CN106943629B - Nano artificial bone carrying Thai-energy hydroxyapatite-enzymolysis ossein protein - Google Patents
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Abstract
The invention discloses a nano artificial bone carrying Thai-energy hydroxyapatite and enzymolysis ossein, which mainly comprises a nano artificial bone loaded with Thai-energy on the hydroxyapatite and enzymolysis ossein, wherein the nano artificial bone of the hydroxyapatite and enzymolysis ossein is a hydroxyapatite crystal mineralized and grown along ossein fibers, and finally forms a bionic bone through biological mineralization; wherein the bone collagen fiber has a characteristic light and shade interval periodic stripe structure, namely a D-Band structure. Aiming at the refractory osteomyelitis infected by drug-resistant gram-negative pathogenic bacteria, the invention combines drug-sensitive highly sensitive energy with artificial bone; synthesizing artificial bone carrying Thai-energy hydroxyapatite and enzymolysis ossein protein. The artificial bone can slowly release imipenem and inhibit infection of drug-resistant gram-negative pathogenic bacteria such as escherichia coli; therefore, the defect that the vancomycin/gentamicin calcium sulfate-loaded artificial bone clinically used at present cannot cover all bacterial spectrums of osteomyelitis is overcome.
Description
Technical Field
The invention belongs to medical materials, and relates to a nano artificial bone carrying Thai-energy hydroxyapatite and enzymolysis ossein protein.
Background
The chronic traumatic osteomyelitis is mostly infected by gram-positive bacteria and gram-negative bacteria such as staphylococcus aureus, escherichia coli and the like, and the drug-resistant bacteria can generate polysaccharide-protein complexes on the cell surface to form biomembrane-coated bacteria and protect the bacteria. To kill the bacteria protected in the bioprotein membrane, the antibiotics reaching the focus of osteomyelitis must be sensitive to the pathogenic bacteria, and the local antibiotic concentration of the focus must be many times higher than the minimum inhibitory concentration of the pathogenic bacteria, so that the systemic intravenous drip of antibiotics is difficult to meet the treatment requirement. Meanwhile, after the focus of osteomyelitis is removed, a cavity is left, and bone grafting materials are needed to fill the bone cavity.
In order to make up for the shortage of systemic administration and achieve the effect of bone grafting, various national scholars have studied antibiotic-loaded artificial bones for many years. At present, the artificial bone which is clinically applied and carries more antibiotics is medical CaSO4It is produced by Wrig, the largest orthopaedic biomaterial production company worldwidehtt, the basic crystal is α -calcium sulfate hemihydrate, and the water-soluble antibiotic can be combined with the calcium sulfate hemihydrate to form a solid implant without affecting the antibacterial effect, so that vancomycin or gentamicin is mixed with α -calcium sulfate hemihydrate to prepare the vancomycin-loaded/gentamicin-loaded calcium sulfate artificial bone which is widely applied to treating osteomyelitis patients clinically.
However, the vancomycin-loaded calcium sulfate artificial bone is only effective to osteomyelitis infected by gram-positive bacteria such as staphylococcus aureus, the gentamicin-loaded artificial bone is also effective to osteomyelitis infected by common non-drug-resistant gram-negative bacteria, the antibiotic-loaded artificial bones are not sensitive to osteomyelitis infected by gram-negative bacteria such as drug-resistant escherichia coli and acinetobacter baumannii, the requirement for clinically treating the osteomyelitis infected by the drug-resistant gram-negative bacteria is nearly 42% of the clinical site, so that the international popular and universal vancomycin-loaded and gentamicin calcium sulfate artificial bones are far away from the clinical site, and the requirement for clinically treating the osteomyelitis infected by the drug-resistant gram-negative bacteria is met.
Moreover, the vancomycin/gentamicin calcium sulfate-loaded artificial bone has a fatal defect that: the chemical components and molecular structure of the artificial bone are not the same as those of the natural human bone tissueAnd the bone grafting can not replace the bone tissue of the self body. Because, the natural bone tissue of human body is formed by biomineralization of organic macromolecular type I collagen and inorganic mineral substance hydroxyapatite calcium crystal; the hydroxyapatite calcium crystal is embedded in the collagen fiber in a sheet shape and is mineralized along the long axis of the collagen fiber. The molecular equation of the artificial bone of calcium sulfate hemihydrate, which is developed by Wright of America, is 1/2H2O.CaSO4The collagen is a pure inorganic substance and does not contain organic macromolecular type I collagen, not to mention the biomineralized molecular structure of the type I collagen and the hydroxyapatite calcium; both in terms of chemical composition and molecular structure, are far from natural bone tissue. Therefore, how to develop a bone grafting material with type I collagen/hydroxyapatite calcium, which has similar components and molecular structures with human autologous bone tissue chemistry, becomes a target for cumin of bone tissue engineering researchers all over the world.
Foreign researchers have used collagen I on soft tissues such as skin and tendon to biomineralize and synthesize collagen I/hydroxyapatite calcium artificial bone material, but the biomineralization degree is inferior to that of human body's own bone tissue because collagen I and collagen on soft tissues such as skin and tendon belong to collagen I and are formed by [ α ]1(I)]2α2(I) But there are differences in amino acid residues in each peptide chain, and bone type I collagen contains a large number of phosphoserine residues, lactate, and glutamate residues; because the ratio of the charge of calcium to the ionic radius is large, the calcium is easy to combine with the phosphate oxygen of phosphoserine, the carboxyl oxygen of carboxyglutamic acid and the sugar hydroxyl oxygen of lactate; thereby enriching high-concentration calcium ions and becoming the part for forming and growing the hydroxyapatite crystal nucleus. Since type I collagen in soft tissues such as tendons contains fewer amino acid residues, type I collagen extracted from tissues such as skin and tendons is biomineralized, and the degree of mineralization in collagen is lower than that in natural bone tissues. Therefore, the invention does not use the type I collagen on the soft tissues such as tendon and the like to carry out biomineralization, but directly extracts the type I collagen from the bone tissues, wherein the existing phosphoserine residues and milk contained in the bone collagenAcid radicals and glutamic acid residues can directly provide crystal nuclei for biomineralization, thereby promoting biomineralization of collagen.
The extraction method of collagen internationally mainly comprises an alkaline method, an acid method, a salt method and an enzymatic method (1) the alkaline method is used for extracting collagen molecules, namely, peptide bonds on a triple helix structure of collagen I are easily hydrolyzed by alkali, so that the obtained hydrolysate has relatively low molecular mass, even gelatin, but not collagen, when the hydrolysis is serious, a racemic mixture of amino acids D and L is generated, because asymmetric carbon atoms are subjected to an intermediate stage in a symmetric state, racemization occurs, and the hydrolysate is converted into a mixture of D and L with equal substance amounts, wherein if the D-type amino acids are more than L, absorption of the L-type amino acids is inhibited, while some D-type amino acids are toxic, some have carcinogenic, teratogenic and mutagenic effects, so the alkaline method is not suitable for extracting the collagen proteins for the medical use of biological common medical materials in a large scale, (2) the salt method is used for extracting the collagen proteins with neutral salts such as sodium chloride, sodium acetate, potassium chloride and citrate, while the extraction method generally can not use the neutral salts such as sodium chloride, sodium acetate, potassium chloride and citrate to reduce the conformational stability of the collagen molecules of the collagen extracted collagen molecules, so that the collagen is not suitable for extracting collagen molecules, the collagen I, the collagen extraction method for extracting collagen molecules is not suitable for the collagen extraction of the collagen extraction method for extracting collagen production of collagen with a low-free collagen I, the collagen with a collagen production of collagen with a low-free alkali method, the collagen I-free collagen protein extraction method, the collagen molecule extraction of a collagen I-free collagen-.
The immunogenicity of the type I collagen is derived from three points, namely a proline-hydroxyproline-glycine repeated sequence in α spiral polypeptide chain, and α spiral polypeptide chain three-dimensional spatial structure, and the differences between the two species are very small, and the most important immunogenicity is derived from the third point, namely non-spiral structures at two ends of a type I collagen molecular chain, namely-N-terminal residues and-C-terminal residues.
Therefore, the invention hydrolyzes pig bones by pepsin; hydrolyzing pig bone with enzyme to obtain bone collagen; then loading Tylen (imipenem/cilastatin), and then carrying out biomineralization on the ossein protein, thereby forming the artificial bone carrying the Tylen hydroxyapatite and the zymolytic ossein protein, aiming at the infection of gram-negative pathogenic bacteria with osteomyelitis drug resistance and the requirement of bone grafting.
Disclosure of Invention
The invention aims to solve the problem that the chemical components and molecular mechanisms of the calcium sulfate artificial bone loaded with vancomycin/gentamicin clinically applied at present do not conform to the natural bone tissues of human beings; and antibiotics sensitive to drug-resistant gram-negative pathogens, imipenem/cilastatin, cannot be loaded. Meanwhile, aiming at the type I collagen for preparing the bionic bone internationally at present, the type I collagen is extracted from soft tissues such as skin, tendon and the like, but not from bone tissues really. The invention hydrolyzes pig bones by pepsin; hydrolyzing pig bone with enzyme to obtain bone collagen; then loading the Tyenergy (imipenem/cilastatin), and then carrying out biomineralization on the ossein protein, thereby forming the artificial bone carrying the Tyenergy hydroxyapatite and the enzymolysis ossein protein. The chemical components and molecular mechanisms of the compound are consistent with those of natural bone tissues, and antibiotics sensitive to osteomyelitis drug-resistant gram-negative pathogenic bacteria can be released.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the nano artificial bone loaded with the Thai energy hydroxyapatite and the enzymolysis ossein protein mainly comprises the Thai energy loaded on the hydroxyapatite and the enzymolysis ossein protein nano artificial bone, wherein the hydroxyapatite and the enzymolysis ossein protein nano artificial bone mineralizes and grows hydroxyapatite crystals along ossein fibers and finally forms bionic bone through biological mineralization; wherein the bone collagen fiber has a characteristic light and shade interval periodic stripe structure, namely a D-Band structure.
Further, the mass ratio of the tylosin to the hydroxyapatite-enzymolysis bone collagen nano artificial bone is 7-10: 100.
Further, the mass ratio of the bone collagen fibers to the hydroxyapatite crystals is 0.5-1: 4.
The nano artificial bone carrying the Thai-energy hydroxyapatite and the enzymolysis bone collagen is prepared by the following method:
step (1), preparation of ossein protein
Carrying out degreasing and decalcification treatment on fresh tissue rich in ossein, and crushing to obtain required aggregate;
adding the pretreated aggregate into 0.1M acetic acid solution with the pH value of 3, and carrying out enzymolysis to obtain transparent and sticky collagen; the mass volume ratio of the aggregate to the acetic acid solution is 1 g: 10-20 ml;
the enzymolysis condition is that 200-250 mg of pepsin is added into every gram of aggregate, the enzymolysis temperature is 30-37 ℃, and the enzymolysis time is 30-60 days;
step (2), loading solar energy:
mixing the ossein protein obtained in the step (1) with the Tyener solution, fully stirring, placing in a 37 ℃ thermostat, and standing for 24 hours to obtain the Tyener-loaded ossein protein.
The tyloxapol solution is prepared by mixing 0.5-1 g of tyloxapol with 100ml of 0.9wt% of normal saline;
the volume ratio of the ossein protein to the tylosin solution is 10-9: 1;
step (3), preparing collagen colloid:
diffusing the bone collagen loaded with the energy from the step (2) for 1 hour by ammonia gas, and then adding glutaraldehyde for crosslinking; after crosslinking for 1 hour, the ossein protein self-assembles into jelly-like colloid; and finally, placing the colloid in deionized water to be soaked for 1 hour, then repeatedly washing for 3 times, and washing away redundant glutaraldehyde to obtain the collagen colloid loaded with the tylene.
The mass-volume ratio of the collagen loaded with the taffeta to the glutaraldehyde is 1 g: 10 to 20 ml.
The crosslinking temperature is 20-25 ℃.
Step (4), biomineralization:
and (4) soaking the collagen colloid carrying the Tyenergy obtained in the step (3) in a mineralization liquid, performing biomineralization, and mineralizing for 7-9 days, thereby forming the artificial bone carrying the Tyenergy hydroxyapatite and the enzymatic hydrolysis collagen.
Preparing a mineralized liquid: pouring 25ml of calcium solution into a beaker, and dripping the PAA with the concentration of 350 ug/ml; 1M HCl was added dropwise to adjust the pH to 7.4. Then, 25ml of phosphorus solution is slowly dropped into the mixture, and about 16 drops/min is added to prepare the mineralized solution.
25ml of calcium solution consists of the following components: 10mM CaCl2.2H2O、150mM NaCl、50mM.Tris、0.02wt％NaN3；
25ml of phosphorus solution 6mM Na2HPO4；
The experiment of the inhibition zone shows that: placing the prepared artificial bone carrying the Tyenergy hydroxyapatite-enzymolysis ossein protein in a culture medium of drug-resistant gram-negative bacteria escherichia coli, and forming a bacteriostatic zone around the artificial bone carrying the Tyenergy hydroxyapatite-enzymolysis ossein protein. In simulated body fluid sustained release experiments: the slow-release imipenem medicine concentration is 1ug/ml, is 8 times of the minimum inhibitory concentration of imipenem/cilastatin to escherichia coli, and is far greater than the minimum inhibitory concentration.
The invention has the beneficial effects that:
1. the invention carries out enzymolysis on pig bones by pepsin; the pig bone is hydrolyzed into type I collagen which is suitable for biomineralization of hydroxyapatite calcium, and the-N terminal residue and the-C terminal residue at the two ends of the molecular chain of the type I collagen are cut off by enzymolysis, so that the antigenicity of the collagen I molecule is greatly reduced. The artificial bone carrying the Tyenergy hydroxyapatite and the enzymolysis ossein protein contains collagen which is directly extracted from bone tissues and can be assembled into collagen fibers, and the collagen fibers have a characteristic light and shade interval periodic stripe structure, namely a D-Band structure; is consistent with the D-Band structure of the type I collagen fiber of human bone tissue and can promote biomineralization.
2. The human bone tissue is composed of organic macromolecular type I collagen and inorganic hydroxyapatite calcium, and in the molecular structure, the hydroxyapatite calcium crystal takes type I collagen fiber as a template, is embedded in the molecular gap of collagen fiber, and is mineralized and grown longitudinally along the long axis of the collagen fiber. The invention synthesizes the type I collagen colloid which is consistent with the D-Band structure of type I collagen fiber in human bone tissue. Then, the biomineralization is simulated, hydroxyapatite crystals grow along collagen fibers in a mineralized mode, and finally biomineralization bionic bones are formed. The chemical components and the molecular structure of the material are consistent with the bone tissue of human, and the material can be used as bone grafting material to fill the residual cavity after the bone marrow focus is removed.
3. Aiming at refractory osteomyelitis infected by drug-resistant gram-negative pathogenic bacteria, combining carbapenem antibiotics (imipenem/cilastatin) with high drug sensitivity with artificial bone; synthesizing artificial bone carrying Thai-energy hydroxyapatite and enzymolysis ossein protein. The artificial bone can slowly release imipenem and inhibit infection of drug-resistant gram-negative pathogenic bacteria such as escherichia coli; therefore, the defect that the vancomycin/gentamicin calcium sulfate-loaded artificial bone clinically used at present cannot cover all bacterial spectrums of osteomyelitis is overcome.
Drawings
FIG. 1 shows the results of the bacteriostatic test of artificial bone carrying Thai-energy hydroxyapatite-enzymolysis ossein protein;
FIG. 2 is a transmission electron microscope image of an artificial bone carrying Thailand hydroxyapatite-enzymolysis bone collagen;
FIG. 3 is an electron diffraction image of an artificial bone carrying Tynenerg hydroxyapatite-enzymolysis bone collagen.
Detailed Description
The present invention is further analyzed with reference to the following specific examples.
Example 1:
step (1), preparation of ossein protein
1.1 removing soft tissues around joints at two ends of fresh pig thighbone, cutting long bone, removing periosteum and cancellous bone at bone ends, scraping bone marrow, washing with water to remove a large amount of fat and hemoglobin in the bone, and remaining tubular long bone. And (3) putting the cleaned aggregate in a closed degreasing container, controlling the temperature to be 60-65 ℃, and refluxing for 3-4 h in a petroleum ether countercurrent circulation mode to remove grease in the aggregate.
1.2 decalcification, namely placing the pig bones treated in the step 1.1 into 0.5-1 mol/L hydrochloric acid for decalcification, adding 10-20 ml of hydrochloric acid into every 1g of pig bone particles, changing the hydrochloric acid every 48-72 hours, decalcification for 14-16 days, softening the aggregates, and cutting the decalcification-softened aggregates into small pieces.
1.3 degreasing: and (3) filling the crushed aggregates into a closed degreasing container, controlling the temperature to be 60-65 ℃, refluxing for 3-4 hours in a petroleum ether countercurrent circulation mode, and removing residual grease in the aggregates to obtain the decalcified and degreased aggregates.
1.4 enzymolysis: the decalcified and defatted bone particles were placed in 0.1M acetic acid at pH 3 and pepsin was added. Soaking 1g of pig bone particles in 20ml of acetic acid solution, and adding 200-250 mg of pepsin; standing in a thermostat with the temperature of 30-37 ℃, and carrying out enzymolysis on the pig bones for 30-60 days under an acidic condition; the bone particles were enzymatically hydrolyzed to become transparent, viscous collagen (fig. 3).
Step (2), loading solar energy:
taking 0.5-1 g of tyloxapol, and mixing with 100ml of 0.9% NaCl; the tyloxapol is dissolved in physiological saline to form a tyloxapol solution. Mixing 9-10 ml of bone collagen with 1ml of Tynen solution, and fully stirring; placing the mixture in a 30-37 ℃ thermostat, and standing for 24-48 hours; thai can be loaded into collagen.
Step (3), preparing collagen colloid:
diffusing the bone collagen loaded with the tyloxapol by ammonia gas for 1-2 hours, and then adding 0.05% glutaraldehyde for crosslinking; soaking 1ml of the Tynenerg-loaded ossein in glutaraldehyde, and crosslinking for 1 hour to self-assemble the ossein into jelly-like colloid; and finally, placing the colloid in deionized water for soaking for 1 hour, then repeatedly washing for 3 times, and washing away redundant glutaraldehyde to finish the preparation of the colloid.
Step (4), biomineralization:
sucking the prepared mineralized liquid, pouring the mineralized liquid into a small culture dish, soaking the Tynen-loaded bone collagen colloid in the mineralized liquid, and adding 50ml of mineralized liquid into every 5ml of bone collagen colloid for biological mineralization; and (3) mineralizing for 7 days to form the artificial bone carrying the Tyenergy hydroxyapatite and the enzymolysis bone collagen.
Preparing a mineralized liquid: pouring 25ml of calcium solution into a beaker, and dripping the PAA with the concentration of 350 ug/ml; 1M HCl was added dropwise to adjust the pH to 7.4. Then, 25ml of phosphorus solution is slowly dropped into the mixture, and about 16 drops/min is added to prepare the mineralized solution.
25ml of calcium solution consists of the following components: 10mM CaCl2.2H2O、150mM NaCl、50mM.Tris、0.02wt％NaN3；
25ml of phosphorus solution 6mM Na2HPO4
The experiment of the inhibition zone shows that: the prepared artificial bone carrying the tyloxapol-hydroxyapatite and the enzymolysis bone collagen is placed in a culture medium of drug-resistant gram-negative bacteria escherichia coli, and a bacteriostatic zone is formed around the artificial bone carrying the tyloxapol-hydroxyapatite and the enzymolysis bone collagen (figure 1). In simulated body fluid sustained release experiments: the slow-release imipenem medicine concentration is 1ug/ml, is 8 times of the minimum inhibitory concentration of imipenem/cilastatin to escherichia coli, and is far greater than the minimum inhibitory concentration.
Fig. 2 transmission electron microscopy shows: the artificial bone collagen fiber prepared by the embodiment has a characteristic light and shade interval periodic stripe structure, namely a D-Band structure; consistent with the D-Band structure of type I collagen fibers of natural bone tissue.
Fig. 3 electron diffraction image shows: in the artificial bone prepared in this example, calcium hydroxyapatite crystals were formed, and each bright ring corresponds to a calcium hydroxyapatite crystal plane, which is 002,211 in turn. The artificial bone was shown under transmission electron microscopy: the hydroxyapatite crystal is embedded in the collagen fiber to grow longitudinally, and the molecular structure of the hydroxyapatite crystal is consistent with that of the human bone tissue.
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (8)
1. The nano artificial bone carrying the Thai energy hydroxyapatite and the enzymolysis ossein protein is characterized by mainly comprising the nano artificial bone loaded with the Thai energy on the hydroxyapatite and the enzymolysis ossein protein, wherein the hydroxyapatite and the enzymolysis ossein protein nano artificial bone mineralizes hydroxyapatite crystals along ossein fibers to grow and finally forms bionic bone through biological mineralization;
the preparation method comprises the following steps:
step (1), preparation of ossein protein
Carrying out degreasing and decalcification treatment on fresh tissue rich in ossein, crushing to obtain required aggregate, and carrying out enzymolysis by pepsin to obtain transparent and viscous ossein;
the enzymolysis condition is that the pH value is 3-5, the enzymolysis temperature is 30-37 ℃, and the enzymolysis time is 30-60 days;
step (2), loading solar energy:
mixing the ossein protein obtained in the step (1) with a Tyenergy solution, fully stirring, placing in a 30-37 ℃ thermostat, and standing for 24-48 hours to obtain the Tyenergy-loaded ossein protein;
step (3), preparing collagen colloid:
diffusing the collagen loaded with the energy from the step (2) for 1-2 hours by ammonia gas, and then adding glutaraldehyde for crosslinking; after crosslinking for 1-2 hours, self-assembling the ossein into jelly-like colloid; finally, placing the colloid in deionized water for soaking, and repeatedly washing to remove redundant glutaraldehyde to obtain the collagen colloid loaded with the Tyenergy;
step (4), biomineralization:
soaking the collagen colloid carrying the Thai energy obtained in the step (3) in a mineralization liquid, performing biomineralization, and mineralizing for 7-9 days, thereby forming the artificial bone carrying the Thai energy hydroxyapatite and the enzymatic hydrolysis collagen;
the preparation process of the mineralized liquid comprises adding PAA 350ug/M L dropwise into 25M L calcium liquid, adding 1M HCl dropwise to adjust pH to 7.4, and adding phosphorus liquid 25M L dropwise, wherein the 25M L calcium liquid comprises 10mM CaCl2·2H2O、150mMNaCl、50mM Tris、0.02wt% NaN325m of L phosphorus solution is 6mM Na2HPO4。
2. The nano artificial bone carrying tylenehydroxy apatite-enzymolysis ossein protein of claim 1, wherein the mass ratio of tylenehydroxy apatite to the nano artificial bone carrying tylenehydroxy apatite-enzymolysis ossein protein is 7-10: 100.
3. The nano artificial bone carrying the tylated hydroxyapatite-enzymolysis ossein protein according to claim 1, wherein the mass ratio of the ossein fiber to the hydroxyapatite crystal is 0.5-1: 4.
4. The nano artificial bone carrying tylated hydroxyapatite-enzymatically hydrolyzed collagen according to claim 1, wherein the collagen fiber has a D-Band structure.
5. The nano artificial bone carrying the tylenehydroxyapatite-enzymolyzed bone collagen according to claim 1, wherein the mass ratio of the aggregate and the pepsin in the step (1) is 5-4: 1.
6. The nano artificial bone carrying tyloxapol-hydroxyapatite-enzymolyzed bone collagen of claim 1, wherein the tyloxapol solution in the step (2) is prepared by mixing 0.5-1 g of tyloxapol with 100m L0.9.9 wt% of normal saline;
the volume ratio of the ossein protein to the tylosin solution is 10-9: 1.
7. the nano artificial bone carrying the tylenehydroxy apatite-enzymolyzed ossein protein as claimed in claim 1, wherein the mass-to-volume ratio of the ossein protein loaded with tylenehydroxy to glutaraldehyde in step (3) is 1g (10-20) m L, and the cross-linking temperature is 20-25 ℃.
8. The use of the nano artificial bone carrying tylenehydroxyapatite-enzymatically hydrolyzed collagen according to claim 1 as a bone grafting material.
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