CN115970051A - Degradable tissue engineering filling material and preparation method and application thereof - Google Patents
Degradable tissue engineering filling material and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a degradable tissue engineering filling material and a preparation method and application thereof. The degradable tissue engineering filling material enhances the function of hydrophobic polymers such as PLGA and the like for promoting bone repair by using hyaluronic acid with small molecular weight, and reduces inflammatory reaction caused by PLGA in vivo degradation. In addition, the degradation rate in the body of the filling material is regulated by using low molecular weight PLGA, thereby achieving a degradation rate consistent with bone repair.
Description
Technical Field
The invention relates to the field of tissue engineering, in particular to a degradable tissue engineering filling material and a preparation method and application thereof.
Background
At present, the main clinical application of bone repair is expensive imported tissue engineering filling materials, most of which are animal sources, and certain immunogenicity, cross infection and other risks exist. The applied artificial bone powder is in a fine sand shape or a bone block shape, has no in-situ solidification effect, cannot be molded, has certain tension after mucosa suture, has tissue edema and the like in a healing process, increases the tension, is difficult to maintain a three-dimensional space, causes slow formation rate and poor shape of new bone, and cannot meet clinical requirements.
Degradable polymers such as polylactic acid (PLA), polyglycolic acid (PGA) and copolymers thereof (PLGA) have excellent biodegradable absorbability, good biocompatibility, no toxicity, good properties of encapsulation and film formation, and are widely applied to the fields of pharmacy, medical engineering materials and modern industry. PLA, PGA and PLGA all belong to aliphatic polyesters, and the principle of degradation is that the random hydrolysis of ester bonds leads to the breakage of molecular chains, and the molecular chains are degraded into lactic acid and glycolic acid in a human body and finally metabolized into products of CO 2 And H 2 And O. The degradation rates of different materials vary widely. Wherein, the time of the levorotatory polylactic acid (PLLA) with weak hydrophilicity and high crystallinity completely degraded in vivo reaches 3 to 5 years, delayed noninfectious inflammatory tissue reaction is easily caused in the late stage of implantation, and PGA with strong hydrophilicity is degraded in 2 to 4 weeks. And the PLGA copolymer of PLLA and PGA can realize the regulation and control of degradation speed by controlling the proportion of the PLLA and the PGA. At present, PLGA is mainly used for in vivo fixing materials, such as bone plates, internal fixing screws, internal fixing rods, bone nails, sutures and the like used for fracture fixation or repair, and PLGA polymers used for tissue engineering filling materials are not available, and the requirements on degradation speed are different due to different purposes.
The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.
Disclosure of Invention
In order to solve at least part of technical problems in the prior art, the invention provides a degradable tissue engineering filling material and a preparation method and application thereof. Specifically, the present invention includes the following.
In a first aspect of the present invention, a method for preparing a degradable tissue engineering filling material is provided, which at least comprises the following steps:
(1) Carrying out ultrasonic treatment on a hyaluronic acid raw material in a first solvent to obtain an oligomer with an ultralow molecular weight, and further obtaining an aqueous phase liquid of the oligomer;
(2) Dissolving the hydrophobic copolymer in a second solvent to obtain an oil phase liquid; and
(3) Mixing the aqueous phase liquid and the oil phase liquid in an amount enough to obtain emulsion, and performing alcohol washing, suction filtration and drying to obtain the degradable tissue engineering filling material;
wherein the first solvent is a weakly basic organic solvent, and the second solvent comprises at least one of N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.
In certain embodiments, the method for preparing a degradable tissue engineering filler material according to the first aspect, wherein the ultra-low molecular weight is 1000-10000Da.
In certain embodiments, the method for preparing a degradable tissue engineering filler material according to the first aspect, wherein the hydrophobic copolymer is a polylactic acid-glycolic acid copolymer with a molecular weight of 2000-7000.
In certain embodiments, the method for preparing a degradable tissue engineering filler material according to the first aspect, wherein the polylactic acid-glycolic acid copolymer consists of 90-55 parts by weight of lactic acid and 10-45 parts by weight of glycolic acid.
In certain embodiments, the method for preparing a degradable tissue engineering filler material according to the first aspect, wherein the weakly basic organic solvent is N, N-dimethylformamide.
In certain embodiments, the method for preparing a degradable tissue engineering filler material according to the first aspect, wherein the amount of the aqueous phase liquid and the oil phase liquid is controlled such that the weight ratio of the ultra-low molecular weight oligomer and the hydrophobic copolymer is (20-80): (80-20).
In certain embodiments, the method for preparing a degradable tissue engineering filler material according to the first aspect further comprises the step of grinding the lumps in the emulsion to a predetermined size.
In a second aspect of the present invention, there is provided a degradable tissue engineering filler material prepared by the method of the first aspect.
In certain embodiments, the degradable tissue engineering filler material according to the second aspect has a porous structure and is in the form of a powder, a block or a film.
In a third aspect of the invention, there is provided the use of a degradable tissue engineering filler material in the preparation of a bone repair material.
The invention enhances the function of hydrophobic polymers such as PLGA and the like for promoting bone repair by using hyaluronic acid with small molecular weight, and reduces inflammatory reaction caused by PLGA degradation in vivo. In addition, the degradation rate in the body of the filling material is regulated by using low molecular weight PLGA, thereby achieving a degradation rate consistent with bone repair.
Drawings
FIG. 1 is a photograph of a product obtained in the first embodiment;
FIG. 2 is a photograph of the product obtained in example four;
FIG. 3 is a diagram of the process of implanting the filling material into the tissues of femur and tendon of a white rat;
FIG. 4 is a section of tissue after one week of implantation of the resulting filling material of example one;
FIG. 5 shows a two-week tissue section after implantation of the first resulting filling material of example one;
FIG. 6 shows a tissue section of the first embodiment after four weeks of implantation of the resulting filling material;
FIG. 7 is a section of the tissue obtained from the filling materials obtained in examples two, three and four at different times after implantation in mice.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
Herein, the terms "first", "second", and the like are used for distinguishing purposes only and do not denote a sequential order. For example, "first solvent" and "second solvent" merely indicate that the two solvents are different or at least partially different in composition.
Herein, the term "tissue engineering filling material" refers to a material for filling to a bone defect site to promote bone regeneration, and belongs to a bone repair material, which is different from a soft gel filling material for soft tissue augmentation and the like. In addition, it is different from the conventional bone filling material in that not only is the bone repair function stronger, but also the inflammatory response is lower. Sometimes simply referred to herein as "the material of the present invention" or "the filler material of the present invention".
Herein, the term "hyaluronic acid raw material" generally refers to hyaluronic acid or a salt thereof having a molecular weight of more than 10000Da, the source of which is not particularly limited, and includes, but is not limited to, hyaluronic acid or a salt thereof extracted from animal tissues, hyaluronic acid or a salt thereof obtained by fermentation of microorganisms, or hyaluronic acid or a salt thereof obtained by artificial synthesis, and also includes degraded hyaluronic acid or a salt thereof obtained by further processing or processing of hyaluronic acid or a salt thereof of the above-mentioned source. Such treatments include enzymatic hydrolysis, chemical digestion, sonication, and the like. Examples of animal tissue include cockscomb, umbilical cord, pigskin, cowhide, skin of fish or other animals, aorta, and the like. The molecular weight of a hyaluronic acid starting material of natural origin is generally greater than 2 to 200 million Da, typically greater than 100 million Da. In certain embodiments, the hyaluronic acid starting material of the invention is a starting material obtained by chemical structural modification, such as acetylation, of hyaluronic acid of natural origin.
As used herein, the term "ultra-low molecular weight oligomer" is an oligosaccharide or salt thereof consisting of 2-30 saccharide monomers, generally having a molecular weight of less than 10000Da, preferably having a molecular weight of 1X 10 3 -9×10 3 Da range, more preferably 2X 10 molecular weight 3 -6×10 3 Da range, e.g. 2X 10 3 -4×10 3 Da. The salt herein refers to a compound formed by at least one carboxyl group of hyaluronic acid and a metal salt ion. Examples of the metal salt ion include alkali metal salt ions such as sodium, potassium, lithium and the like; alkaline earth metal salt ions such as calcium, magnesium, etc.
Herein, the term "first solvent" refers to a weakly basic organic solvent, preferably a small molecule amide solvent, examples of which include, but are not limited to, substituted or unsubstituted formamides, acetamides, and propionamides. Examples of substituted formamides include methylformamides, such as dimethylformamide and the like.
Herein, the term "second solvent" refers to an organic solvent capable of dissolving the hydrophobic copolymer, particularly PLGA, and examples thereof include, but are not limited to, N-methylpyrrolidone, dichloromethane, chloroform, and acetonitrile. One of these, or a combination of more, may be used herein. In the case of using a plurality of solvents in combination, the mixing ratio of the solvents is not limited and may be any ratio as long as the object of the present invention is not impaired. In certain embodiments, the second solvent is a mixed solvent, which further includes other organic solvents in addition to the above-mentioned organic solvents, and the other organic solvents are generally water-soluble organic solvents, and examples thereof include small molecule amide solvents such as substituted or unsubstituted formamide, acetamide, and the like, for example, methylformamide, dimethylformamide, and the like.
[ production method ]
In a first aspect of the present invention, a method for preparing a degradable tissue engineering filling material is provided, which at least comprises the following steps:
(1) Carrying out ultrasonic treatment on a hyaluronic acid raw material in a first solvent to obtain an oligomer with an ultralow molecular weight, and further obtaining an aqueous phase liquid of the oligomer;
(2) Dissolving the hydrophobic copolymer in a second solvent to obtain an oil phase liquid; and
(3) And mixing the aqueous phase liquid and the oil phase liquid in an amount enough to obtain emulsion, and carrying out alcohol washing, suction filtration and drying to obtain the degradable tissue engineering filling material.
It is understood by those skilled in the art that the step numbers (1), (2), etc. are only for the purpose of distinguishing different steps, and do not indicate the sequential order of the steps. The order of the above steps is not particularly limited as long as the object of the present invention can be achieved. In addition, two or more of the above steps may be combined and performed simultaneously. In addition, it will be understood by those skilled in the art that other steps or operations may be included before, after, or between any of the above steps (1) - (3), such as to further optimize and/or improve the methods of the present invention.
In the invention, the step (1) is a step for preparing the ultralow molecular weight oligomer, and unlike the existing filling material, the ultralow molecular weight oligomer obtained by processing the hyaluronic acid raw material is used in the filling material, so that the filling material has stronger angiogenesis promoting effect, further has stronger osteogenesis effect and has lower inflammatory reaction.
Herein, the aqueous phase liquid is a water-based liquid phase, immiscible with the oil phase liquid, obtained by adding water to the separated ultra-low molecular weight oligomer, or by directly adding water to the ultra-low molecular weight oligomer-containing solution after the ultrasonic treatment. In the case of obtaining an aqueous liquid by adding water to the separated ultra-low molecular weight oligomer, the volume of water used for dissolving the ultra-low molecular weight oligomer is smaller than the volume of the second solvent on a volume basis, and in general, the ratio of the volume of water to the volume of the second solvent is 1 (2-10), preferably 1 (2-5).
In a preferred embodiment, the water phase liquid is obtained by directly adding water into the ultra-low molecular weight oligomer-containing solution after ultrasonic treatment, so that the step of recovering or removing the first solvent after ultrasonic treatment is omitted, and the purposes of comprehensively utilizing the solvent, reducing the cost and improving the benefit are achieved. For this purpose, it is necessary to select a specific first solvent, second solvent and optimize the amount thereof. Specifically, in order to achieve the purpose of dissolving the raw materials while the aqueous phase and the oil phase are immiscible with each other, it is preferable that the first solvent is dimethylformamide and the second solvent is dichloromethane, so that the binding between the hydrophilic supramolecular oligomer and the hydrophobic copolymer can be promoted, which is advantageous for obtaining a filler having an excellent effect. In addition, it is also necessary to control the concentration of the hyaluronic acid raw material in the first solvent, and an appropriate concentration facilitates the ultrasonic treatment and the dissolution of the hyaluronic acid raw material. If the concentration is too high, the dissolution of the hyaluronic acid raw material is not facilitated, the degradation of the hyaluronic acid raw material during the ultrasonic treatment is not facilitated, and the degradation to an ultra-low molecular weight level is particularly not facilitated. On the other hand, if the concentration is too low, an increased amount of the first solvent is required, thereby adversely affecting the formation of the emulsion. Generally, the concentration of the hyaluronic acid raw material in the first solvent is 0.01-0.5g/ml, preferably 0.05-0.3g/ml, such as 0.1g/ml,0.2g/ml, etc. Preferably, the amount of water is further controlled, and in general, the volume of water is larger than the volume of the first solvent. Typically the volume ratio of first solvent to water is 1:3-50, preferably 1:5-10. If the amount of water is too low, the formation of an emulsion is not favored. On the other hand, if the amount of water used is too high, the concentration of the ultra-low molecular weight oligomer in the aqueous liquid becomes low, so that the resulting filling material tends to have an increased inflammatory reaction, and the bone-repairing effect tends to be weak. It is also necessary to ensure that the volume of the aqueous liquid phase is less than the volume of the second solvent, and in general, the ratio of the volume of water to the volume of the second solvent is 1 (5-50), preferably 1 (10-30). The content of the hydrophobic copolymer in the second solvent is not limited as long as the weight ratio of the ultralow molecular weight oligomer to the hydrophobic copolymer can be ensured to be in the range of (20-80) to (80-20). Preferably, the ratio of the amount of ultra-low molecular weight oligomer to the amount of hydrophobic copolymer is 55 to 95, further preferably 60 to 70.
In the present invention, the molecular weight of the ultra-low molecular weight oligomer is in a suitable range. If the molecular weight is too large, the tissue repair effect becomes poor, including angiogenesis or osteogenesis, and even an effective filler material cannot be formed. On the other hand, if the molecular weight is too low, the production cost increases, and the tissue compatibility tends to be poor. Preferably, the ultra-low molecular weight oligomer is a chemically modified oligomeric hyaluronic acid, in particular an acetylated modified oligomeric hyaluronic acid, so that the lipophilicity of the oligomeric hyaluronic acid can be improved, and the binding with the hydrophobic copolymer can be improved.
In the present invention, the hydrophobic copolymer has an appropriate molecular weight. If the molecular weight is too large, effective mixing of the hydrophobic copolymer with the ultra-low molecular weight oligomer is not facilitated, and the hardness of the resulting composite filler material is too high. On the other hand, if the molecular weight is too small, the resulting filler tends to have increased viscosity, which is disadvantageous in forming a porous structure, and is further disadvantageous in particular in the generation of bone tissue within the porous structure. Preferably, the molecular weight of the hydrophobic copolymer is in the range 2000 to 5000, more preferably 2500 to 4000, such as 3000.
The content of lactic acid and glycolic acid in the PLGA of the present invention is not particularly limited, and generally consists of 90 to 55 parts by weight of lactic acid and 10 to 45 parts by weight of glycolic acid. Illustratively, the polylactic acid-glycolic acid copolymer of the present invention is composed of 80% lactic acid and 20% glycolic acid, i.e., PLGA8020. In order to maintain higher hydrophobicity and degradation time necessary for bone repair, it is preferred to select a higher lactic acid content, such as 75%, 80%, or 90%, etc., in the range of, for example, 60-90%. Although higher lactic acid content can prolong the degradation time, the filling material with high lactic acid content generates an intermediate product lactic acid in vivo, which has stronger local inflammatory immune response reaction and can trigger inflammatory reaction after tissue aggregation. However, the inventors have found that the filler material of the present invention is less inflammatory. The reason for this may be that ultra-low molecular weight hyaluronic acid after sonication has an immunomodulatory function, thereby eliminating or counteracting the inflammatory reaction caused by lactic acid aggregation.
The hydrophobic copolymer of the present invention can be prepared by using a known copolymer or by a synthetic method. Illustratively, the hydrophobic copolymers of the present invention are synthesized by a ring-opening polymerization process. Illustratively, the ring-opening polymerization method comprises the steps of taking Lactic Acid (LA) and Glycolic Acid (GA) to respectively dehydrate, cyclize and dimerize into lactide and glycolide monomers, and then opening and copolymerizing under the action of an initiator according to different proportions. The PLGA thus obtained is a random copolymer, the composition of which can be controlled with different dosage ratios.
In certain embodiments, the preparation of the hydrophobic copolymers of the present invention comprises:
(1) A certain amount of viscous liquid stannous isooctanoate (Sn (Oct)) is measured and dissolved in dichloromethane to prepare solution with the concentration of about l-2g/mL, and the solution is centrifuged and homogenized. Placing into a brown bottle, sealing and storing in dark.
(2) The tube is sealed and added with methylene chloride solution of monomer lactide, glycolide, t-butyldimethylsilyl alcohol (t-butyldimethylsilyl ilan 01) as a molecular weight regulator and Sn (Oct) as an initiator according to a certain proportion.
(3) Vacuum treatment, argon protection, and the environment reaches the pressure of 10-3pa.
(4) Catalyzing, placing in a thermostat at 160 ℃, and melting. Homogenizing and reacting for 5-l0h. And (5) carrying out suction filtration and drying. Low molecular weight PLGA of different molecular weight are obtained.
In the step (3) of the present invention, the mixing of the aqueous phase liquid and the oil phase liquid is preferably performed in a gentle manner, and stirring is performed at the time of mixing, thereby obtaining an emulsion. The stirring can be carried out by a conventional mechanical stirring, and the stirring speed is not particularly limited and may be 1 to 50 revolutions/second, preferably 2 to 30 revolutions/second, more preferably 5 to 10 revolutions/second. Illustratively, the aqueous phase is added dropwise to the oil phase, and the resulting emulsion is a white reaction solution.
In certain embodiments, the preparation methods of the present invention further comprise a washing step, which titrates the white reaction solution, for example, by alcohol. Examples of the alcohol are not limited, and may be methanol, ethanol, propanol, etc. After cleaning, further cold storage and standing treatment can be carried out.
In certain embodiments, the preparation method of the present invention further comprises a suction filtration step comprising suction filtration of the refrigerated solution with a circulating vacuum suction filtration pump to obtain a milky white solid.
[ degradable tissue engineering filling Material ]
In a second aspect of the present invention, there is provided a degradable tissue engineering filler material prepared by the method of the first aspect.
In the present invention, the filler material of the present invention generally refers to an organic material composite comprising both an ultra-low molecular weight oligomer and a hydrophobic copolymer. The method of the present invention can form effective combination between the hydrophilic ultralow molecular weight oligomer and the hydrophobic copolymer, and further form a solid material with a porous structure, and the specific form of the solid material is not limited, and the solid material may be, for example, a powder, a block, or a film.
[ use ]
In a third aspect of the invention, there is provided the use of a degradable tissue engineering filler material in the preparation of a bone repair material.
Example one
1. 10g of hyaluronate solid powder (molecular weight 500 ten thousand Da, drying weight loss less than 5%) is dispersed in 200ml of N, N-Dimethylformamide (DMF) under 1MHz ultrasonic to form suspension, and the suspension is treated by ultrasonic for 20 hours. Mixing acetyl chloride and formamide in the same volume ratio under inert atmosphere, slowly adding 50mL of the mixed solution into the ultrasonic treatment solution, and treating at 70-90 ℃ under 1MHz ultrasonic until the acetyl chloride and the formamide are completely dissolved. Adding 2L of water into the solution, fully stirring, separating white solid, washing with water to neutrality, and drying to obtain the ultralow molecular weight oligomer with the molecular weight of about 6 kDa.
3g of the above ultra-low molecular weight oligomer was weighed. 20ml of distilled water was put in a container. Putting the container on a magnetic stirrer, opening the magnetic stirrer, setting the speed to be 5-10 r/s, uniformly adding the mixture into distilled water in a dropping mode, and homogenizing for 3-5h after the mixture enters the solution.
2. 40ml of N, N-Dimethylformamide (DMF) and 10ml of methylene chloride were added to a closed vessel, and magnetic stirring was conducted while adjusting the magnetic stirring to 10 to 20 revolutions/sec, and heating was conducted by means of a magnetic stirrer. Then 2g of PLGA8218 (MW 4000) was added uniformly to the sealed container and homogenized.
3. And (3) dropwise adding the solution obtained in the step (1) into the solution obtained in the step (2) in a sealed container under the condition of mechanical stirring at the speed of 5-10 revolutions per second to obtain white milky liquid.
4. And a cleaning step, adding excessive absolute ethyl alcohol, stirring, and standing in a refrigerating chamber for 5-10h.
5. And (4) a suction filtration step, namely taking out the refrigerated solution, performing suction filtration by using a circulating vacuum suction filtration pump to obtain a milky white solid, and cleaning for 5-10 times at high temperature. And (5) drying. As shown in fig. 1, the product was a fixed powder.
Example two
1. 20g of hyaluronate solid powder (with the molecular weight of 50 ten thousand Da and the drying weight loss of less than 5 percent) is dispersed in 200ml of N, N-Dimethylformamide (DMF) under the ultrasonic of 1MHz to form a suspension, and the suspension is treated by ultrasonic for 25 hours. 1.5L of water is added into the solution and fully stirred to obtain aqueous phase liquid, wherein the ultra-low molecular weight compound is about 4 kDa.
2. Adding 50ml dichloromethane into a sealed container, adjusting magnetic stirring to 10-20 rpm, adding 2g PLGA8218 (molecular weight 4000) into the sealed container uniformly, and homogenizing.
3. In a closed container, 20ml of the above aqueous phase was added dropwise to the solution of step 2 under mechanical stirring at a speed of 5-10 rpm, to give a white milky liquid.
4. And a cleaning step, adding excessive absolute ethyl alcohol, stirring, and standing in a refrigerating chamber for 5-10h.
5. And (4) a suction filtration step, namely taking out the refrigerated solution, performing suction filtration by using a circulating vacuum suction filtration pump to obtain a milky white solid, and cleaning for 5-10 times at high temperature. And (5) drying.
EXAMPLE III
1. 10g of hyaluronate solid powder (molecular weight 500 ten thousand Da, drying weight loss less than 5%) is dispersed in 200ml of N, N-Dimethylformamide (DMF) under 1MHz ultrasonic to form suspension, and ultrasonic treatment is carried out for 15 hours. Mixing acetyl chloride and formamide in the same volume ratio under inert atmosphere, slowly adding 50mL of the mixed solution into the ultrasonic treatment solution, and treating at 70-90 ℃ under 1MHz ultrasonic until the acetyl chloride and the formamide are completely dissolved. 2L of water was added to the solution, and after stirring well, a white solid was isolated, washed with water to neutrality, and dried. Adding alkali liquor into the dried solid to make the pH value of the solution be 9-12, and carrying out 1MHz ultrasonic treatment at 40-60 ℃ for 1 hour to obtain the ultralow molecular weight oligomer with the molecular weight of 6.5kDa.
3g of the above ultra-low molecular weight oligomer was weighed. 20ml of distilled water was put in a container. Putting the container on a magnetic stirrer, opening the magnetic stirrer, setting the speed to be 5-10 r/s, uniformly adding the mixture into distilled water in a dropping mode, and homogenizing for 3-5h after the mixture enters the solution.
2. 40ml of N, N-Dimethylformamide (DMF) and 10ml of methylene chloride were added to a closed vessel, and magnetic stirring was conducted while adjusting the magnetic stirring to 10 to 20 revolutions/sec, and heating was conducted by means of a magnetic stirrer. Then 2g of PLGA8218 (molecular weight 4000) was added uniformly to the sealed container and homogenized.
3. And (3) dropwise adding the solution obtained in the step (1) into the solution obtained in the step (2) in a sealed container under the condition of mechanical stirring at the speed of 5-10 revolutions per second to obtain white milky liquid.
4. And (3) a cleaning step, namely adding excessive absolute ethyl alcohol, stirring, and standing in a refrigerating chamber for 5-10h.
5. And (4) a suction filtration step, namely taking out the refrigerated solution, performing suction filtration by using a circulating vacuum suction filtration pump to obtain a milky white solid, and cleaning for 5-10 times at high temperature. And (5) drying.
Example four
1. The solid obtained in example two was added to the solvent dichloromethane and mixed by magnetic stirring until a homogeneous dispersion was formed. Wherein the mass ratio of the solid to the solvent is 18.
2. Loading the dispersion into an injector in an electrostatic spinning machine for electrostatic spinning to form a fiber membrane with the thickness of 130 mu m; the parameters of electrostatic spinning are as follows: the voltage is 20kV, the injection rate is 0.5ml/h, the receiving distance is 15cm, the rotating speed of a receiving roller is 1000 rpm, the spinning temperature is normal temperature, the humidity is 30 percent, and the spinning time is 6 hours. The product is shown in fig. 2, which is a film material with a rough structure on the surface.
Comparative example 1
1. 10g of hyaluronate solid powder (molecular weight 500 ten thousand Da, drying weight loss less than 5%) is dispersed in 200ml of NaCl aqueous solution (0.2 mol/L) under 1MHz ultrasonic to form suspension, and the suspension is treated by ultrasonic for 20 hours. The molecular weight of the obtained hyaluronic acid degradation product is about 25 kDa.
5g of the above degradation product was weighed. 20ml of distilled water was put in a container. Putting the container on a magnetic stirrer, opening the magnetic stirrer, setting the speed to be 5-10 r/s, uniformly adding the mixture into distilled water in a dropping mode, and homogenizing for 3-5h after the mixture enters the solution.
2. 40ml of N, N-Dimethylformamide (DMF) and 10ml of dichloromethane were added to a closed vessel, and magnetic stirring was conducted while adjusting the magnetic stirring to 10 to 20 revolutions per second, and heating was conducted by means of a magnetic stirrer. Then 2g of PLGA8218 (MW 4000) was added uniformly to the sealed container and homogenized.
3. In a sealed container, the solution of step 1 is added dropwise to the solution of step 2 under mechanical stirring at a speed of 5-10 rpm to obtain a white milky liquid in which more aggregates or lumps are formed in the liquid.
Comparative example II
1. 20g of hyaluronate solid powder (molecular weight 50 ten thousand Da, drying weight loss less than 5%) is dispersed in 200ml of N, N-Dimethylformamide (DMF) under the ultrasonic of 1MHz to form a suspension, and the suspension is treated by ultrasonic for 25 hours. Wherein the ultralow molecular weight compound is about 4 kDa.
2. 50ml of methylene chloride was put into a sealed container, magnetic stirring was adjusted to 10 to 20 rpm, and 2g of PLGA8218 (molecular weight 4000) was uniformly added into the sealed container, followed by homogenization.
3. In a sealed container, 20ml of the solution obtained in step 1 is added dropwise to the solution in step 2 under mechanical stirring at a speed of 5-10 rpm, and the ultra-low molecular weight compound forms a solid precipitate in the mixture.
Comparative example III
1. 20g of hyaluronate solid powder (molecular weight 500 ten thousand Da, drying weight loss less than 5%) is dispersed in 200ml of mixed solvent of N, N-dimethylformamide and water (volume ratio 1:8) under 1MHz ultrasonic to form suspension, and the suspension is subjected to ultrasonic treatment for 25 hours. Wherein the ultra-low molecular weight compound is about 23 kDa.
2. 50ml of methylene chloride was put into a sealed container, magnetic stirring was adjusted to 10 to 20 rpm, and 2g of PLGA8218 (molecular weight 4000) was uniformly added into the sealed container, followed by homogenization.
3. In a closed container, 20ml of the solution obtained in step 1 was added dropwise to the solution obtained in step 2 under mechanical stirring at a speed of 5-10 rpm, to give a white milky liquid in which more aggregates or lumps were formed in the liquid.
In the preparation process, when the PLGA content is increased, the toughness of the resulting filling material is increased, and agglomeration may occur in the solution, in which case the agglomerated portion needs to be pulverized using a tissue pulverizer, or the resulting composite material needs to be pulverized to a desired particle size using a pulverizer. In order to obtain the bone meal with required mesh number, the prepared composite material needs to be sieved.
Test example
As shown in FIG. 3 below, the filling material according to the first example was implanted into the tissues of femur and tendon of a rat, which is an experimental animal, and the change of the tissue section with the lapse of time was shown in FIGS. 4, 5 and 6. As can be seen from fig. 4-6, the material did not degrade after one week of implantation of the filling material, no bone formation was seen, and only a small amount of inflammatory cells were present. Two weeks after implantation new bone is formed with angiogenesis, the material starts to degrade and only a few inflammatory cells are present. After four weeks of implantation, a large amount of new bone formation was observed, the material was degraded by about 50%, and inflammatory cells were few.
Fig. 7 shows the bone repair results of the filling materials obtained in examples two, three and four. Wherein, a corresponds to the second embodiment, B corresponds to the third embodiment, and C corresponds to the fourth embodiment.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (10)
1. A preparation method of a degradable tissue engineering filling material is characterized by comprising the following steps:
(1) Carrying out ultrasonic treatment on a hyaluronic acid raw material in a first solvent to obtain an ultralow molecular weight oligomer, and further obtaining an aqueous phase liquid;
(2) Dissolving the hydrophobic copolymer in a second solvent to obtain an oil phase liquid; and
(3) Mixing the aqueous phase liquid and the oil phase liquid in an amount enough to obtain emulsion, and performing alcohol washing, suction filtration and drying to obtain the degradable tissue engineering filling material;
wherein the first solvent is a weakly basic organic solvent and the second solvent comprises at least one of N-methylpyrrolidone, dichloromethane, chloroform, and acetonitrile.
2. The method for preparing the degradable tissue engineering filler material according to claim 1, wherein the ultra-low molecular weight is 1000-10000Da.
3. The method for preparing the degradable tissue engineering filling material according to claim 1, wherein the hydrophobic copolymer is polylactic acid-glycolic acid copolymer, and the molecular weight of the copolymer is 2000-6000.
4. The method for preparing the degradable tissue engineering filling material according to claim 3, wherein the polylactic acid-glycolic acid copolymer is composed of 90-55 parts by weight of lactic acid and 10-45 parts by weight of glycolic acid.
5. The method for preparing the degradable tissue engineering filling material according to claim 1, wherein the weakly basic organic solvent is N, N-dimethylformamide.
6. The method for preparing the degradable tissue engineering filling material according to claim 1, wherein the amount of the aqueous phase liquid and the oil phase liquid is controlled to make the weight ratio of the ultra-low molecular weight oligomer to the hydrophobic copolymer (20-80) to (80-20).
7. The method of claim 1, further comprising the step of grinding the agglomerates in the emulsion to a predetermined size.
8. A degradable tissue engineering filler material, prepared by the method according to any one of claims 1 to 7.
9. The degradable tissue engineering filler material of claim 8, wherein the degradable tissue engineering filler material has a porous structure and is in a powder, block or film form.
10. Use of the degradable tissue engineering filler material according to claim 8 or 9 for the preparation of a bone repair material.
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