CN102936407B - A kind of energy biological material based on tricarboxylic acid cycle and preparation method thereof - Google Patents

Abstract

An energy biomaterial based on a tricarboxylic acid cycle and a preparation method thereof. The material is a porous material based on the tricarboxylic acid cycle pathway, which can produce a series of bioactive molecules, and then provide energy for tissue cells through the tricarboxylic acid cycle pathway. The porous scaffold further prepared from the energy biomaterial obtained by the method of the present invention not only has good biocompatibility, but also exerts its tissue repair function, and as its own degradation process continues, the degradation products continue to enter Cells, participate in the tricarboxylic acid cycle, accelerate the generation of ATP in the cells, and play a role in the cells in the form of energy supply, thereby promoting cell growth, proliferation and tissue repair. In addition, the molecular structure characteristics of the material endow it with reactive side chain groups, which provides a basis for further surface modification and immobilization of active molecules.

Description

A kind of energy biomaterial based on tricarboxylic acid cycle and its preparation method

technical field

The invention belongs to the field of biomedical materials, and relates to a preparation method of a porous tissue engineering scaffold, more specifically, an energy biomaterial based on a tricarboxylic acid cycle and applied to rapid repair of bone tissue and a preparation method thereof.

Background technique

Bone defect is a common clinical disease, because tumors, trauma, surgical debridement of osteomyelitis, and various congenital diseases are the main causes of bone defects. Bone transplantation is the most traditional and effective method for repairing bone defects clinically, but the source of autologous bone graft materials is limited, while allogeneic bone grafts have the defects of poor immunogenicity and osteoinductivity. Therefore, using the basic principles of regenerative medicine and tissue engineering to develop new bone substitute materials is the development trend of bone defect repair.

In the field of tissue construction and regenerative medicine, biomaterials play an irreplaceable role. For a long time, a large number of literatures have conducted extensive research on various inorganic mineral materials, organic polymer materials, metals and their composite materials, and have developed from the initial biologically inert materials to biologically active and degradable materials, and then to cell regulatory factors and gene activity. Materials (Linda G. Griffith, Gail Naughton. Biomimetic materials for tissue engineering. Advanced Drug Delivery Review, 2008, 2: 184-198.), but so far, no application of this type of biomaterials involving energy (ATP) has been seen Based on the reports on tissue reconstruction and regeneration, it is a new concept to design and prepare energy bioremediation materials. It is expected that such materials can also function as energy supply or promote endogenous ATP generation while playing the supporting role of cell tissue scaffolding. . Based on this new idea, combined with the important clinical needs of early rapid regeneration of bone repair, this topic proposes the construction of energy biomaterials based on the ATP pathway.

ATP, also known as adenosine triphosphate, is an energy substance in living organisms. The phosphate group at the end of ATP is unstable and can be easily removed by hydrolysis, while releasing a lot of heat. Studies have shown that ATP can be divided into intracellular ATP (i.e. endogenous ATP) and extracellular ATP (exogenous ATP) in terms of sources, and the two play the role of energy supply, signal transduction and protein activation respectively. In cells, almost all forms of energy are directly converted from ATP and utilized by cells, including mechanical energy, thermal energy, chemical potential energy, and electrical potential energy. In addition, ATP directly participates in various biochemical reactions in cells. Outside the cell, ATP also plays an important role in intercellular communication and is a common extracellular communication medium. Promote the rapid repair of bone through the following two ways: 1) Mediate the activation of nucleic acid receptors on the cell membrane surface to promote bone mineralization (Bo Huo, et al. An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation. Cell Calcium, 2010, 47: 234-241.). 2) Promote the formation of hydroxyapatite (hydroxyapatite, HA) by regulating the concentration of extracellular calcium and phosphorus (Lovisa Hessle, et al. Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization . PNAS, 2002, 99: 9445-9449.). In summary, on the one hand, intracellular ATP, as an energy molecule, can significantly promote the growth and proliferation of cells; on the other hand, exogenous ATP secreted by cells can mediate P2 receptors on the surface of osteoblast membranes and regulate extracellular Calcium and phosphorus concentration, improve the osteogenic ability of osteoblasts, inhibit bone resorption of osteoclasts, and play a key role in the process of bone formation and mineralization.

Studies have found that the tricarboxylic acid cycle (TCA) is an important part of the ATP generation process in the mitochondria, and the generation of ATP by cellular respiration can be significantly accelerated by the addition of succinic acid, fumaric acid, malic acid, etc. from external sources. The exogenous addition of any of these dicarboxylic acids will lead to a large consumption of oxygen and a large production of CO2 and ATP. Subsequent studies have proved that succinic acid, fumaric acid, malic acid, etc. are all important intermediates in the tricarboxylic acid cycle, and their exogenous addition will greatly increase the rate of TCA, thereby promoting the generation of ATP. Therefore, the small molecular fragments generated by the degradation of the polymer chain segment containing the following formula 1 are easily absorbed by cells and promote the production of endogenous ATP. These small fragments have some things in common, that is, they are all derived from succinic acid, and the H atoms are replaced by other groups (represented by W, X, Y in the structure of Formula 1), and different group combinations represent different derived substances. According to the research method of molecular design, different combinations of W, X, Y and R are selected as reactants for the synthesis of polyester materials, and a series of macromolecules with different structures can be obtained. After comprehensive consideration, the optimal combination of reactants is determined, and then through a series of molding and modification processes, the system of energy biomaterials is finally constructed.

After the energy material is implanted in the body, while exerting its bone defect repair function, as its own degradation process continues, the degradation products (that is, the small molecule fragments of the tricarboxylic acid cycle intermediates) continue to enter the cells and actively participate in the three processes. The carboxylic acid cycle accelerates the generation of ATP in the cells, and plays a role in the cells in the form of energy supply to promote the growth, proliferation and mineralization of osteoblasts and other life activities.

Contents of the invention

In order to solve the deficiency that the existing biological materials do not have energy functions, the present invention provides an energy biological material based on the tricarboxylic acid cycle and a preparation method thereof. The energy biological material prepared according to the method can produce a series of biologically active molecules, and then through The tricarboxylic acid cycle pathway provides energy for tissue cells, and the present invention proposes the concept of energy biomaterials accordingly.

The purpose of the present invention is also to further prepare the energy biomaterial obtained by the method of the present invention into a porous scaffold, which not only has good biocompatibility, but also exerts its tissue repair function, as its self-degradation process continues, The degradation products continue to enter the cells, participate in the tricarboxylic acid cycle, accelerate the generation of ATP in the cells, and play a role in the cells in the form of energy supply, thereby promoting cell growth, proliferation and tissue repair.

The purpose of the present invention is also to implement the preparation method of an energy biomaterial provided by the present invention, endow the molecular structure with reactive side chain groups, and provide a basis for further surface modification and anchoring of active molecules.

The purpose of the present invention is achieved through the following technical solutions:

An energy biomaterial based on the tricarboxylic acid cycle, its material chemical structure is a cross-linked polyurethane material obtained by the reaction of low molecular weight polyester polyol and diisocyanate, with the following structure,

              

Where W is: -OH, =CHCOOH, -COCOOH, =CO or -H; X is -CH2COOH, =CHCOOH or -H; Y is -H or -CH2COOH; R is -CH3, -H, -COC2H5, - CONH2, -COC17H35, phosphate group) or polyethoxyethyl; n is a natural number from 10 to 100.

The material can produce a series of biologically active molecules, and then provide energy for tissue cells through the tricarboxylic acid cycle pathway. This material is biodegradable, and the degraded fragments can enter the mitochondria of cells, participate in the tricarboxylic acid cycle, and accelerate the generation of adenosine triphosphate ATP.

A method for preparing energy biomaterials based on the tricarboxylic acid cycle, comprising: step 1, preparation of prepolymers, mainly including the selection of prepolymer precursors and their proportions; step 2, pouring of reaction liquids, mainly including Preparation of porogen sugar balls and material ratio; step 3, polyaddition reaction and material molding.

A method for preparing energy biomaterials based on the tricarboxylic acid cycle of the present invention is specifically carried out in accordance with the following steps: through molecular design, the reaction precursor is selected to synthesize a prepolymer, and diisocyanate is added after being dissolved in a solvent; The reaction liquid is added into the mold containing the porogen; then, after the polyaddition reaction is solidified, it is taken out, and the porous energy biomaterial is obtained after the porogen is removed.

A kind of preparation method of the energy biological material based on tricarboxylic acid cycle of the present invention, concrete steps are as follows:

Step 1, the preparation of prepolymer:

Add dibasic acid A(COOH) ₂ , dibasic alcohol B(OH) ₂ and tribasic alcohol C(OH) ₃ into a 250ml round bottom flask, the molar ratio of which is 10:10-n:n; Measure organic solvent a as the water-carrying agent; heat the oil bath for 12 hours, replenish the water-carrying agent during the reaction; vacuumize and continue the reaction for 12 hours, then remove the reaction device, and obtain a transparent viscous prepolymer at the bottom of the flask, which can be dissolved in polar solvent b to obtain 25% ( w/v) Solution c for later use;

Step 2, pouring of reaction solution:

Prepare sugar spheres as a porogen; measure the molecular weight of the prepolymer obtained in step 1; add diisocyanate to the aforementioned solution c, the molar ratio of which to the prepolymer is 1:1, and this is solution d; add the sugar spheres Shake the sugar balls tightly after reaching the mold, then pour the solution d into the mold, and air-dry at room temperature for 24 hours to obtain the preformed compound e;

Step 3, polyaddition reaction and material molding;

Heat the composite e obtained in step 2 to 80°C, react and solidify for 48 hours. After the reaction, take it out and soak it in deionized water for 48 hours. biomaterials;

Wherein, the dibasic acid is one or more of succinic acid, citric acid, cis-aconitic acid, isocitric acid, fumaric acid, malic acid, cis-aconitic acid, ketoglutaric acid, and oxaloacetic acid; Described dibasic alcohol is one or more in ethylene glycol, ethylene glycol diethyl ester, butanediol, propylene glycol and hexanediol; Described tribasic alcohol is one or more in glycerol, glycidol Various.

In the solution of the present invention, the organic solvent a is toluene, benzene or xylene; the polar solvent b is acetone, tetrahydrofuran, dioxane, dichloromethane or chloroform; the diisocyanate is hexa One of methylene diisocyanate (HMDI), toluene diisocyanate (TDI), lysine diisocyanate (LDI), and isophorone diisocyanate.

As a preferred version of the present invention, the dibasic acid is succinic acid, the dibasic alcohol is ethylene glycol, the tribasic alcohol is glycerol, and the organic solvent a is toluene; The polar solvent b is acetone; the diisocyanate is lysine diisocyanate (LDI), and the sugar spheres have a diameter of 200-400 μm.

As a preferred solution of the present invention, the mass ratio of the mixed liquid solute to the sugar spheres in the step 2 is 1:6, and the porous energy biomaterial obtained by the method has a porosity of 80-90%.

In the preparation method of the present invention, the value of n in the step 1 is: 2<n<10, and the preferred value of n is 6-10.

As a preferred solution of the present invention, the method for preparing energy biomaterials based on the tricarboxylic acid cycle is characterized in that: the dibasic acid is succinic acid (succinic acid), and the dibasic alcohol is ethylene glycol, the trihydric alcohol is glycerin, the value of n is 8, and the diisocyanate is hexamethylene diisocyanate; 10:2:8 was added to a 250ml three-necked flask, and then 20ml of toluene was added; after continuous heating and reaction for 12 hours, the reaction was continued for 12 hours under a negative pressure of 1333Pa; after cooling, it was dissolved in acetone to form a 25% (w/v) solution; in the above solution, 1 : After adding hexamethylene diisocyanate at a molar ratio of 1, pour it into a mold containing 200-400 μm sugar sphere porogen; after air-drying at room temperature, continue curing reaction at 80°C for 24 hours; after taking it out, soak it in deionized water for 48 hours, and freeze-dry it. Porous energy biomaterials.

The application of the energy biomaterial based on the tricarboxylic acid cycle of the present invention is applied to the field of bone tissue engineering to prepare porous scaffolds.

Utilizing the technical scheme of the present invention, not only succinic acid, ethylene glycol and glycerin can be selected to prepare porous scaffolds for energy biomaterials, but also other derivatives can be selected as reaction precursors, which can be prepared with other dibasic acids on the basis of the present invention. Porous scaffolds for energetic biomaterials. The reaction precursor used can be selected from other derivatives of succinic acid such as citric acid, cis-aconitic acid, isocitric acid, fumaric acid or malic acid. After selecting the desired reaction precursor, adjust the corresponding reaction conditions, use the method provided by the present invention to mix the reaction prepolymer with the crosslinking agent diisocyanate, cast the mold, continue to react and solidify, and wash away the porogen microspheres The porous scaffold of the energy biomaterial can be obtained by freeze-drying.

Compared with the prior art, the present invention has the advantage that the material is a new type of porous material based on the tricarboxylic acid cycle pathway, which can produce a series of bioactive molecules, and then provide tissue cells through the tricarboxylic acid cycle pathway. energy. The porous scaffold further prepared from the energy biomaterial obtained by the method of the present invention not only has good biocompatibility, but also exerts its tissue repair function, and as its own degradation process continues, the degradation products continue to enter Cells, participate in the tricarboxylic acid cycle, accelerate the generation of ATP in the cells, and play a role in the cells in the form of energy supply, thereby promoting cell growth, proliferation and tissue repair. In addition, the molecular structure characteristics of the material endow it with reactive side chain groups, which provides a basis for further surface modification and immobilization of active molecules.

Description of drawings

Figure 1 is a scanning electron microscope (SEM) photo of a porous scaffold based on a tricarboxylic acid cycle-based energy biomaterial.

Figure 2 is a fluorescence micrograph of pre-osteoblasts (MC3T3-E1) cultured on the porous scaffold of energy biomaterials for 7 days.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

Add 5.8g of succinic acid, 2.78ml of ethylene glycol and 3.35ml of glycerin into a 250ml round-bottomed flask, then add 10ml of toluene as a water-carrying agent, heat and react in an oil bath for 12 hours under continuous stirring, replenish the water-carrying agent during the period, and vacuumize (1333Pa) Continue to react for 12 hours and then remove the reaction device. A transparent viscous prepolymer is obtained at the bottom of the flask. After cooling, 25ml of acetone is added to fully dissolve the above viscous prepolymer to obtain a 25% (w/v) transparent solution. Add 1ml of hexamethylene diisocyanate to the solution, mix well and pour it into a mold equipped with sugar balls with a close-packed diameter of 200-400μm, air-dry at room temperature, heat to 80°C and continue curing reaction for 24h, after the reaction is over Take it out, soak it in deionized water for 48 hours, change the water every 12 hours during this period, soak the fructose and freeze-dry to obtain the formed porous scaffold of the energy biomaterial.

Characterize the performance of the prepared porous scaffold of energy biomaterials: the apparent size is 5mm high, the diameter is 8mm cylindrical, the porosity is 89%, the compression simulation is 1.7MPa, and the compressive strength is 80KPa. The SEM photo of the obtained porous scaffold of the energy biomaterial is shown in FIG. 1 .

The fluorescence micrographs of preosteoblasts (MC3T3-E1) cultured on the porous scaffold of the prepared energy biomaterial for 7 days are shown in Fig. 2 . After the cells were cultured on the energy material for 7 days, they were fixed with paraformaldehyde, and then treated with fluorescein isothiocyanate-labeled phalloidin (a fluorescent dye for cell microfilaments) and DAPI (a fluorescent dye for cell nuclei). After fluorescent staining, the cells were observed with a fluorescent microscope. By staining the microfilaments in the cells, it can be seen that the cells stretch well and have regular shapes on the porous scaffold of the energy material, which proves that the material has good biocompatibility.

The present invention has been described as an example above, and it should be noted that, without departing from the core of the present invention, any simple deformation, modification, or other equivalent replacements that can be made by those skilled in the art without creative labor fall within the scope of the present invention. protection scope of the invention.

Claims (7)

1. The preparation method of the energy biomaterial based on tricarboxylic acid cycle, is characterized in that, specifically comprises the steps: Step 1, the preparation of prepolymer: Add polyacids and their derivatives, diols and their derivatives, trihydric alcohols and their derivatives in a 250ml round bottom flask, the molar ratio of which is 10:10-n:n; add enough organic solvent a As a water-carrying agent; heat the oil bath for 12 hours, replenish the water-carrying agent during the reaction; vacuumize and continue the reaction for 12 hours, then remove the reaction device, and obtain a transparent viscous prepolymer at the bottom of the flask, which can be dissolved in a polar solvent b to obtain a concentration of 25% by mass volume , the solution c in g/mL is for later use; Step 2, pouring of reaction solution: Prepare sugar spheres as a porogen; measure the molecular weight of the prepolymer obtained in step 1; add diisocyanate to the aforementioned solution c, the molar ratio of which to the prepolymer is 1:1, and this is solution d; add the sugar spheres Shake the sugar balls tightly after reaching the mold, then pour the solution d into the mold, and air-dry at room temperature for 24 hours to obtain the preformed compound e; Step 3, polyaddition reaction and material molding; Heat the composite e obtained in step 2 to 80°C, react and solidify for 48 hours. After the reaction, take it out and soak it in deionized water for 48 hours. Biological materials, the materials are biodegradable, and the degraded fragments can enter the mitochondria of cells, participate in the tricarboxylic acid cycle, and accelerate the generation of adenosine triphosphate ATP; Wherein, the polybasic acid and its derivatives are one or more of succinic acid, citric acid, isocitric acid, fumaric acid, malic acid, cis-aconitic acid, ketoglutaric acid, and oxaloacetic acid; The dibasic alcohols and derivatives thereof are one or more of ethylene glycol, ethylene glycol diethyl ester, butanediol, propylene glycol, hexylene glycol and polyethylene glycol; the tribasic alcohols and derivatives thereof The substance is one or more of glycerin and glycidol. 2. the preparation method of the energy biological material based on tricarboxylic acid cycle according to claim 1, is characterized in that, described organic solvent a is toluene, benzene or xylene; Described polar solvent b is acetone, Tetrahydrofuran, dioxane, methylene chloride or chloroform; the diisocyanate is one of hexamethylene diisocyanate, toluene diisocyanate, lysine diisocyanate or isophorone diisocyanate. 3. the preparation method of the energy biological material based on tricarboxylic acid cycle according to claim 1, is characterized in that, described polybasic acid and derivative thereof are succinic acid, and described dibasic alcohol and derivative thereof are Ethylene glycol, the trihydric alcohol and its derivatives are glycerol. 4. the preparation method of the energy biological material based on tricarboxylic acid cycle according to claim 2, is characterized in that, described organic solvent a is toluene; Described polar solvent b is acetone; Described diisocyanate It is lysine diisocyanate, and the diameter of the sugar sphere is 200-400 μm. 5. the preparation method of the energy biological material based on tricarboxylic acid cycle according to claim 1, is characterized in that, the ratio of the mass of mixed solution solute and sugar ball in described step 2 is 1:6, and described The porous energy biomaterial obtained by the method has a porosity of 80-90%. 6 . The preparation method of energy biomaterials based on tricarboxylic acid cycle according to claim 1 , characterized in that, the value of n in the step 1 is: 2<n<10. 7. The preparation method of energy biomaterials based on tricarboxylic acid cycle according to claim 1, characterized in that: said polybasic acid and its derivatives are succinic acid, and said dibasic alcohol and its derivatives are Ethylene glycol, described trihydric alcohol and its derivatives are glycerol, described n is taken as 8, and described diisocyanate is hexamethylene diisocyanate; Add 10:2:8 to a 250ml three-neck flask, then add 20ml toluene; continue heating for 12 hours, then continue to react for 12 hours under a negative pressure of 1333Pa; after cooling, dissolve in acetone to a concentration of 25% by mass volume, and the unit is g/mL solution; after adding hexamethylene diisocyanate at a molar ratio of 1:1 to the above solution, pour it into a mold containing 200-400 μm sugar ball porogen; after air-drying at room temperature, continue curing reaction at 80°C for 24 hours; after taking it out, deionize Soak in water for 48 hours, during which the water is changed every 12 hours, and then freeze-dried to form a porous energy biomaterial.