development of bone tissue engineering in recent years, various novel scaffold materials provide new choices for the treatment of bone defects. The application of the bone tissue engineering technology mainly depends on the reconstruction of a blood vessel network after the implantation of the bracket material, namely whether the vascularization can be quickly realized in the early stage of the implantation. Insufficient vascularization can result in the formation of fibrous capsule structures between the scaffold material and surrounding tissue, limiting nutrient uptake, and hindering the excretion of metabolic waste products, thereby affecting the formation of new bone. Therefore, whether the stent material can be quickly vascularized in an early stage after being implanted directly influences the final osteogenesis effect. To overcome the above problems, a pre-formed microvascular network may be created within the material prior to implantation of the tissue structure material. The pre-formed microvascular network is interconnected with the host's microvasculature after implantation, thereby achieving rapid perfusion, i.e., prevascularization. In order to achieve the goal of good prevascularization and thus promoting tissue healing, prevascularization materials constructed by combining composite scaffold materials containing multiple growth factors and multiple cells are the hot spots of research in recent years. However, due to the complexity and time consuming in vitro procedures, such a preventive vascularization method is difficult to apply in a clinical setting, and therefore, there is still a need to develop new preventive vascularization reconstruction strategies.
Disclosure of Invention
The present invention has been made to solve the above problems, and provides a bone repair material and a method for preparing the same, which promotes bone tissue repair by providing pluripotent stem cells and releasing various growth factors to accelerate vascularization, based on the extraction of microvessels from autologous adipose tissue.
According to the technical scheme, the bone repair material comprises an ad-MVF (microvascular tissue-derived microvascular fragments) segment and a matrix carrier, wherein the microvascular segment is extracted from Adipose tissues, and the matrix carrier is made of a biocompatible material.
The invention adopts fat-derived microvascular segments as a matrix. The fragment can be obtained by extracting a microvascular fragment containing growth factors favorable for accelerating vascularization and tissue repair, such as Vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bFGF), etc., multipotent mesenchymal stem cells having multiple differentiation potential, such as Endothelial Progenitor Cells (EPCs), and a lymphatic vessel fragment having an immunoregulatory effect, by using autologous adipose tissue.
Furthermore, the microvascular fragments are obtained by adding collagenase type I into adipose tissues, digesting and separating, and the length of the microvascular fragments is 40-180 mu m.
Specifically, 40000-50000 microvascular segments per ml of adipose tissue were extracted on average.
Further, the biocompatible material is methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA) acid, or methacrylated silk fibroin (SilMA).
The second aspect of the invention provides a preparation method of a bone repair material containing microvascular tissue, comprising the following steps:
s1: adding collagenase type I into adipose tissue, digesting, and removing excessive collagenase type I to obtain a mixture;
s2: separating the mixture to obtain microvascular fragments;
s3: and adding the microvascular segments into a matrix carrier solution, and crosslinking by ultraviolet or blue light to obtain the bone repair material containing the microvascular tissue, wherein the matrix carrier is methacryloylated gelatin, methacryloylated hyaluronic acid or methacryloylated silk fibroin, and the matrix carrier solution further comprises a photoinitiator.
Further, the step S1 includes, before the collagenase I is added, cutting the adipose tissue and washing the contaminants such as blood with physiological saline.
Further, in the step S1, the collagenase type I is added in an amount of 1-6mg per 1mL of the adipose tissue.
Specifically, the collagenase I is dissolved in a DMEM/F12 culture medium to obtain a collagenase I solution with the concentration of 1-2mg/mL, and then the collagenase I solution is added into adipose tissues; the volume ratio of the collagenase I solution to the adipose tissue is 1-3: 1.
further, in step S1, the digestion is performed under humidified atmospheric conditions (37 ℃).
Further, in the step S1, the digestion time is 8-12 min.
Further, in the step S1, the excessive collagenase type I is removed by adding Phosphate Buffered Saline (PBS) containing Fetal Bovine Serum (FBS).
Further, in the step S2, separating the mixture includes removing undigested fat mass and miscellaneous cells in the mixture.
Specifically, the operation of separating the mixed solution is as follows: filtering the suspension through a screen to remove undigested fat mass; centrifuging to remove fat supernatant; filtering the remainder with a filter screen to remove the rest of the mixed cells, flushing the filtrate on the filter screen with normal saline, and centrifuging to obtain the ad-MVF.
Further, the pore diameter of the filter screen for the first filtration is 400-600 μm; the aperture of the filter screen for the second filtration is 30-50 μm; the speed of the two centrifugations is 600-12000rpm, and the time is 3-8 min.
Further, in the step S3, the content of the microvascular fragments per 1mL of the matrix carrier is 4000-60000.
Specifically, after the micro-vessel fragments are resuspended, a micro-vessel fragment solution with the concentration of 40000-; the concentration of the matrix carrier in the matrix carrier solution is 5-20% w/v; 1: 1-10.
Further, the solvent of the microvascular fragment solution and the matrix carrier solution is the same, and is PBS or 0.9% physiological saline.
Further, the preparation method of the matrix carrier solution may be: adding PBS into GelMA, HAMA or SilMA solid sponge, dissolving in water bath (37 deg.C) to obtain transparent liquid, adding photoinitiator, and dissolving to obtain the matrix carrier solution
Further, the photoinitiator is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate or I2959 (2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone).
Further, the concentration of the photoinitiator in the matrix carrier solution is 2-10 mg/mL.
Further, in the step S3, the time of the crosslinking reaction is 60 to 120S.
Compared with the prior art, the technical scheme of the invention has the following advantages:
1) the ad-MVF is a microvascular segment derived from autologous adipose tissues, and can secrete a large amount of angiogenesis promoting factors such as VEGF and bFGF and multipotential mesenchymal stem cells in addition to the preservation of the complete vascular structure, so that the vascularization and the bone tissue repair are promoted;
2) the ad-MVF contains a large number of lymphatic vessel segments and has an immunoregulation effect;
3) the ad-MVF is a source of autologous adipose tissues, has the advantages of wide source, easy acquisition, low cost, small immune rejection, high biocompatibility and the like, and has very good application prospect in biological tissue engineering;
4) the preparation method of the ad-MVF loaded composite hydrogel is simple, has good stability and good operability;
5) the composite hydrogel loaded with the ad-MVF has an intercommunicated macroporous structure, excellent biocompatibility, short preparation period and strong plasticity, and can be used for obtaining base materials with different shapes and sizes according to requirements and simulating a natural tissue structure.
Drawings
FIG. 1 is an optical microscope photograph of ad-MVF extracted in example 1.
FIG. 2 is a topographical view of the ad-MVF/HAMA composite hydrogel of example 2.
FIG. 3 is a graph showing the mechanical strength of the ad-MVF/SilMA composite hydrogel in example 3 at different volume ratios.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
The embodiment relates to a preparation method of an ad-MVF/GelMA composite hydrogel, which comprises the following steps:
s11, taking 10mL of adipose tissue, mechanically cutting, washing dirt such as blood on the adipose tissue by using 0.9% physiological saline, and adding 2mg/mL type I collagenase solution with double volume (20 mL);
s12, digesting with rapid stirring at 37 ℃ for 8min under humidified atmospheric conditions, adding an equal volume (30mL) of PBS containing 20% FBS to neutralize collagenase type I;
s13, filtering the suspension by the aid of a 500-micron filter screen, removing undigested fat blocks on the filter screen, centrifuging the filtrate at 10000rpm for 5min, and removing fat supernatant;
s14, filtering the suspension of the remainder through a 30-micron filter screen, filtering out the rest mixed cells, flushing the filtrate on the filter screen with 0.9% of normal saline, centrifuging at 10000rpm for 5min, removing the supernatant, and leaving the precipitate to obtain ad-MVF;
s15, adding 10mL PBS into ad-MVF extracted from 10mL of adipose tissue to obtain ad-MVF solution;
s16, adding 10mL of PBS into 0.5g of GelMA solid sponge, then placing the GelMA solid sponge in a water bath kettle at 37 ℃ to be dissolved into transparent liquid to obtain a GelMA solution with the mass percentage of 5%, then adding 0.025g of photoinitiator (phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate, LAP), adding 10mL of ad-MVF solution into 10mL of GelMA solution with the mass concentration of 10%, and irradiating 60S with blue light with the wavelength of 405nm to obtain the ad-MVF/GelMA composite hydrogel with the mass concentration of 2.5%.
The extracted ad-MVF is shown in figure 1 under an optical microscope, and is a blood vessel fragment with different lengths, and the length of the extracted ad-MVF is about 40-180 mu m.
The number of isolated ad-MVF per ml of adipose tissue was 40000 and 50000 on average.
Example 2
The embodiment relates to a preparation method of ad-MVF/HAMA composite hydrogel, which comprises the following steps:
s11, taking 5mL of adipose tissue, mechanically cutting, washing dirt such as blood on the adipose tissue by using 0.9% physiological saline, and adding 1.5-time volume of 1.5mg/mL type I collagenase solution;
s12, digesting the mixture for 10min at 37 ℃ under the humidified atmosphere by rapid stirring, and adding PBS containing 20% FBS in the same volume to neutralize collagenase type I;
s13, filtering the suspension by a 600-micron filter screen, removing undigested fat blocks on the filter screen, centrifuging the filtrate at 12000rpm for 3min, and removing fat supernatant;
s14, filtering the remainder through a 50-micron filter screen to remove the suspension, filtering out the rest miscellaneous cells, flushing the filtrate on the filter screen with 0.9% physiological saline, centrifuging at 12000rpm for 3min, removing the supernatant, and leaving the precipitate to obtain ad-MVF;
s15, adding 5mL of PBS into ad-MVF extracted from 5mL of adipose tissue to obtain ad-MVF solution;
s16, adding 5mL of PBS into 0.5g of HAMA solid sponge, then placing the HAMA solid sponge in a water bath kettle at 37 ℃ to dissolve the HAMA solid sponge into a transparent liquid to obtain a HAMA solution with the mass concentration of 10%, then adding 0.025g of photoinitiator (phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate, LAP), adding 5mL of ad-MVF solution into 5mL of HAMA solution with the mass concentration of 10%, and irradiating 120S with blue light with the wavelength of 405nm to obtain the ad-MVF/HAMA composite hydrogel with the mass concentration of 5%.
Example 3
The embodiment relates to a preparation method of an ad-MVF/SilMA composite hydrogel, which comprises the following steps:
s21, taking 2.5mL of adipose tissue, mechanically cutting, washing dirt such as blood on the adipose tissue by using 0.9% physiological saline, and adding 1mg/mL I type collagenase solution with three times of volume;
s22, digesting for 12min under the condition of humidifying atmosphere and at 37 ℃ by rapid stirring, and adding PBS with 20% FBS in the same volume to neutralize collagenase type I;
s23, filtering the suspension by the aid of a 450-micron filter screen, removing undigested fat blocks on the filter screen, centrifuging the filtrate at 800rpm for 6min, and removing fat supernatant;
s24, filtering the suspension of the remainder through a filter screen with the diameter of 45 mu m, filtering out the rest mixed cells, flushing the filtrate on the filter screen with 0.9 percent of normal saline, centrifuging at 800rpm for 6min, removing the supernatant, and leaving the precipitate to obtain the ad-MVF;
s25, adding 2.5mL PBS into ad-MVF extracted from 2.5mL of adipose tissue to obtain ad-MVF solution;
s26, adding 5mL of PBS into 0.5g of SilMA solid sponge, then placing the SilMA solid sponge in a water bath kettle at 37 ℃ to dissolve the SilMA solid sponge into transparent liquid to obtain 10% of mass concentration, then adding 0.025g of photoinitiator (phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate, LAP), adding 2.5mL of ad-MVF solution into 5mL of 10% of mass concentration SilMA solution, and irradiating 120S with blue light with the wavelength of 405nm to obtain 6.7% of ad-MVF/SilMA composite hydrogel.
In specific application, the volume ratio of the ad-MVF solution to the SilMA solution is preferably 1:1-1: 10; when the content of the ad-MVF in the composite material is gradually increased, the mechanical strength of the material is gradually reduced, the mechanical strength of the ad-MVF/SilMA composite hydrogel at different volume ratios is shown in figure 3, and the appropriate concentration can be selected according to the type and the area of the defect tissue.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. The bone repair material containing the microvascular tissue is characterized by comprising microvascular segments and a matrix carrier, wherein the microvascular segments are extracted from adipose tissue, and the material of the matrix carrier is a biocompatible material.
2. The bone repair material of claim 1 wherein the microvascular segments are 40-180 μ ι η in length.
3. The bone repair material of claim 1, wherein the biocompatible material is a methacrylated gelatin, a methacrylated hyaluronic acid, or a methacrylated silk fibroin.
4. The preparation method of the bone repair material is characterized by comprising the following steps:
s1: adding collagenase type I into adipose tissue, digesting, and removing excessive collagenase type I to obtain a mixture;
s2: separating the mixture to obtain microvascular fragments;
s3: and adding the microvascular segments into a matrix carrier solution, and crosslinking by ultraviolet or blue light to obtain the bone repair material containing the microvascular tissue, wherein the matrix carrier is methacryloylated gelatin, methacryloylated hyaluronic acid or methacryloylated silk fibroin, and the matrix carrier solution further comprises a photoinitiator.
5. The method of claim 4, wherein in step S1, the collagenase type I is added in an amount of 1-6mg per 1mL of the adipose tissue.
6. The method according to claim 4, wherein in step S1, the excessive collagenase type I is removed by adding phosphate buffered saline containing fetal bovine serum.
7. The method of claim 4, wherein in step S2, separating the mixture includes removing undigested fat clumps and miscellaneous cells from the mixture.
8. The method of claim 4, wherein the amount of the microvascular fragments per 1mL of the matrix carrier in step S3 is 4000-60000.
9. The method of claim 4, wherein the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate or I2959.
10. The method of claim 4, wherein in step S3, the time for the crosslinking reaction is 60-120S.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210356618.6A CN114652894A (en) | 2022-04-06 | 2022-04-06 | Bone repair material and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210356618.6A CN114652894A (en) | 2022-04-06 | 2022-04-06 | Bone repair material and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114652894A true CN114652894A (en) | 2022-06-24 |
Family
ID=82035279
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210356618.6A Pending CN114652894A (en) | 2022-04-06 | 2022-04-06 | Bone repair material and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114652894A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101282652A (en) * | 2005-06-02 | 2008-10-08 | 亚利桑那董事会（代表亚利桑那大学） | Prevascularized devices and related methods |
| CN110607271A (en) * | 2018-06-14 | 2019-12-24 | 中国科学院大连化学物理研究所 | Preparation method of in vitro vascularized 3D tissue based on micromachining technology |
| CN112107731A (en) * | 2020-09-24 | 2020-12-22 | 武汉理工大学 | Injectable double-layer drug-loaded osteochondral repair hydrogel scaffold and preparation method thereof |
-
2022
- 2022-04-06 CN CN202210356618.6A patent/CN114652894A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101282652A (en) * | 2005-06-02 | 2008-10-08 | 亚利桑那董事会（代表亚利桑那大学） | Prevascularized devices and related methods |
| CN110607271A (en) * | 2018-06-14 | 2019-12-24 | 中国科学院大连化学物理研究所 | Preparation method of in vitro vascularized 3D tissue based on micromachining technology |
| CN112107731A (en) * | 2020-09-24 | 2020-12-22 | 武汉理工大学 | Injectable double-layer drug-loaded osteochondral repair hydrogel scaffold and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Optimization of a natural collagen scaffold to aid cell–matrix penetration for urologic tissue engineering | |
| Tian et al. | Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering | |
| US20190247541A1 (en) | Preparation of artificial tissues by means of tissue engineering using fibrin and agarose biomaterials | |
| Chen et al. | Transplantation of amniotic scaffold-seeded mesenchymal stem cells and/or endothelial progenitor cells from bone marrow to efficiently repair 3-cm circumferential urethral defect in model dogs | |
| KR20060110637A (en) | Transplantation of differentiated immature adipocytes and biodegradable scaffold for tissue augmentation | |
| JP2005524426A (en) | Graft composition with enhanced angiogenesis | |
| CN107050517B (en) | Vascularized tissue engineering bone without exogenous support and its prepn | |
| Patel et al. | Tissue engineering of the penis | |
| Ebrahimi Sadrabadi et al. | Decellularized extracellular matrix as a potent natural biomaterial for regenerative medicine | |
| Zou et al. | Tissue engineering for urinary tract reconstruction and repair: Progress and prospect in China | |
| Xiao et al. | Bladder acellular matrix prepared by a self-designed perfusion system and adipose-derived stem cells to promote bladder tissue regeneration | |
| CN112755250A (en) | Tissue engineering peripheral nerve tissue and preparation method thereof | |
| CN114652894A (en) | Bone repair material and preparation method thereof | |
| CN110755174A (en) | Biological mixed type artificial blood vessel and preparation method thereof | |
| Moghimi et al. | Adipose-derived human mesenchymal stem cells seeded on denuded or stromal sides of the amniotic membrane improve angiogenesis and collagen remodeling and accelerate healing of the full-thickness wound | |
| Sundaram et al. | Tissue engineering and regenerative medicine | |
| CN111450321A (en) | Artificial skin substitute and scaffold-free self-assembly construction method and application thereof | |
| CN111548988B (en) | Medical rinsing liquid and preparation method and application thereof | |
| EP2145635B1 (en) | Method for preparing three-dimensional structures for tissue engineering | |
| Cao et al. | A Preliminary Study of Constructing the Tissue-Engineered Corpus Cavernosum With Autologous Adipose Stem Cells In Vivo | |
| CN1369555A (en) | Engineered scaffold of amniotic membrane tissue and process for removing cells from aniotic membrane | |
| Huang et al. | Frozen bean curd-inspired xenogeneic acellular dermal matrix with triple pretreatment approach of freeze–thaw, laser drilling and ADSCs pre-culture for promoting early vascularization and integration | |
| US20240091411A1 (en) | Ear cartilage tissue engineering complex and use thereof | |
| RU2661738C2 (en) | Method of obtaining tissue engineering structure | |
| Zhang et al. | Bioprinted dermis with human adipose tissue‐derived microvascular fragments promotes wound healing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220624 |
|
| RJ01 | Rejection of invention patent application after publication |