CN111671977A - Superparamagnetic nanoparticle bone repair membrane and preparation method thereof - Google Patents
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Abstract
The invention discloses a superparamagnetic nanoparticle bone repair membrane and a preparation method thereof, wherein the superparamagnetic nanoparticle modified bone repair membrane is prepared by taking collagen extracted from bovine tendon and obtained by an enzymolysis method as a matrix and assembling superparamagnetic nanoparticles (gamma-Fe)2O3). After the surgical operation of bone trauma, since the proliferation speed of the connective tissue is faster than that of the bone tissue in the bone healing process, the proliferated connective tissue occupies the bone defect area too early to block the growth of the bone tissue, thus leading to bone nonunion or slow healing. Therefore, the bone repair membrane modified by the superparamagnetic nano particles is placed at the bone defect part and can be used as a mechanical barrier to control the peripheral tissues to grow into the bone defect area,providing a guide space for the growth of bone cells; and secondly, after the bone trauma surgery, the superparamagnetic nanoparticles have electromagnetic effect and other indirect effects under the action of an external specific magnetic field, so that the repair of the bone tissue injury can be promoted, and the effect of accelerating recovery is achieved.
Description
Technical Field
The invention belongs to the field of medical materials, and particularly relates to a superparamagnetic nanoparticle bone repair membrane and a preparation method thereof.
Background
Periosteum is a dense connective tissue membrane covering the surface of bone (except joint surfaces), and fresh periosteum is pink and rich in blood vessels, nerves and osteoblasts. The periosteum is divided into an inner layer and an outer layer, and the fibrous layer contains fibroblasts, collagen and elastic fibers, and a network of nerves and capillaries, so that the mechanical stability of the periosteum is provided. The cambium stores a large number of different types of cells, providing a source of cells for bone formation and repair. The role of natural periosteum in maintaining normal skeletal blood circulation and metabolism has been widely recognized by anatomical, physiological, pathological and clinical orthopedics medicine. Clinically, the protection and reconstruction of periosteum directly affect the success or failure of bone trauma or bone disease treatment. Aiming at defective bone grafting, the application of a bone repair membrane guided regeneration technology can effectively prevent connective tissues from occupying a bone defect area too early and promote bone healing. The artificial periosteum can effectively prevent connective tissues from growing into a junction area between the bone grafting material and the host bone defect by wrapping the bone defect part with the artificial periosteum, and creates good conditions for guiding the bone tissues with slower growth speed to migrate into the bone defect area.
The magnetic field is a non-invasive physical factor and can directly act on organisms to generate corresponding biological effects, and when the magnetic field of the organisms is changed, the activity of cell molecules and the activity of the organisms are influenced. A tissue of a living body contains a large number of free electrons, and these free electrons are stationary in a natural state. However, when the organism tissue is in an electromagnetic environment, the free electrons in the organism tissue are excited to move continuously, so that the organism in the changing electromagnetic field can generate a very strong response under very small stimulation. At present, the research on treating bone diseases by utilizing the electromagnetic effect is carried out in China, and the feasibility of the electromagnetic effect for exerting the treatment effect is proved. The magnetic nanoparticles are a chemical compound formed by combining nanoparticles and magnetic elements such as iron, nickel and the like, and have the advantages of special magnetic effect, magnetic guidance, good biocompatibility, low toxicity, long blood circulation time, specific advantages due to small size and the like. In one aspect, the gamma-Fe used in the present invention2O3The iron oxide particles have passed the safety certification of the ISO international quality control system and have low cytotoxicity to organisms; on the other hand, the remanence Mr and the coercive force Hc of the superparamagnetic nanoparticles are basicallyTends to zero, resulting in only very weak magnetic dipole interactions from particle to particle, enabling the formation of stable magnetic fluids. At present, the iron oxide nanoparticles are widely applied biomedical nano materials, have good biocompatibility, in-vivo stability and special electromagnetic properties, can generate a plurality of unique effects under an external electromagnetic field, and are increasingly researched and paid attention to magnetic drug carriers, tumor thermotherapy and brain deep magnetic stimulation.
Disclosure of Invention
The technical problem is as follows: in order to solve the problems, the invention discloses a superparamagnetic nanoparticle bone repair membrane and a preparation method thereof.
The technical scheme is as follows: in order to achieve the aim, the superparamagnetic nanoparticle bone repair film is a natural biological inactivated acellular dermal matrix collagen film with gamma-Fe on the surface2O3A coating of superparamagnetic nanoparticles.
The bone repair membrane is prepared by extracting collagen from tendon of cattle, taking collagen obtained by enzymolysis as matrix, and assembling superparamagnetic nanoparticle gamma-Fe on the surface2O3。
The preparation method of the superparamagnetic nanoparticle bone repair membrane comprises the following steps:
step 1: removing sarcolemma and fascia from adult bovine achilles tendon, washing with normal saline, soaking in 75% alcohol solution for 15-25min, freezing in a low temperature refrigerator of-60 deg.C, cutting into 0.6-1.2mm slices, and soaking in acetic acid solution for 1-3 hr;
step 2: taking out the thin slice, repeatedly washing with PBS and soaking for 1-2h, washing with distilled water, taking out, further digesting with trypsin, placing in an ultrasonic cleaning instrument, ultrasonically treating for 44-52h, taking out, washing with PBS for 20-30 times, and washing with distilled water for 30-40 times to obtain acellular dermal matrix;
and step 3: placing the acellular dermal matrix into a formaldehyde solution for 2-3h, taking out, washing with PBS, and washing with distilled water;
and 4, step 4: after the above steps are completed, the acellular dermal matrix collagen membrane is placed on gamma-Fe2O3And (3) putting the nano-particle colloidal solution into a freeze dryer for freeze-drying at the temperature of 30-40 ℃ for 15-20 min, taking out the nano-particle colloidal solution, and then irradiating and disinfecting by cobalt-60 (Co) to obtain the superparamagnetic nano-particle bone repair membrane for later use.
Wherein;
in step 1, taking the adult bovine achilles tendon, removing the sarcolemma and fascia, and flushing 8-12 times with physiological saline.
And (2) taking out the slices, repeatedly washing the slices with PBS for 20-30 times, and washing the slices with distilled water for 20-30 times.
And 3, putting the acellular dermal matrix into a formaldehyde solution for 2-3h, taking out, washing with PBS for 30-35 times, and washing with distilled water for 20-30 times.
The application method of the superparamagnetic nanoparticle bone repair membrane comprises the following steps: mixing superparamagnetic gamma-Fe2O3The nano particles are assembled on the bone repair membrane, and under the action of an external magnetic field, the superparamagnetic iron oxide magnetic nano particles can generate an electromagnetic effect and other indirect effects to promote the repair of bone tissue damage.
Has the advantages that: the superparamagnetic nanoparticle acellular dermal matrix collagen membrane is implanted into a bone injury part, and performs a specific action on bone tissues by utilizing the electromagnetic effect and other indirect effects of the magnetic nanoparticles under an externally applied electromagnetic field, such as a magnetocaloric effect, a magnetoelastic effect and the like. Because the magnetic material can gather the electromagnetic field, the action intensity of the electromagnetic field is locally enhanced in the bone tissue, the electromagnetic action energy at the interface of the magnetic nanoparticle assembly structure and the tissue cells is greatly increased, and the point can ensure that a stronger electromagnetic effect can be generated at the interface of the nano material even if only the electromagnetic field with lower intensity exists in the external environment. Meanwhile, the electromagnetic field generated by the polarization or magnetization of the nanoparticle interface is quickly attenuated along with the increase of the distance, so that the electromagnetic effect is a local electromagnetic effect, the in-vivo electromagnetic effect is only acted on the local part of the bone injury tissue, and the simultaneous influence on multiple parts of the whole body is avoided.
Detailed Description
The invention is further illustrated below with the understanding that the following detailed description is illustrative of the invention only and is not intended to limit the invention
The scope of the invention.
The bone repair membrane is a natural inactivated acellular dermal matrix collagen membrane, can be collagen extracted from bovine tendon and obtained by enzymolysis, and is assembled with superparamagnetic nanoparticles (gamma-Fe) on the surface2O3)。
The application method of the superparamagnetic nanoparticle bone repair membrane comprises the following steps: the superparamagnetic iron oxide magnetic nano particle is assembled on a bone repair membrane, and can generate an electromagnetic effect and other indirect effects under the action of an external magnetic field to promote the repair of bone tissue damage.
The preparation method of the superparamagnetic nanoparticle bone repair membrane comprises the following steps:
example 1
Step 1: removing sarcolemma and fascia from adult bovine achilles tendon, washing with normal saline for 10 times, soaking in 75% alcohol solution for 20min, freezing in a low temperature refrigerator of-60 deg.C, cutting into 1mm slices, and soaking in acetic acid solution for 2 hr.
Step 2: the slices were taken out and washed with PBS for 25 times and soaked for 1.5h, washed with distilled water for 25 times, further digested with trypsin after being taken out, placed in an ultrasonic cleaner, treated with ultrasound for 48h, taken out and washed with PBS for 25 times and washed with distilled water for 35 times.
And step 3: the decellularized slice is placed in a formaldehyde solution for 2.5h, taken out and washed with PBS for 33 times and distilled water for 25 times.
And 4, step 4: after the above steps are completed, the collagen membrane is placed on the gamma-Fe2O3Adding the nano-particle colloid solution at 35 deg.C for 17min, freeze-drying in a freeze-drying machine, and irradiating with cobalt-60 (Co) after taking outAnd (6) sterilizing.
Example 2
Step 1: removing sarcolemma and fascia from adult bovine achilles tendon, washing with normal saline for 8 times, soaking in 75% alcohol solution for 25min, freezing in a low temperature refrigerator of-60 deg.C, cutting into 0.6mm slices, and soaking in acetic acid solution for 3 hr.
Step 2: the slices were taken out and repeatedly washed with PBS for 20 times and soaked for 2h, washed with distilled water for 20 times, then further digested with trypsin after being taken out, placed in an ultrasonic cleaner, ultrasonically treated for 52h, taken out and washed with PBS for 20 times and washed with distilled water for 40 times.
And step 3: the cell-free slice is put into a formaldehyde solution for 2 hours, taken out and washed with PBS for 35 times and distilled water for 20 times.
And 4, step 4: after the above steps are completed, the collagen membrane is placed on the gamma-Fe2O3And (3) putting the nano-particle colloidal solution into a freeze dryer for freeze drying at the temperature of 30 ℃ for 20min, and taking out the nano-particle colloidal solution for irradiation disinfection by cobalt-60 (Co).
Example 3
Step 1: removing sarcolemma and fascia from adult bovine achilles tendon, washing with normal saline for 12 times, soaking in 75% alcohol solution for 15min, freezing in a low temperature refrigerator of-60 deg.C, cutting into 1.2mm slices, and soaking in acetic acid solution for 1 h.
Step 2: the slices are taken out and repeatedly washed by PBS for 30 times and soaked for 1h, and washed by distilled water for 30 times, then taken out and further digested by trypsin, and then placed into an ultrasonic cleaner for ultrasonic treatment for 44h, and then taken out and washed by PBS for 30 times and distilled water for 30 times.
And step 3: the cell-free slice is put into a formaldehyde solution for 3 hours, washed 30 times by PBS after being taken out, and washed 30 times by distilled water.
And 4, step 4: after the above steps are completed, the collagen membrane is placed on the gamma-Fe2O3And (3) putting the nano-particle colloidal solution into a freeze dryer for freeze drying at the temperature of 40 ℃ for 15min, and taking out the nano-particle colloidal solution for irradiation disinfection by cobalt-60 (Co).
The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features.
Claims (7)
1. A superparamagnetic nanoparticle bone repair membrane is characterized in that: the bone repair membrane is a natural biological inactivated acellular dermal matrix collagen membrane with gamma-Fe on the surface2O3A coating of superparamagnetic nanoparticles.
2. The superparamagnetic nanoparticulate bone repair film according to claim 1, wherein: the bone repair membrane is prepared by extracting collagen from tendon of cattle, taking collagen obtained by enzymolysis as matrix, and assembling superparamagnetic nanoparticle gamma-Fe on the surface2O3。
3. A preparation method of a superparamagnetic nanoparticle bone repair membrane is characterized by comprising the following steps: the preparation method comprises the following steps:
step 1: removing sarcolemma and fascia from adult bovine achilles tendon, washing with normal saline, soaking in 75% alcohol solution for 15-25min, freezing in a low temperature refrigerator of-60 deg.C, cutting into 0.6-1.2mm slices, and soaking in acetic acid solution for 1-3 hr;
step 2: taking out the thin slice, repeatedly washing with PBS and soaking for 1-2h, washing with distilled water, taking out, further digesting with trypsin, placing in an ultrasonic cleaning instrument, ultrasonically treating for 44-52h, taking out, washing with PBS for 20-30 times, and washing with distilled water for 30-40 times to obtain acellular dermal matrix;
and step 3: placing the acellular dermal matrix into a formaldehyde solution for 2-3h, taking out, washing with PBS, and washing with distilled water;
and 4, step 4: after the above steps are completed, the acellular dermal matrix collagen membrane is placed on gamma-Fe2O3And (3) putting the nano-particle colloidal solution into a freeze dryer for freeze-drying at the temperature of 30-40 ℃ for 15-20 min, taking out the nano-particle colloidal solution, and then irradiating and disinfecting by cobalt-60 (Co) to obtain the superparamagnetic nano-particle bone repair membrane for later use.
4. The method for preparing a superparamagnetic nanoparticle bone repair film according to claim 3, wherein: in step 1, taking the adult bovine achilles tendon, removing the sarcolemma and fascia, and flushing 8-12 times with physiological saline.
5. The method for preparing a superparamagnetic nanoparticle bone repair film according to claim 3, wherein: and (2) taking out the slices, repeatedly washing the slices with PBS for 20-30 times, and washing the slices with distilled water for 20-30 times.
6. The method for preparing a superparamagnetic nanoparticle bone repair film according to claim 3, wherein: and 3, putting the acellular dermal matrix into a formaldehyde solution for 2-3h, taking out, washing with PBS for 30-35 times, and washing with distilled water for 20-30 times.
7. A method of using the superparamagnetic nanoparticulate osteochondral repair membrane of claim 1, wherein: the using method of the bone repair membrane comprises the following steps: mixing superparamagnetic gamma-Fe2O3The nano particles are assembled on the bone repair membrane, and under the action of an external magnetic field, the superparamagnetic iron oxide magnetic nano particles can generate an electromagnetic effect and other indirect effects to promote the repair of bone tissue damage.
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Application publication date: 20200918 |
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