WO2023006113A1 - 一种生物能量活性材料及其应用 - Google Patents

一种生物能量活性材料及其应用 Download PDF

Info

Publication number
WO2023006113A1
WO2023006113A1 PCT/CN2022/109458 CN2022109458W WO2023006113A1 WO 2023006113 A1 WO2023006113 A1 WO 2023006113A1 CN 2022109458 W CN2022109458 W CN 2022109458W WO 2023006113 A1 WO2023006113 A1 WO 2023006113A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
hydroxybutyric acid
bone
bioenergy
active material
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/109458
Other languages
English (en)
French (fr)
Inventor
李健
张鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to US18/574,727 priority Critical patent/US20240299620A1/en
Priority to CN202280004789.0A priority patent/CN115884799A/zh
Publication of WO2023006113A1 publication Critical patent/WO2023006113A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • A61L27/14—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
    • A61L27/50—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
    • A61L27/54—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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the invention belongs to biomedical tissue engineering, and specifically relates to a bioenergy active material and its application.
  • Biodegradable polymer materials are widely used in bone tissue engineering research because of their good biocompatibility and degradability.
  • Biodegradable polymer materials include natural biodegradable polymers (such as collagen, chitosan) and synthetic biodegradable polymers (such as PLA, PLGA, PCL), because of their good biocompatibility and degradability, they are Currently commonly used bone tissue engineering scaffold materials ⁇ F.Asghari, M.Samiei, K.Adibkia, A.Akbarzadeh, S.Davaran, Biodegradable and biocompatible polymers for tissue engineering application: a review, ArtifCells Nanomed Biotechnol 45(2)(2017 )185-192.].
  • biodegradable polymers have certain bone defect repair functions, they still have a series of shortcomings that are difficult to overcome, such as uncontrollable degradation rate, poor mechanical properties, and acidic degradation products causing inflammation [Y.X.Lai, Y.Li, H.J.Cao , J.Long, X.L.Wang, L.Li, C.R.Li, Q.Y.Jia, B.Teng, T.T.Tang, J.Peng, D.Eglin, M.Alini, D.W.Grijpma, G.Richards, L.Qin, Osteogenic magnesium incorporated into PLGA/TCP porous scaffold by 3D printing for repairing challenging bone defect, Biomaterials 197(2019) 207-219.], hindering its widespread clinical application.
  • the bone regeneration process is an energy-consuming process, and cellular energy metabolism plays a vital role in tissue repair and regeneration.
  • Adenosine triphosphate (ATP) is the main source of cellular energy and plays a role in many biological processes, including cell proliferation, migration and differentiation [I. Gadjanski, S. Yodmuang, K. Spiller, S. Bhumiratana, G. Vunjak-Novakovic, Supplementation of Exogenous Adenosine 5-Triphosphate Enhances Mechanical Properties of 3D Cell-Agarose Constructs for Cartilage Tissue Engineering, Tissue Eng Pt A 19(19-20)(2013)2188-2200.].
  • the present invention proposes a bioenergy active material and its application.
  • the specific plan is as follows:
  • the first aspect of the present invention provides a bioenergy active material
  • the bioenergy active material is a biodegradable polymer
  • the degradation product of the bioenergy active material is the metabolism in the tricarboxylic acid cycle and/or glycolysis pathway mid product
  • the degradation product of the bioenergy active material is a polymer monomer that can be converted into a metabolic intermediate in the citric acid cycle and/or glycolysis pathway;
  • the degradation product of the bioenergy active material is a polymer monomer that can be converted into acetyl-CoA.
  • the metabolic intermediates of the tricarboxylic acid cycle include citric acid, cis-aconitic acid, isocitric acid, oxalylsuccinic acid, ⁇ -ketoglutarate, succinyl-CoA , succinic acid, fumaric acid, malic acid and adenosine triphosphate;
  • the metabolic intermediates of the glycolytic pathway include glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, glycerol 1,3-bisphosphate
  • glucose-6-phosphate fructose-6-phosphate
  • fructose-1,6-diphosphate fructose-1,6-diphosphate
  • glyceraldehyde-3-phosphate dihydroxyacetone phosphate
  • glycerol 1,3-bisphosphate One or more of acid, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate PEP and pyruvate;
  • the polymer monomer capable of converting to acetyl-CoA is 3-hydroxybutyric acid.
  • 3-hydroxybutyrate will generate acetoacetate under the action of 3-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase), and then succinyl-CoA (succinyl-CoA) in 3-succinyl-CoA transferase (3
  • Acetoacetyl-CoA (Acetoacetyl-CoA) is synthesized under the action of -oxoacid CoA-transferase) enzyme, acetoacetyl-CoA reacts with a coenzyme A (CoA), and is generated in acetyl-CoA-acyltransferase (acetyl-CoAC-acetyltransferases)
  • Two acetyl-coenzyme A (acetyl-CoA)
  • the bioenergy active material is polyhydroxyalkanoate whose degradation product is 3-hydroxybutyric acid.
  • polyhydroxyalkanoate includes 3-hydroxybutyrate-4-hydroxybutyrate copolyester (P34HB), poly-3-hydroxybutyrate (PHB), 3-hydroxybutyrate-3-hydroxy One or more of valeric acid copolyester (PHBV) and 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolyester (PHBHHx).
  • the second aspect of the present invention provides the application of the bioenergy active material in the field of bone tissue regeneration and repair.
  • the third aspect of the present invention provides the application of the bioenergy active material in preparing a porous scaffold for bone tissue repair.
  • the fourth aspect of the present invention provides a porous scaffold for bone tissue repair prepared from the bioenergy active material. Furthermore, the porous scaffold for bone tissue repair can be prepared by traditional preparation methods or by 3D printing technology.
  • the fifth aspect of the present invention provides a method for preparing a 3D porous scaffold for bone tissue repair, comprising the following steps:
  • the bioenergy active material is synthesized by microbial method or chemical synthesis method, and may also be synthesized by other methods.
  • 3-hydroxybutyrate-4-hydroxybutyrate copolyester can be produced by fermentation of the microorganism Halomonas spp.
  • the fifth aspect of the present invention provides the 3D porous scaffold for bone tissue repair prepared by the above preparation method.
  • the sixth aspect of the present invention provides the use of degradation products including polyhydroxyalkanoate esters of 3-hydroxybutyric acid as a bioenergy active material with both bone tissue regeneration and angiogenesis functions, and the 3-hydroxybutyric acid is metabolized by tricarboxylic acid Circulates and participates in bone formation in the form of citrate, the 3-hydroxybutyrate induces angiogenesis.
  • polyhydroxyalkanoate includes 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester, poly-3-hydroxybutyrate, 3-hydroxybutyric acid-3-hydroxyvaleric acid copolyester and One or more types of 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolyesters.
  • the seventh aspect of the present invention provides the use of degradation products including polyhydroxyalkanoate esters of 3-hydroxybutyric acid in the preparation of vascularized bone regeneration materials.
  • polyhydroxyalkanoate includes 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester, poly-3-hydroxybutyrate, 3-hydroxybutyric acid-3-hydroxyvaleric acid copolyester and One or more types of 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolyesters.
  • the eighth aspect of the present invention provides the application of degradation products including polyhydroxyalkanoate esters of 3-hydroxybutyric acid in the preparation of large-segment bone defect repair materials or critical bone defect repair materials.
  • polyhydroxyalkanoate includes 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester, poly-3-hydroxybutyrate, 3-hydroxybutyric acid-3-hydroxyvaleric acid copolyester and One or more types of 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolyesters.
  • the ninth aspect of the present invention provides a vascularized bone regeneration material, a large bone defect repair material or a critical bone defect repair material, which is characterized in that it is prepared from a degradation product including polyhydroxyalkanoate of 3-hydroxybutyric acid.
  • the material is a porous scaffold prepared from degradation products including polyhydroxyalkanoate of 3-hydroxybutyric acid.
  • polyhydroxyalkanoate includes 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester, poly-3-hydroxybutyrate, 3-hydroxybutyric acid-3-hydroxyvaleric acid copolyester and One or more types of 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolyesters.
  • the degradation products of bioenergy active materials provided by the present invention are metabolic intermediates in the tricarboxylic acid cycle and/or glycolytic pathway, or can be converted into metabolic intermediates in the tricarboxylic acid cycle and/or glycolytic pathway.
  • the polymer monomer of the product, or the polymer monomer that can be converted into acetyl-CoA, the degradation product of the bioenergy active material provides bioenergy for tissue cells through the tricarboxylic acid metabolic cycle or glycolysis pathway, solving the traditional biological
  • the problem that degradable materials cannot continuously improve the stability of ATP in cells and the activity of related biomass has shown broad application prospects in the field of bone tissue regeneration, especially in the repair of large bone defects.
  • the bioenergy active material 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester (P34HB) provided by the present invention is a kind of degradable polymer polyester, which has unique bioenergy activity, mechanical properties, biodegradable Degradability and biocompatibility.
  • the main product of its degradation, 3-hydroxybutyric acid (3HB) is one of the main components of ketone bodies in mammals. It is not only non-toxic to the body, but also can be used as an energy substance to promote cell adhesion, proliferation and differentiation.
  • the 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester material can maintain a longer degradation time, and the content of 3-hydroxybutyric acid in the material can be adjusted according to the requirements of tissue repair.
  • the ratio can achieve a long-term degradation of 6-12 months.
  • 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester material can solve the problems that traditional biodegradable materials cannot continuously improve the stability of ATP in cells and related biomass activity, and the acidic degradation products cause inflammatory reactions. It is a kind of A bone repair scaffold active material with excellent performance, bioenergy activity and bone formation promoting function.
  • the porous scaffold for bone tissue repair provided by the present invention is based on bioenergy active materials and 3D printing technology.
  • the porous scaffold structure is controllable and has good biocompatibility, biodegradability and mechanical properties.
  • the scaffold can be implanted in the body With the continuous degradation of itself, the bioenergy active substances produced by its degradation can promote the proliferation, differentiation and mineralization of bone marrow mesenchymal stem cells (hBMSCs), and have the function of promoting bone formation, which is not available in traditional polymer scaffold materials. of.
  • hBMSCs bone marrow mesenchymal stem cells
  • the 3D porous scaffold for bone tissue repair further provided by the present invention is prepared based on the bioenergy active material 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester (P34HB) and 3D printing technology.
  • the porous scaffold structure is controllable and has Good biocompatibility, biodegradability and mechanical properties.
  • the bioenergy active substance 3-hydroxybutyric acid (3HB) produced by the degradation after the tricarboxylic acid metabolic cycle, not only Provide bioenergy for tissue cells, promote the proliferation, differentiation and mineralization of bone marrow mesenchymal stem cells (hBMSCs), and produce intermediate metabolites in the form of citrate to participate in bone formation, which is beneficial to osteogenesis and bone formation in the process of bone repair.
  • hBMSCs bone marrow mesenchymal stem cells
  • hBMSCs bone marrow mesenchymal stem cells
  • hBMSCs bone marrow mesenchymal stem cells
  • vascular function shortens the repair time of bone defects, and shows great application prospects in the field of bone tissue regeneration and repair, especially in the repair of large bone defects.
  • the bioenergy active substance 3-hydroxybutyric acid (3HB) produced by the degradation of the bioenergy active material of the present invention 3HB not only passes through the tricarboxylic acid metabolic cycle and participates in bone formation in the form of citric acid, but also can induce angiogenesis. It has important application prospects in the field of bone regeneration materials, large bone defect repair materials or critical bone defect repair materials.
  • Fig. 1 is the gel permeation chromatogram of 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester; Wherein, A: standard curve, B: 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester;
  • Fig. 2 is the porous support (A) prepared by 3D printing and the scanning electron microscope picture (B) of the cross-section of the porous support;
  • Fig. 3 is the effect result of scaffold degradation product 3-hydroxybutyric acid on the proliferation of human bone marrow mesenchymal stem cells (hBMSCs);
  • Fig. 4 is the impact result of scaffold degradation product 3-hydroxybutyric acid on hBMSCs alkaline phosphatase expression activity
  • Figure 5 is the result of the impact of scaffold degradation product 3-hydroxybutyric acid on the formation of hBMSCs extracellular calcium nodules
  • Figure 6 shows the formation of extracellular apatite in hBMSCs analyzed by laser copolymerization Raman spectroscopy
  • Figure 7 is the result of the effect of scaffold degradation product 3-hydroxybutyric acid on the expression of genes related to hBMSCs osteogenic differentiation
  • Figure 8 is the result of the impact of scaffold degradation product 3-hydroxybutyric acid on the potential energy of the mitochondrial membrane of cells
  • Figure 9 shows the LC-MS/MS metabolic flow analysis of 13 C-labeled 3HB involved in the formation of TCA cycle intermediate metabolites
  • Figure 10 is the quantitative analysis of TCA cycle intermediate metabolites by LC-MS/MS;
  • Figure 11 is the analysis of the citric acid content in the cell supernatant
  • Figure 12 is the body weight and uterus changes of osteoporosis model rats
  • Figure 13 is Micro-CT analysis of the impact of 3HB on the bone mass of the osteoporosis model
  • Figure 14 is silver nitrate staining (a) and double fluorescent labeling analysis of bone formation (c-e);
  • Figure 15 is an in vivo analysis of the way 3HB participates in bone formation (a-c) and the content determination of citrate (d) and calcium (e) in serum;
  • Fig. 16 is to establish the critical bone defect model of rat skull
  • Figure 17 is a scratch test to analyze the effect of 3HB on the migration of EA.hy926 cells
  • Figure 18 is an in vitro analysis of the effect of 3HB on the formation of the microvascular network of EA.hy926 cells
  • Figure 19 is high-resolution two-photon in vivo microscopic imaging analysis of angiogenesis in bone defects (a, b);
  • Figure 20 is micro-CT evaluation of new bone tissue regeneration at the bone defect site 12 weeks after surgery.
  • microorganism Halomonas that can synthesize 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester was fermented and cultured for 72 hours in 60MMG medium at 37°C and 400-800rpm, and collected after 72 hours The cells are placed at 70° C. to ventilate and dry the cells to obtain dry cell powder containing 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester.
  • composition of 60MMG medium is: glucose 30g/L, yeast extract 1g/L, ammonium sulfate 0.25g/L, magnesium sulfate 0.2g/L, disodium hydrogen phosphate 9.65g/L, potassium dihydrogen phosphate 1.5g/L, Trace element I 10ml/L, trace element II 1ml/L.
  • step (4) Collect the precipitate in step (4) by filtration, and place the collected precipitate in a vacuum oven at 40°C for 24 hours. After the solvent is completely evaporated, 3-hydroxybutyric acid-4-hydroxy Butyric copolyester.
  • the 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester material synthesized in Example 1 was loaded into a melt 3D printer (180° C.) to prepare a porous scaffold for bone tissue repair.
  • Figure 2 The results are shown in Figure 2, in which Figure 2-A is the porous scaffold prepared by 3D printing, and Figure 2-B is the electron microscope scanning image of the cross-section of the scaffold, and the pore size of the porous scaffold is about 350-400 ⁇ m.
  • the 3D porous scaffold in Example 2 was soaked in phosphate buffer for 8 weeks, and the degradation products were collected.
  • the degradation product of 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester is mainly 3-hydroxybutyric acid (3HB).
  • the concentration of the collected degradation products was determined, and then the in vitro experiment was carried out.
  • composition of the phosphate buffer is: sodium chloride 7.9g/L, potassium chloride 0.2g/L, potassium dihydrogen phosphate 0.24g/L.
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Figure 3 shows the effect of scaffold degradation product 3-hydroxybutyric acid on the proliferation of human bone marrow mesenchymal stem cells (hBMSCs).
  • hBMSCs human bone marrow mesenchymal stem cells
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Composition of osteogenic differentiation medium low-sugar DMEM medium + 10% fetal bovine serum + 2mM L-glutamine + 100U/mL penicillin + 100 ⁇ g/mL streptomycin + 100nM dexamethasone + 0.2mML-ascorbic acid + 10mM ⁇ - Sodium Glycerophosphate.
  • Figure 4 shows the effect of scaffold degradation product 3-hydroxybutyric acid on the expression activity of hBMSCs alkaline phosphatase, and the results show that the scaffold degradation product can promote the expression of hBMSCs alkaline phosphatase.
  • 3HB significantly enhanced the expression of alkaline phosphatase after stimulating the differentiation of bone marrow mesenchymal stem cells for 14 days, while the control group showed an inhibitory effect.
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Figure 5 shows the effect of scaffold degradation product 3-hydroxybutyric acid on the formation of extracellular calcium nodules in hBMSCs.
  • LA control group
  • 3HB stimulated bone marrow mesenchymal stem cells to induce differentiation for 10 and 14 days, and significantly enhanced the extracellular Ca nodule formation.
  • the deposition of calcium nodules showed that 3-hydroxybutyrate promoted the formation of calcium nodules, a marker of osteogenic differentiation in bone marrow mesenchymal stem cells.
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Figure 6 shows the formation of extracellular apatite in hBMSCs analyzed by laser co-polymerization Raman spectroscopy. After bone marrow mesenchymal stem cells were induced to differentiate for 21 days, a large amount of extracellular apatite was formed and its content increased with the increase of 3HB concentration , indicating that 3-hydroxybutyric acid can promote the formation of apatite in osteogenic differentiation of bone marrow mesenchymal stem cells.
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Figure 7 shows the effect of scaffold degradation product 3-hydroxybutyric acid on the expression of genes related to osteogenic differentiation of hBMSCs.
  • LA control group
  • 3HB stimulated the differentiation of bone marrow mesenchymal stem cells for 7 days and promoted the genes related to osteogenic differentiation
  • the control group inhibited gene expression to a certain extent, indicating that the scaffold degradation product 3-hydroxybutyric acid can promote the expression of genes related to osteogenic differentiation of hBMSCs.
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Figure 8 shows the effect of the scaffold degradation product 3-hydroxybutyric acid on the mitochondrial membrane potential energy of the cells.
  • the results show that the scaffold degradation product 3-hydroxybutyric acid can provide cells with ATP and maintain the mitochondrial membrane potential energy ( ⁇ m), and compared with the LA group , 3HB can provide more biological energy (ATP) for cells and increase the potential energy of cell membrane.
  • 13 C-labeled 3HB was traced to analyze the metabolic flow of 3HB and the way it participates in bone formation.
  • the control group was treated without 13 C-labeled 3HB.
  • Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on culture plates, osteogenic differentiation medium containing 1 mM 13 C-3HB was added to the above cells, and metabolomics analysis was performed on the 14th day (LC-MS/ MS).
  • metabolomics analysis found that multiple intermediate metabolites (Citrate-citric acid, succinate-succinate) were detected in the TCA cycle acid, fumarate-fumaric acid, malate-malic acid) contained carbon atoms derived from 13 C-labeled 3HB, while no carbon atoms derived from 13 C-labeled 3HB were found in the control group (untreated with 13 C-labeled 3HB) (Fig. 9), thus proving that the bioenergy active substance 3HB produced by the degradation of the P34HB scaffold can participate in metabolism through the TCA cycle.
  • bioenergy active substance 3HB participates in the formation of in vitro biomineralization in the form of intermediate metabolite citric acid through the TCA cycle.
  • 3HB was used as the medium
  • the castrated rat osteoporosis model was used as the research object to illustrate the mechanism of 3HB in promoting bone regeneration in vivo.
  • tissue volume (TV) showed no statistical difference among the groups, it also increased to varying degrees compared with the OVX group (Fig. 13e).
  • Tb.Sp trabecular spacing
  • Fig. 13g the trabecular spacing
  • the low-dose and middle-dose 3HB treatment groups showed significant MAR and BFR/BS (Fig. 14d, e).
  • the E2 treatment group showed the highest MAR and BFR/BS, while the sham group maintained a normal level of MAR and BFR/BS.
  • bioenergy active substance 3HB produced by the degradation of P34HB can increase the rate of bone mineralization and bone formation in vivo, reduce the loss of bone mass in osteoporotic rats, and participate in bone formation in the form of citric acid, improving osteoporosis symptom.
  • P34HB is an excellent candidate material for bone regeneration and shows great application potential in the field of bone tissue engineering regeneration.
  • the repair of bone injury is a pathological and physiological process in which stem/progenitor cells, vascular endothelial cells and other cells are proliferated, migrated and differentiated by bioenergy, which enables bone regeneration and angiogenesis to proceed in an orderly manner through a complex signal regulation network .
  • the 3D printed P34HB bioenergy scaffold was used as the medium
  • the human umbilical vein fusion cell (EA.hy926) was used as the cell model in vitro
  • the critical bone defect of the rat skull was used as the animal model in vivo to explore the promotion of vascularization by the P34HB bioenergy scaffold The role and function of bone regeneration.
  • EA.hy926 cells were seeded in a 6-well plate at a density of 5 ⁇ 10 4 /ml, and cultured in a cell culture incubator;
  • the skin of the surgical site was disinfected with tincture of iodine and 75% alcohol, and an incision of about 10 mm was made on the top of the head along the sagittal suture to expose the left parietal bone, and the periosteum was pushed away with a sterile cotton swab.
  • the high-speed dental drill removes the skull with a diameter of 5mm under the cooling of normal saline, and the dura mater cannot be damaged when removing the bone fragments. After giving different experimental treatments, the positions of the periosteum and head skin were restored, and the skin was sutured.
  • the rat skull critical bone defect model was randomly divided into three groups, 6 rats in each group, group A: no treatment at the bone defect (Empty); group B: PLLA stent grafting group (PLLA); group C: P34HB stent Transplantation group (P34HB). After stent transplantation, normal feed and water were given.
  • the two-photon microscopic imaging technology established by the research group was used to monitor and quantify the dynamic changes of angiogenesis and development in the process of bone defect repair in vivo, including the number of angiogenesis, vascular diameter, vascular density, and vascular density. changes in shape, etc.
  • 300 ⁇ l, 2 mg/ml FITC-Dextran was injected into the tail vein of rats.
  • the pixels of each imaging picture are 256 ⁇ 256, the size of the picture is 512 ⁇ m ⁇ 512 ⁇ m, and it takes 8s to acquire each image.
  • micro-CT was used to analyze bone regeneration at the bone defect site, including bone-related indicators such as bone mineral density (BMD), BV/TV, Tb.N, and Tb.Th, to evaluate the efficiency of P34HB scaffolds in repairing bone defects.
  • BMD bone mineral density
  • BV/TV BV/TV
  • Tb.N BV/TV
  • Tb.Th Tb.Th
  • Bone regeneration is a complex physiological process involving a variety of cells, and cell migration plays an important role in regulating tissue regeneration.
  • the migration of vascular endothelial cells will be beneficial to promote the regeneration and functional reconstruction of bone defect tissue.
  • the cell scratch test showed that, compared with the control group, 3HB at different concentrations showed a promoting effect on cell migration to a certain extent after treating EA.hy926 cells for 10 hours; when 3HB treated EA.hy926 cells for 20 hours, this The ability to promote migration was further improved, and 1.0mM 3HB showed a significant ability to promote the migration of EA.hy926 cells (Figure 17).
  • the above experiments show that the bioenergy active substance 3HB produced by the degradation of P34HB can promote the migration of cells in the process of bone regeneration and participate in the regeneration and repair of tissues.
  • Bone tissue regeneration is a complex process based on the interaction of osteogenesis and angiogenesis.
  • Angiogenesis is an essential part of bone formation, skeletal development, and osseointegration, and is a prerequisite for cell survival and function. Due to the destruction of the normal vascular network function at the defect site, the necessary growth factors and nutrients cannot be provided for tissue regeneration, thus hindering tissue regeneration and functional repair.
  • Bioscaffolds with vascularization function will be beneficial to tissue regeneration and functional reconstruction of bone defects. The occurrence and development of blood vessels is also an energy-consuming event. The generation of bioenergy (ATP) is beneficial to the vascularization of bioscaffolds.
  • ATP bioenergy
  • Ideal bone repair materials not only need to have osteoinductive regeneration ability, but also need to meet the requirements of early vascularization. Bone graft materials without the ability to vascularize can lead to graft necrosis due to ischemia.
  • the critical bone defect of rat skull was used as a model, and the coupled angiogenesis during bone regeneration was observed by high-resolution two-photon intravital microscopy.
  • P34HB bioenergy scaffolds can couple angiogenesis in the process of bone regeneration, promote vascularized bone formation to repair critical bone defects and achieve functional reconstruction.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Materials For Medical Uses (AREA)

Abstract

本发明公开了一种生物能量活性材料及其应用，具体公开一种生物能量活性材料，其为生物可降解聚合物；所述生物能量活性材料的降解产物为三羧酸循环和/或糖酵解途径中的代谢中间产物，或者为能够转换生成三羧酸循环和/或糖酵解途径中代谢中间产物的聚合物单体，或者为能够转换生成乙酰辅酶A的聚合物单体。该生物能量活性材料的降解产物经过三羧酸代谢循环或者糖酵解途径为组织细胞提供生物能量，解决传统生物可降解材料无法持续提高细胞中ATP的稳定性和相关生物质活性的问题，在骨组织再生领域尤其是在大段骨缺损修复方面表现出广阔的应用前景。

Description

一种生物能量活性材料及其应用 技术领域
本发明属于生物医学组织工程，具体涉及一种生物能量活性材料及其应用。
背景技术
在临床上，因创伤、感染、骨肿瘤切除等原因造成的各种类型骨缺损十分常见。全世界范围内每年进行骨移植患者的数量达到220万【J.Van der Stok,E.M.Van Lieshout,Y.El-Massoudi,G.H.Van Kralingen,P.Patka,Bone substitutes in the Netherlands-a systematic literature review,Acta Biomater 7(2)(2011)739-50.】。据美国骨科医师协会(AAOS)统计，在美国每年有630万人发生骨折，其中需要接受骨移植物的患者就有50万之多，每年仅在骨折治疗方面的费用支出高达2000亿美元【D.C.Lobb,B.R.DeGeorge,Jr.,A.B.Chhabra,Bone Graft Substitutes:Current Concepts and Future Expectations,J Hand Surg Am 44(6)(2019)497-505e2.】。我国每年骨损伤患者超过350万，每年新增病例达到数十万之多，创伤住院年增长率达7.2％，高居住院人数的第二位，骨缺损的高发生率使得骨移植物成为仅次于输血之后需求量最大的医疗耗材【G.H.Brundtland,A WHO Scientific Group on the Burden of Musculoskeletal Conditions at the Start of the New Millennium met in Geneva from 13to 15January 2000.,Who Tech Rep Ser 919(2003)1-218.】，给社会带来沉重的医疗负担。
自体骨的移植可以较好的进行骨修复，但是“供体有限”限制了其更好的应用。生物可降解高分子材料因具有良好的生物相容性和降解性而广泛用于骨组织工程研究。生物可降解高分子材料包括天然可生物降解聚合物(如胶原、壳聚糖)和合成生物可降解聚合物(如PLA、PLGA、PCL)，因具有良好的生物相容性和降解性，是目前常用的骨组织工程支架材料【F.Asghari,M.Samiei,K.Adibkia,A.Akbarzadeh,S.Davaran,Biodegradable and biocompatible polymers for tissue engineering application:a review,ArtifCells Nanomed Biotechnol 45(2)(2017)185-192.】。虽然可生物降解聚合物具有一定的骨缺损修复功能，但仍然存在降解速率不可控、力学性能较差、酸性降解产物引起炎症反应等系列自身难以克服的缺点【Y.X.Lai,Y.Li,H.J.Cao,J.Long,X.L.Wang,L.Li,C.R.Li,Q.Y.Jia,B.Teng,T.T.Tang,J.Peng,D.Eglin,M.Alini,D.W.Grijpma,G.Richards,L.Qin,Osteogenic magnesium incorporated into PLGA/TCP porous scaffoldby 3D printing for repairing challenging bone defect,Biomaterials 197(2019)207-219.】，阻碍了其在临床上的广泛应用。
骨再生过程是一个能量消耗的过程，细胞能量代谢对组织修复和再生起着至关重要的作 用。三磷酸腺苷(ATP)是细胞能量的主要来源，在许多生物过程中发挥作用，包括细胞的增殖、迁移及分化【I.Gadjanski,S.Yodmuang,K.Spiller,S.Bhumiratana,G.Vunjak-Novakovic,Supplementation of Exogenous Adenosine 5-Triphosphate Enhances Mechanical Properties of 3D Cell-Agarose Constructs for Cartilage Tissue Engineering,Tissue Eng Pt A 19(19-20)(2013)2188-2200.】。研究表明，生物能量作为一种潜在的治疗方法，已在体外模型或相对薄的浅表组织(如皮肤)再生方面取得了成功。到目前为止，用于复杂骨组织缺损修复的具有长期生物能量释放效应的3D支架鲜有报道。这在很大程度上是由于使用现有的生物支架材料无法持续提高细胞中ATP的稳定性和相关生物质活性，阻碍了生物能量活性材料在骨组织工程中的应用和发展。
发明内容
本发明为了解决现有技术中的不足和缺点，提出一种生物能量活性材料及其应用。具体方案如下：
本发明第一方面提供一种生物能量活性材料，所述生物能量活性材料为生物可降解聚合物；所述生物能量活性材料的降解产物为三羧酸循环和/或糖酵解途径中的代谢中间产物；
或者所述生物能量活性材料的降解产物为能够转换生成三羧酸循环和/或糖酵解途径中代谢中间产物的聚合物单体；
或者所述生物能量活性材料的降解产物为能够转换生成乙酰辅酶A的聚合物单体。
进一步地，本发明的上述技术方案中，所述三羧酸循环的代谢中间产物包括柠檬酸、顺乌头酸、异柠檬酸、草酰琥珀酸、a-酮戊二酸、琥珀酰辅酶A、琥珀酸、延胡索酸、苹果酸和三磷酸腺苷中的一种以上；
所述糖酵解途径的代谢中间产物包括葡萄糖-6-磷酸、果糖-6-磷酸、果糖-1,6-二磷酸、3-磷酸甘油醛、磷酸二羟丙酮、1,3-二磷酸甘油酸、3-磷酸甘油酸、2-磷酸甘油酸、磷酸烯醇式丙酮酸PEP和丙酮酸中的一种以上；
所述能够转换生成乙酰辅酶A的聚合物单体为3-羟基丁酸。3-羟基丁酸会在3-羟基丁酸脱氢酶(3-hydroxybutyrate dehydrogenase)的作用下生成乙酰乙酸，再和琥珀酰辅酶A(succinyl-CoA)在3-琥珀酰辅酶A转移酶(3-oxoacid CoA-transferase)酶的作用下合成乙酰乙酰辅酶A(Acetoacetyl-CoA)，乙酰乙酰辅酶A与一个辅酶A(CoA)反应，在乙酰辅酶A-酰基转移酶(acetyl-CoAC-acetyltransferases)生成两个乙酰辅酶A(acetyl-CoA)，从而进入三羧酸循环，为组织细胞提供生物能量。
优选地，本发明的上述技术方案中，所述生物能量活性材料为降解产物为3-羟基丁酸的聚羟基脂肪酸酯。
进一步地，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯(P34HB)、聚-3-羟基丁酸酯(PHB)、3-羟基丁酸-3-羟基戊酸共聚酯(PHBV)和3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)中的一种以上。
本发明第二方面提供所述的生物能量活性材料在骨组织再生及修复领域中的应用。
本发明第三方面提供所述的生物能量活性材料在制备骨组织修复多孔支架中的应用。
本发明第四方面提供一种骨组织修复多孔支架，由所述的生物能量活性材料制备得到。进一步地，骨组织修复多孔支架可以通过传统制备方法，也可以通过3D打印技术制备。
本发明第五方面提供一种骨组织修复3D多孔支架的制备方法，包括如下步骤：
(1)合成所述的生物能量活性材料；
(2)结合3D打印技术制备骨组织修复3D多孔支架。
进一步地，本发明的上述制备方法中，所述生物能量活性材料通过微生物法或化学合成法合成，也可以通过其他方式合成。例如，可以通过微生物嗜盐单胞菌发酵生产3-羟基丁酸-4-羟基丁酸共聚酯。
本发明第五方面提供上述制备方法制备的骨组织修复3D多孔支架。
本发明第六方面提供降解产物包括3-羟基丁酸的聚羟基脂肪酸酯作为兼具骨组织再生及血管生成功能的生物能量活性材料的应用，所述3-羟基丁酸通过三羧酸代谢循环并以柠檬酸形式参与骨形成，所述3-羟基丁酸诱导血管生成。
进一步地，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
本发明第七方面提供降解产物包括3-羟基丁酸的聚羟基脂肪酸酯在制备血管化骨再生材料中的应用。
进一步地，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
本发明第八方面提供降解产物包括3-羟基丁酸的聚羟基脂肪酸酯在制备大段骨缺损修复材料或临界型骨缺损修复材料中的应用。
进一步地，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
本发明第九方面提供一种血管化骨再生材料、大段骨缺损修复材料或临界型骨缺损修复材料，其特征在于，由降解产物包括3-羟基丁酸的聚羟基脂肪酸酯制备得到。
进一步地，所述材料为由降解产物包括3-羟基丁酸的聚羟基脂肪酸酯制备得到的多孔支架。
进一步地，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
本发明的有益效果：
1、本发明提供的生物能量活性材料的降解产物为三羧酸循环和/或糖酵解途径中的代谢中间产物，或者为能够转换生成三羧酸循环和/或糖酵解途径中代谢中间产物的聚合物单体，或者为能够转换生成乙酰辅酶A的聚合物单体，该生物能量活性材料的降解产物经过三羧酸代谢循环或者糖酵解途径为组织细胞提供生物能量，解决传统生物可降解材料无法持续提高细胞中ATP的稳定性和相关生物质活性的问题，在骨组织再生领域尤其是在大段骨缺损修复方面表现出广阔的应用前景。
2、本发明提供的生物能量活性材料3-羟基丁酸-4-羟基丁酸共聚酯(P34HB)是一类可降解高分子聚酯，具有独特的生物能量活性、机械力学特性、生物可降解性和生物相容性，其降解产生的主要产物3-羟基丁酸(3HB)是哺乳动物体内酮体的主要组成之一，不仅对机体无毒性作用，而且可作为一种能量物质促进细胞的黏附、增殖以及分化。此外，与聚乳酸相比，在相同条件下，3-羟基丁酸-4-羟基丁酸共聚酯材料可以维持较长的降解时间，根据组织修复的要求调整材料中3-羟基丁酸的比例可以实现6-12月较长时间的降解。3-羟基丁酸-4-羟基丁酸共聚酯材料能够解决传统生物可降解材料无法持续提高细胞中ATP的稳定性和相关生物质活性，以及酸性降解产物引起炎症反应的问题，是一种性能优异的具有生物能量活性兼具骨形成促进功能的骨修复支架活性材料。
3、本发明提供的骨组织修复多孔支架基于生物能量活性材料和3D打印技术，该多孔支架结构可控，且具有良好的生物相容性、生物可降解性和机械力学特性，支架植入体内后随着自身不断的降解，其降解产生的生物能量活性物质可以促进骨髓间充质干细胞(hBMSCs)细胞的增殖、分化及矿化，具有骨形成促进功能，是传统高分子支架材料所不具备的。
4、本发明进一步提供的骨组织修复3D多孔支架基于生物能量活性材料3-羟基丁酸-4-羟基丁酸共聚酯(P34HB)和3D打印技术制备，该多孔支架结构可控，且具有良好的生物 相容性、生物可降解性和机械力学特性，植入体内后随着自身不断的降解，降解产生的生物能量活性物质3-羟基丁酸(3HB)，经过三羧酸代谢循环不仅为组织细胞提供生物能量，促进骨髓间充质干细胞(hBMSCs)细胞的增殖、分化及矿化，而且产生的中间代谢物以柠檬酸盐的形式参与骨形成，有利骨修复过程中成骨和成血管功能，缩短了骨缺损修复时间，在骨组织再生及修复领域尤其是在大段骨缺损修复方面表现出巨大的应用前景。
5、本发明生物能量活性材料降解产生的生物能量活性物质3-羟基丁酸(3HB)，3HB不仅通过三羧酸代谢循环并以柠檬酸形式参与骨形成，而且能诱导血管形成，在制备血管化骨再生材料、大段骨缺损修复材料或临界型骨缺损修复材料领域，具备重要的应用前景。
附图说明
图1为3-羟基丁酸-4-羟基丁酸共聚酯的凝胶渗透色谱图；其中，A：标准曲线，B：3-羟基丁酸-4-羟基丁酸共聚酯；
图2为3D打印制备的多孔支架(A)及多孔支架横截面电镜扫描图(B)；
图3为支架降解产物3-羟基丁酸对人源骨髓间充质干细胞(hBMSCs)增值的影响结果；
图4为支架降解产物3-羟基丁酸对hBMSCs碱性磷酸酶表达活性的影响结果；
图5为支架降解产物3-羟基丁酸对hBMSCs胞外钙结节形成的影响结果；
图6为激光共聚拉曼光谱分析hBMSCs胞外磷灰石的形成情况；
图7为支架降解产物3-羟基丁酸对hBMSCs成骨分化相关基因表达的影响结果；
图8为支架降解产物3-羟基丁酸对细胞线粒体膜势能的影响结果；
图9为LC-MS/MS代谢流分析 13C标记3HB参与TCA循环中间代谢物形成；
图10为LC-MS/MS定量分析TCA循环中间代谢物；
图11为细胞上清柠檬酸含量分析；
图12为骨质疏松模型大鼠体重及子宫变化；
图13为Micro-CT分析3HB对骨质疏松模型骨量的影响；
图14为硝酸银染色(a)及双荧光标记分析骨形成(c-e)；
图15为体内分析3HB参与骨形成的方式(a-c)及血清中柠檬酸(d)和钙(e)的含量测定；
图16为建立大鼠颅骨临界性骨缺损模型；
图17为划痕实验分析3HB对EA.hy926细胞迁移的影响；
图18为体外分析3HB对EA.hy926细胞微血管网络形成的影响；
图19为高分辨双光子活体显微成像分析骨缺损部位血管生成(a,b)；
图20为术后12周micro-CT评估骨缺损部位新生骨组织再生情况。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
(1)将能合成3-羟基丁酸-4-羟基丁酸共聚酯的微生物嗜盐单胞菌在60MMG培养基中，37℃，400-800rpm条件下发酵培养72小时，72小时后收集菌体，置于70℃对菌体进行通风干燥，得到含有3-羟基丁酸-4-羟基丁酸共聚酯的干菌体粉末。
60MMG培养基组成为：葡萄糖30g/L，酵母提取物1g/L，硫酸铵0.25g/L，硫酸镁0.2g/L，磷酸氢二钠9.65g/L，磷酸二氢钾1.5g/L，微量元素I 10ml/L，微量元素II 1ml/L。
(2)用氯仿对干菌体粉末中的3-羟基丁酸-4-羟基丁酸共聚酯进行提取(1g干菌体粉末加入20ml氯仿)，搅拌均匀后装入高压反应釜中，在100℃条件下反应4小时。
(3)待高压反应釜冷却后，采用过滤或者抽滤的方法去掉细胞碎片，得到澄清氯仿溶液。
(4)在60℃条件下将氯仿溶液浓缩(按照100ml氯仿溶液浓缩到60ml的比例进行)，然后加入到15倍体积的预冷无水乙醇中，然后置于4℃冰箱中过夜，进行沉淀析出。
(5)过滤收集步骤(4)中的沉淀析出物，并将收集到的沉淀析出物置于40℃真空干燥箱中24h，待溶剂挥发完全后，即可得3-羟基丁酸-4-羟基丁酸共聚酯。
称取50mg 3-羟基丁酸-4-羟基丁酸共聚酯溶解于氯仿中，静置1小时形成均匀的溶液，然后取10ul进行凝胶渗透色谱，测试分子量。结果如图1所示。
分子量计算结果如下：
Figure PCTCN2022109458-appb-000001
实施例2
将实施例1中合成得到的3-羟基丁酸-4-羟基丁酸共聚酯材料装入熔融3D打印机(180℃)，进行骨组织修复多孔支架制备。
结果如图2所示，其中，图2-A为3D打印制备的多孔支架，图2-B为支架横截面电镜扫描图，多孔支架的孔径大约在350-400μm。
实验例1
将实施例2中3D多孔支架浸泡于磷酸盐缓冲液8周，收集降解产物。在磷酸盐缓冲液中，3-羟基丁酸-4-羟基丁酸共聚酯的降解产物主要是3-羟基丁酸(3HB)。对收集到的降解产物进行浓度测定，然后进行体外实验。
磷酸盐缓冲液的组成为：氯化钠7.9g/L，氯化钾0.2g/L，磷酸二氢钾0.24g/L。
(1)取生长状态良好的人源骨髓间充质干细胞(hBMSCs)，按照2×10 4细胞密度接种于48孔板，4小时后向上述细胞中加入不同浓度的3-羟基丁酸(0μM，10μM，40μM，80μM，160μM，320μM)，在第1，5，7天用CCK-8分别测定细胞增殖情况。同时以乳酸(LA)作为对照组。
结果如图3所示。图3为支架降解产物3-羟基丁酸对人源骨髓间充质干细胞(hBMSCs)增值的影响结果，与对照组(LA)相比，在第5天，第7天3HB刺激后显著促进了骨髓间充质干细胞的增殖，表明支架降解产物能够促进hBMSCs增值。
(2)将人源骨髓间充质干细胞(hBMSCs)，按照1×10 5细胞密度接种于6孔板，培养12小时后，向上述细胞中加入含有不同浓度的3-羟基丁酸(0μM，40μM，160μM，320μM)的成骨诱导分化液，在第7，14天分别检测碱性磷酸酶的表达情况。根据碱性磷酸酶活性检测试剂盒的使用说明进行碱性磷酸酶表达的检测。
成骨诱导分化液的组成：低糖DMEM培养基+10％胎牛血清+2mM L-谷氨酰胺+100U/mL青霉素+100μg/mL链霉素+100nM地塞米松+0.2mML-抗坏血酸+10mMβ-甘油磷酸钠。
结果如图4所示。图4为支架降解产物3-羟基丁酸对hBMSCs碱性磷酸酶表达活性的影响结果，结果表明支架降解产物能够促进hBMSCs碱性磷酸酶表达。且与对照组(LA)相比，3HB刺激骨髓间充质干细胞诱导分化14天后，显著增强了碱性磷酸酶的表达，而对照组则表现出抑制作用。
(3)将人源骨髓间充质干细胞(hBMSCs)，按照1×10 5细胞密度接种于6孔板，培养12小时后，向上述细胞中加入含有不同浓度的3-羟基丁酸(0μM，40μM，160μM，320μM)的成骨诱导分化液，在第10，14天分别用茜素红然检测胞外钙结节的形成。
结果如图5所示。图5为支架降解产物3-羟基丁酸对hBMSCs胞外钙结节形成的影响结果，与对照组(LA)相比，3HB刺激骨髓间充质干细胞诱导分化10，14天后显著增强了胞外钙结节的沉积，表明3-羟基丁酸促进了骨髓间充质干细胞成骨分化标志物钙结节的形成。
(4)将人源骨髓间充质干细胞(hBMSCs)，按照1×10 5细胞密度接种于6孔板，培养12小时后，向上述细胞中加入含有不同浓度的3-羟基丁酸的成骨诱导分化液(0μM，40μM，160μM，320μM)，进行21天诱导分化，诱导21天后，利用激光共聚焦拉曼光谱检测胞外磷灰石的形成。
结果如图6所示。图6为激光共聚拉曼光谱分析hBMSCs胞外磷灰石的形成情况，骨髓间充质干细胞诱导分化21天后，胞外有大量的磷灰石形成且其含量随着3HB浓度的变大而增多，表明3-羟基丁酸能够促进骨髓间充质干细胞成骨分化磷灰石的形成。
(5)将人源骨髓间充质干细胞(hBMSCs)，按照1×10 5细胞密度接种于6孔板，培养12小时后，向上述细胞中加入含有不同浓度的3-羟基丁酸(0μM，40μM，160μM，320μM) 的成骨诱导分化液，进行7天诱导分化，诱导7天后，利用实时荧光定量PCR技术检测成骨分化相关基因(Runx相关转录因子2基因、骨钙素基因、骨保护素基因)表达情况。
结果如图7所示。图7为支架降解产物3-羟基丁酸对hBMSCs成骨分化相关基因表达的影响结果，与对照组(LA)相比，3HB刺激骨髓间充质干细胞诱导分化7天后促进了成骨分化相关基因的表达，而对照组则在一定程度上抑制了基因的表达，表明支架降解产物3-羟基丁酸能够促进hBMSCs成骨分化相关基因表达。
(6)将人源骨髓间充质干细胞(hBMSCs)，按照1×10 4细胞密度接种于6孔板，培养12小时后，实验分为阳性对照组，阴性对照组以及实验组，阳性对照组用高糖培养基(HG)处理细胞6小时；阴性对照组用含有氧化磷酸化解偶联剂(CCCP)的高糖培养基处理细胞6小时；实验组用含有不同浓度(40μM，160μM，320μM)的3-羟基丁酸和乳酸(LA)的无糖培养基(GF)处理细胞6小时。
结果如图8所示。图8为支架降解产物3-羟基丁酸对细胞线粒体膜势能的影响结果，结果表明支架降解产物3-羟基丁酸能够为细胞提供ATP及维持线粒体膜势能(ΔΨm)，且与LA组相比，3HB能为细胞提供较多的生物能量(ATP)并提高细胞膜势能。
实验例2
本实验例以 13C标记3HB示踪实验，解析3HB的代谢流向及参与骨形成的方式，以未用 13C标记3HB处理为对照组。将人源骨髓间充质干细胞(hBMSCs)接种于培养板中，向上述细胞中加入含有1mM的 13C-3HB的成骨诱导分化液，在第14天进行代谢组学分析(LC-MS/MS)。
当用1mM的 13C-3HB刺激诱导hBMSCs成骨分化14天后，代谢组学分析(LC-MS/MS)发现，在TCA循环中检测到多个中间代谢物(Citrate-柠檬酸、succinate-琥珀酸、fumarate-延胡索酸、malate-苹果酸)含有来源于 13C标记3HB的碳原子，而在对照组(未用 13C标记3HB处理)并未发现含有来源于 13C标记3HB的碳原子(图9)，由此证明P34HB支架降解产生的生物能量活性物质3HB能够通过TCA循环参与代谢。
进一步，通过对胞内TCA循环代谢物总含量相对定量，结果显示，与对照组相比，经过1mM  13C标记3HB处理后，胞内的Citrate、isocitrate、fumarate、malate等多种中间代谢物含量显著低于未处理组(图10)这一结果似乎与 13C标记3HB参与TCA代谢循环提高中间代谢物含量相矛盾；也与上述3HB能够上调氧化磷酸化功能，促进hBMSCs成骨分化，增强胞外钙沉积形成相矛盾。
进一步，分析细胞成骨诱导分化上清，与对照组相比，发现 13C标记3HB处理组在上清中检测到更多柠檬酸的存在(图11)这一结果解释了生物能量活性物质3HB虽然参与TCA循环，却并没有在胞内提高中间代谢物含量。原因是因为 13C标记3HB处理后，通过TCA形成的柠檬酸从胞内转移到胞外参与钙基质沉积，从而引起胞内柠檬酸含量的降低。然而，柠檬酸是TCA循环中的关键中间体，为了维持基本的细胞代谢活动，必须要有TCA循环中的其他物质例如琥珀酸、延胡索酸等转化而来，从而降低了整个TCA循环中间代谢物含量积累。
以上研究结果证明，生物能量活性物质3HB通过TCA循环以中间代谢物柠檬酸的形式参与体外生物矿化形成。
实验例3
本实验例以3HB为媒介，以去势大鼠骨质疏松模型为研究对象，阐述3HB在体内促进骨再生的机理。
结果显示，去势大鼠体重增加快于sham组，而在E2组其体重增加与sham组体重增加相当(图12a)，这是因为去势后引起体内雌激素失衡不能发挥有效的调控功能，而引起脂肪组织增加所致。在连续灌胃3个月后，对每组实验大鼠的子宫进行了取材。发现OVX组和3HB灌胃组大鼠的子宫呈现出明显的萎缩状态，给予E2组的子宫萎缩有所改善，而sham组大鼠子宫则呈现出正常形态(图12b)。此外，OVX组和3HB灌胃组大鼠的子宫重量也显著低于sham组和E2组(图12c)。以上实验数据说明去势大鼠骨质疏松模型建立成功，为后续研究3HB调控骨再生奠定了基础。
小动物micro-CT扫描发现，在给予去势大鼠连续3个月3HB灌胃后，不同剂量的3HB在一定程度上表现出减缓去势大鼠骨量的丢失(图13a)。与OVX组相比，当给予去势大鼠低剂量(30mg/kg/d)和中剂量(150mg/kg/d)3HB时，则显著提高了骨密度(BMD)，骨体积分数线(BV/TV)以及骨体积(BV)(图13b-d)。然而在中剂量3HB灌胃组中骨小梁数量(Tb.N)表现最多，显著多于OVX组(图13f)。虽然组织体积(TV)在各组之间没有表现出统计学差异，但与OVX组相比，也有不同程度增加(图13e)。此外，骨小梁间距(Tb.Sp)在给予不同剂量的3HB后也发生不同程度降低(图13g)。
胫骨组织切片硝酸银染色发现，骨小梁数量在给予不同剂量3HB后多于OVX组，而不同剂量3HB组之间骨小梁数量并没有明显差异性(图14a)，而给予E2治疗后，其骨小梁数量得到很大程度的恢复，但是其骨小梁数量还是低于假手术组，这一现象也可以从micro-CT 定量分析结果看出(图14f)。随后在体内进行了钙黄绿素/二甲酚橙双荧光标记来分析骨组织再生速率和矿化能力(图14b)。研究发现，在给予不同剂量3HB组，钙黄绿素/二甲酚橙双荧光标记周长百分数均高于OVX组，且中剂量和高剂量3HB的钙黄绿素/二甲酚橙双荧光标记周长百分数与OVX组相比具有统计学差异；然而不同剂量3HB组、sham组和E2之间钙黄绿素/二甲酚橙双荧光标记周长百分数并无差异性(图14c)。另外，在骨矿化沉积率(MAR)和骨形成率(BFR/BS)也表现出类似的结果。与OVX组相比，低剂量和中剂量3HB处理组则表现出显著的MAR和BFR/BS(图14d,e)。然而，E2治疗组则表现出最高的MAR和BFR/BS，sham组的MAR和BFR/BS则维持在一个正常的水平。
13C标记3HB示踪实验结果表明，在低剂量 13C标记3HB灌胃组，LC-MS/MS检测到了骨组织中存在 13C标记的柠檬酸和α-酮戊二酸(图15a,b)。同时，测定了骨组织中的柠檬酸含量，发现3HB提高了骨组织中柠檬酸的形成(图15c)。此外，血清学检测发现生物能量活性物质3HB能够提高血清中柠檬酸的含量(图15d)；然而并没有发现血清中钙含量发生显著改变(图15e)。因此，去势大鼠骨量减少有可能并不是血清中的钙含量降低引起的，而有可能是骨中柠檬酸含量的减少而不能有效结合钙磷而造成的。
以上实验结果表明，P34HB降解产生的生物能量活性物质3HB能够提高体内骨矿化率和骨形成率，降低骨质疏松大鼠骨量的丢失，并柠檬酸的形式参与骨形成，改善骨质疏松症状。
实验例4
上述实验例表明P34HB降解产生的生物能量活性物质3HB能够促进hBMSCs增殖，介导线粒体氧化磷酸化促进成骨分化，并以TCA循环中间代谢物柠檬酸的形式参与骨再生。因此，P34HB是一种优异的骨再生候选材料，在骨组织工程再生领域展现出巨大的应用潜力。骨损伤的修复是由生物能量驱动干/祖细胞、血管内皮细胞等多种细胞发生增殖、迁移及分化的病理和生理过程，其通过复杂的信号调控网络使得骨再生和血管生成得以有序进行。欲使大段骨缺损得以尽快再生和功能重建，必须尽早在移植物与周围组织之间建立起完善的血管网络，为骨再生提供所需要的氧气和营养物质。本实验例以3D打印P34HB生物能量支架为媒介，体外以人脐静脉融合细胞(EA.hy926)为细胞模型，体内以大鼠颅骨临界性骨缺损为动物模型，探究P34HB生物能量支架促进血管化骨再生的作用与功能。
一、实验方法与步骤
(一)P34HB降解产生的生物能量活性物质3HB对EA.hy926细胞迁移的影响
(1)EA.hy926细胞按照5×10 4/ml密度接种于6孔板中，放入细胞培养箱进行培养；
(2)当细胞生长融合度接近100％，移除培养基并用PBS漂洗3次；
(3)用200μl枪头沿着板孔中央划线，PBS漂洗3次，洗去划掉的细胞；
(4)用含有不同浓度3HB的无糖培养基(含1％FBS)分别处理细胞10h和20h；
(5)在设定的时间点，显微镜观察细胞的迁移情况，并拍照；
(6)用ImagJ软件对细胞迁移进行定量分析。
(二)P34HB降解产生的生物能量活性物质3HB对EA.hy926细胞微血管形成的影响
(1)事先将基质胶(Matrigel matrix)放在4℃冰箱过夜解冻，并准备预冷的枪头和血管生成小室；
(2)向血管生成小室中加入解冻的基质胶，按照每孔10μl体积，放入细胞培养箱30min；
(3)制备密度为3×10 5/ml的EA.hy926细胞悬液，按照50μl体积/孔加入到血管生成小室；
(4)在设定的时间点内(6h),用钙黄绿素对形成的微血管网络进行染色，并拍照；
(5)用ImagJ软件对形成的微血管网络进行定量分析。
(三)建立大鼠颅骨临界性骨缺损模型
本实验使用雄性SD大鼠(10周龄)，购买于北京维通利华实验动物技术有限公司。所有实验大鼠饲养于中国科学院深圳先进技术研究院动物中心(SPF)，本论文动物实验经过中国科学院深圳先进技术研究院伦理审查委员会批准(SIAT-IACUC-201010-KYC-ZP-A1416)。SD大鼠颅骨临界性骨缺损建立过程(图16)及步骤如下：手术均在3.5％异氟烷-100％氧气呼吸麻醉下进行，同时有保温及防脱水处理，包括呼吸麻醉管道中加蒸馏水及术前皮下推注生理盐水(500μl)。动物麻醉后，用碘酊及75％酒精对手术部位皮肤进行消毒处理，沿矢状缝在头顶皮肤作约10mm切口以暴露左侧顶骨，用无菌棉签将骨膜推开，用安装有圆锯的高速牙医钻在生理盐水散热下移除直径5mm的颅骨，移除骨片时不能损伤硬脑膜。给予不同实验处置后，恢复颅骨膜、头部皮肤位置，缝合皮肤。
(四)动物分组及支架移植
大鼠颅骨临界性骨缺损模型随机分为三组，每组6只，A组：骨缺损处不做任何处理组(Empty)；B组：PLLA支架移植组(PLLA)；C组：P34HB支架移植组(P34HB)。支架移植后，给予正常的饲料和水。
(五)高分辨双光子活体显微成像检测骨缺损部位血管生成
术后12周，利用课题组前期建立的双光子显微成像技术，活体监测和定量在骨缺损修复过程中血管发生、发展的动态变化过程，包括血管生成数目、血管管径、血管密度、血管形态等的变化情况。实验中为了进一步提高颅骨内血管的荧光信号对比度，给大鼠尾静脉注射300μl，2mg/ml的FITC-Dextran。每张成像图片的像素为256×256，图片的大小为512μm×512μm，获取每张图像耗时为8s。首先对准焦距找到清晰成像部位，先进行X-Y平面成像，然后适度调节焦距，沿Z轴方向进一步扫描成像，扫描深度为200μm。评价P34HB支架在骨再生过程中偶联血管生成的能力。
(六)Micro-CT分析P34HB支架对缺损部位骨再生的影响
术后12周，利用micro-CT分析骨缺损部位骨再生情况，包括骨密度(BMD)、BV/TV、Tb.N、Tb.Th等骨相关指标，评估P34HB支架修复骨缺损的效率。
(七)统计学分析
利用GraphPad Prism 8.0软件对所测得数据进行统计分析。数据采用单因素方差分析(one-wayANOVA)或者T检验进行分析。结果用均数±标准差(SD)表示。*P<0.05代表在统计学上具有差异性。
二、结果与讨论
(一)P34HB降解产生的生物能量活性物质3HB促进EA.hy926细胞迁移
骨再生是一个涉及多种细胞参与的复杂生理过程，细胞迁移对组织再生具有重要的调控作用。骨再生过程中，血管内皮细胞的迁移将有利于促进骨缺损组织再生与功能重建。细胞划痕实验表明，与对照组相比，不同浓度的3HB在处理EA.hy926细胞10h后，均在一定程度上表现出细胞迁移的促进作用；当3HB处理EA.hy926细胞20h后，这种促进迁移的能力进一步提高，其中1.0mM 3HB则表现出显著的促进EA.hy926细胞迁移能力(图17)。以上实验表明，P34HB降解产生的生物能量活性物质3HB能够促进骨再生过程中细胞的迁移，参与组织的再生与修复。
(二)P34HB降解产生的生物能量活性物质3HB促进EA.hy926细胞微血管网络形成
骨组织再生是一种基于成骨和血管生成相互作用的复杂过程。血管形成是骨形成、骨骼发育和骨整合过程中必不可少的一部分，是细胞存活和发挥功能的前提。由于缺损部位正常血管网络功能受到破坏，无法为组织再生提供必需的生长因子和营养物质，从而阻碍了组织再生与功能修复。具有血管化功能的生物支架将有利于骨缺损组织再生与功能重建。血管的 发生与发展过程同样是一个能量消耗的事件。生物能量(ATP)的产生有利于生物支架发生血管化。体外微血管形成实验表明，与对照组相比，P34HB降解产生的生物能量活性物质3HB处理EA.hy926细胞6h后，观察到较多的微血管网络形成(图18a)。ImageJ进一步定量分析发现，EA.hy926细胞经过生物能量活性物质3HB处理后，显著提高了微血管分支点形成数量(Branch points)和微血管形成连接程度(Junctions)(图18b,d)。虽然在微血管网孔形成数量(Number ofmeshes)方面，0.5mM 3HB处理后，并未表现出显著性差异，但是在一定程度上提高了网孔形成数量(图18c)。以上实验结果表明，34HB降解产生的生物能量活性物质3HB能够促进血管内皮细胞的迁移和微血管网络形成，初步表明P34HB支架材料具有血管化潜能。
(三)P34HB生物能量支架促进血管化骨形成修复大鼠颅骨临界性骨缺损
理想的骨修复材料不仅需要具备骨诱导再生能力，而且还需要满足能够早期血管化要求。没有血管化能力的骨移植材料，会导致移植物因缺血而发生坏死。上述研究表明，P34HB生物能量支架具有血管化功能。为了探究P34HB生物能量支架的血管化潜能，以大鼠颅骨临界性骨缺损为模型，利用高分辨双光子活体显微镜观察骨再生过程中偶联血管生成情况。术后12周，双光子活体显微成像观察发现，支架移植组都表现出较多的血管生成，然而P34HB支架移植组血管密度明显多于PLLA支架移植组；Empty组几乎没有血管生成(图19a)。ImageJ定量分析进一步支持了上述所观察到的现象(图19b),表明P34HB能够促进骨再生过程中血管再生。
双光子活体显微成像表明，P34HB生物能量支架能够促进骨缺损部位血管生成。随后，利用小动物micro-CT分析支架移植后骨再生情况。Micro-CT观察发现，移植P34HB支架的大鼠颅骨骨缺损部位被大量新生骨组织所填充，而空白对照组(Empty)和PLLA支架移植组的骨缺损部位仅观察到少量的新生骨组织出现。Micro-CT定量分析发现，P34HB支架移植组其骨缺损部位的BMD、BV/TV、TV、BV、Tb.Th值显著高于空白对照组和PLLA支架移植组，而空白对照组和PLLA支架移植组两者之间并无显著性差异。然而，Tb.N在空白对照组、PLLA支架移植组以及P34HB支架移植组均无显著差异。(图20)
以上实验结果表明，P34HB生物能量支架能够在骨再生过程中偶联血管生成，促进血管化骨形成修复临界性骨缺损并实现功能重建。

Claims (19)

  1. 一种生物能量活性材料，其特征在于，所述生物能量活性材料为生物可降解聚合物；所述生物能量活性材料的降解产物为三羧酸循环和/或糖酵解途径中的代谢中间产物；
    或者所述生物能量活性材料的降解产物为能够转换生成三羧酸循环和/或糖酵解途径中代谢中间产物的聚合物单体；
    或者所述生物能量活性材料的降解产物为能够转换生成乙酰辅酶A的聚合物单体。
  2. 根据权利要求1所述的生物能量活性材料，其特征在于，所述三羧酸循环的代谢中间产物包括柠檬酸、顺乌头酸、异柠檬酸、草酰琥珀酸、a-酮戊二酸、琥珀酰辅酶A、琥珀酸、延胡索酸、苹果酸和三磷酸腺苷中的一种以上；
    所述糖酵解途径的代谢中间产物包括葡萄糖-6-磷酸、果糖-6-磷酸、果糖-1,6-二磷酸、3-磷酸甘油醛、磷酸二羟丙酮、1,3-二磷酸甘油酸、3-磷酸甘油酸、2-磷酸甘油酸、磷酸烯醇式丙酮酸PEP和丙酮酸中的一种以上；
    所述能够转换生成乙酰辅酶A的聚合物单体为3-羟基丁酸。
  3. 根据权利要求1所述的生物能量活性材料，其特征在于，所述生物能量活性材料为降解产物为3-羟基丁酸的聚羟基脂肪酸酯。
  4. 根据权利要求3所述的生物能量活性材料，其特征在于，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
  5. 权利要求1-4任一项所述的生物能量活性材料在骨组织再生及修复领域中的应用。
  6. 权利要求1-4任一项所述的生物能量活性材料在制备骨组织修复多孔支架中的应用。
  7. 一种骨组织修复多孔支架，由权利要求1-4任一项所述的生物能量活性材料制备得到。
  8. 一种骨组织修复3D多孔支架的制备方法，其特征在于，包括如下步骤：
    (1)合成权利要求1-4任一项所述的生物能量活性材料；
    (2)结合3D打印技术制备骨组织修复3D多孔支架。
  9. 根据权利要求8所述的制备方法，其特征在于，所述生物能量活性材料通过微生物法或化学合成法合成。
  10. 权利要求8或9所述制备方法制备的骨组织修复3D多孔支架。
  11. 降解产物包括3-羟基丁酸的聚羟基脂肪酸酯作为兼具骨组织再生及血管生成功能的生物能量活性材料的应用，其特征在于，所述3-羟基丁酸通过三羧酸代谢循环并以柠檬酸形式参与骨形成，所述3-羟基丁酸诱导血管生成。
  12. 根据权利要求11所述的应用，其特征在于，所述聚羟基脂肪酸酯包括3-羟基丁酸-4- 羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
  13. 降解产物包括3-羟基丁酸的聚羟基脂肪酸酯在制备血管化骨再生材料中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
  15. 降解产物包括3-羟基丁酸的聚羟基脂肪酸酯在制备大段骨缺损修复材料或临界型骨缺损修复材料中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
  17. 一种血管化骨再生材料、大段骨缺损修复材料或临界型骨缺损修复材料，其特征在于，由降解产物包括3-羟基丁酸的聚羟基脂肪酸酯制备得到。
  18. 根据权利要求17所述的材料，其特征在于，所述材料为由降解产物包括3-羟基丁酸的聚羟基脂肪酸酯制备得到的多孔支架。
  19. 根据权利要求17所述的材料，其特征在于，所述聚羟基脂肪酸酯包括3-羟基丁酸-4-羟基丁酸共聚酯、聚-3-羟基丁酸酯、3-羟基丁酸-3-羟基戊酸共聚酯和3-羟基丁酸-3-羟基己酸共聚酯中的一种以上。
PCT/CN2022/109458 2021-07-30 2022-08-01 一种生物能量活性材料及其应用 Ceased WO2023006113A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US18/574,727 US20240299620A1 (en) 2021-07-30 2022-08-01 Bioenergetic-active material and use thereof
CN202280004789.0A CN115884799A (zh) 2021-07-30 2022-08-01 一种生物能量活性材料及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110875054.2 2021-07-30
CN202110875054.2A CN113546213A (zh) 2021-07-30 2021-07-30 一种生物能量活性材料及其应用

Publications (1)

Publication Number Publication Date
WO2023006113A1 true WO2023006113A1 (zh) 2023-02-02

Family

ID=78133463

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/109458 Ceased WO2023006113A1 (zh) 2021-07-30 2022-08-01 一种生物能量活性材料及其应用

Country Status (3)

Country Link
US (1) US20240299620A1 (zh)
CN (2) CN113546213A (zh)
WO (1) WO2023006113A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113546213A (zh) * 2021-07-30 2021-10-26 深圳先进技术研究院 一种生物能量活性材料及其应用
CN115444985B (zh) * 2022-09-19 2023-09-26 中鼎凯瑞科技成都有限公司 三磷酸腺苷基高能骨修复材料及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102936407A (zh) * 2012-10-19 2013-02-20 华中科技大学 一种基于三羧酸循环的能量生物材料及其制备方法
CN109676915A (zh) * 2019-02-15 2019-04-26 复旦大学 一种车轮状多孔支架及其制备方法与应用
RU2708396C1 (ru) * 2018-12-19 2019-12-06 Федеральное государственное бюджетное учреждение науки Институт высокомолекулярных соединений Российской академии наук Биосовместимый биоразлагаемый остеокондуктивный полимерный композиционный материал для регенерации костной ткани
CN112773940A (zh) * 2021-01-26 2021-05-11 深圳市创想三维科技有限公司 一种3d打印用人工骨支架材料及其制备方法和应用
CN113546213A (zh) * 2021-07-30 2021-10-26 深圳先进技术研究院 一种生物能量活性材料及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103785059B (zh) * 2014-01-03 2015-12-02 朱小龙 一种骨修复再生材料及其制备方法
US10113160B2 (en) * 2014-01-30 2018-10-30 Easel Biotechnologies, Llc Carbon dioxide fixation via bypassing feedback regulation
CN105771000B (zh) * 2016-02-26 2019-07-09 华中科技大学 一种提供能量的骨修复可降解多孔支架及制备方法、应用
CN107375934B (zh) * 2017-08-10 2020-05-22 浙江大学 含有果糖-1，6-二磷酸的组合物在制备抗肿瘤药物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102936407A (zh) * 2012-10-19 2013-02-20 华中科技大学 一种基于三羧酸循环的能量生物材料及其制备方法
RU2708396C1 (ru) * 2018-12-19 2019-12-06 Федеральное государственное бюджетное учреждение науки Институт высокомолекулярных соединений Российской академии наук Биосовместимый биоразлагаемый остеокондуктивный полимерный композиционный материал для регенерации костной ткани
CN109676915A (zh) * 2019-02-15 2019-04-26 复旦大学 一种车轮状多孔支架及其制备方法与应用
CN112773940A (zh) * 2021-01-26 2021-05-11 深圳市创想三维科技有限公司 一种3d打印用人工骨支架材料及其制备方法和应用
CN113546213A (zh) * 2021-07-30 2021-10-26 深圳先进技术研究院 一种生物能量活性材料及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FENG JING, SHI QINGSHAN, OUYANG YOUSHENG, CHEN YIBEN: "Research Progress on Biodegradation of Several Polymer Materials", SULIAO KEJI - PLASTICS SCIENCE AND TECHNOLOGY, DALIAN SULIAO YANJIUSUO, DALIAN, CN, vol. 39, no. 2, 28 February 2011 (2011-02-28), CN , pages 94 - 99, XP093028413, ISSN: 1005-3360, DOI: 10.15925/j.cnki.issn1005-3360.2011.02.012 *

Also Published As

Publication number Publication date
US20240299620A1 (en) 2024-09-12
CN113546213A (zh) 2021-10-26
CN115884799A (zh) 2023-03-31

Similar Documents

Publication Publication Date Title
Zhang et al. Construction of vascularized tissue-engineered bone with polylysine-modified coral hydroxyapatite and a double cell-sheet complex to repair a large radius bone defect in rabbits
Yoshimi et al. Self-assembling peptide nanofiber scaffolds, platelet-rich plasma, and mesenchymal stem cells for injectable bone regeneration with tissue engineering
Fan et al. A prevascularized nerve conduit based on a stem cell sheet effectively promotes the repair of transected spinal cord injury
Li et al. Graphdiyne-loaded polycaprolactone nanofiber scaffold for peripheral nerve regeneration
Sun et al. Total flavonoids of rhizoma drynariae ameliorates bone formation and mineralization in BMP-Smad signaling pathway induced large tibial defect rats
Mahdavi et al. Bioactive glass ceramic nanoparticles-coated poly (l-lactic acid) scaffold improved osteogenic differentiation of adipose stem cells in equine
WO2023006113A1 (zh) 一种生物能量活性材料及其应用
Wang et al. A study on in vitro and in vivo bioactivity of silk fibroin/nano-hydroxyapatite/graphene oxide composite scaffolds with directional channels
Xiong et al. DLP fabrication of HA scaffold with customized porous structures to regulate immune microenvironment and macrophage polarization for enhancing bone regeneration
Ortiz et al. Potential of fibrin glue and mesenchymal stem cells (MSCs) to regenerate nerve injuries: a systematic review
Fu et al. Enhancing the osteogenic differentiation of aligned electrospun poly (L-lactic acid) nanofiber scaffolds by incorporation of bioactive calcium silicate nanowires
Yang et al. Construction of microunits by adipose-derived mesenchymal stem cells laden with porous microcryogels for repairing an acute achilles tendon rupture in a rat model
Zang et al. Study of osteogenic differentiation of human adipose-derived stem cells (HASCs) on bacterial cellulose
Liu et al. High-strength double-network silk fibroin based hydrogel loaded with Icariin and BMSCs to inhibit osteoclasts and promote osteogenic differentiation to enhance bone repair
Sun et al. Exploring the mechanism of total flavonoids of drynariae rhizoma to improve large bone defects by network pharmacology and experimental assessment
Cao et al. Cascaded controlled delivering growth factors to build vascularized and osteogenic microenvironment for bone regeneration
Blando et al. Can a scaffold enriched with mesenchymal stem cells be a good treatment for spinal cord injury?
Brooker et al. The future of facial fat grafting
Zhang et al. The bio-functionalized membrane loaded with Ta/WH nanoparticles promote bone regeneration through neurovascular coupling
Abazari et al. Poly (glycerol sebacate) and polyhydroxybutyrate electrospun nanocomposite facilitates osteogenic differentiation of mesenchymal stem cells
Wood et al. Current status of tissue engineering in urology
Al-Bishari et al. JK-2 loaded electrospun membrane for promoting bone regeneration
Chang et al. Engineering of MSCs sheet for the prevention of myocardial ischemia and for left ventricle remodeling
Vaegler et al. Tissue engineering in urothelium regeneration
Bahrami et al. Combined application of human amniotic membrane mesenchymal stem cells and a modified PGS-co-PCL film in an experimental model of myocardial ischemia–reperfusion injury

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22848717

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22848717

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 22848717

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 19.09.2024)

122 Ep: pct application non-entry in european phase

Ref document number: 22848717

Country of ref document: EP

Kind code of ref document: A1