Preparation method of human decellularized alveolar bone matrix particles

Abstract

The invention relates to the technical field of acellular matrix repair materials, and discloses a preparation method of human acellular alveolar bone matrix particles, which comprises the following steps: step S1, clinically obtaining fresh and clean human alveolar bone; and S2, treating the obtained human alveolar bone, and cleaning the human alveolar bone twice. According to the preparation method, after the human alveolar bone taken clinically is cleaned, cells, bloodstains and exogenous DNA in the alveolar bone are successfully removed by using an ultrasonic cleaner and a common deionized water treatment fluid in the market under the condition of not depending on nuclease, so that the immunogenicity of the material is effectively reduced, the biocompatibility and the safety are remarkably improved, and the preparation method is suitable for industrial production. The method not only retains the natural structure and collagen integrity of the alveolar bone, but also simplifies the preparation process and reduces the use of chemical reagents, thereby preparing the safe and non-toxic acellular alveolar bone matrix particles suitable for scientific research and clinical application.

Description

Technical Field

[0001] The invention relates to the technical field of acellular matrix repair materials, in particular to a method for preparing human acellular alveolar bone matrix particles. Background Art

[0002] The periodontal tissue is a complex and orderly tissue structure composed of gums, periodontal ligament and alveolar bone. Due to the limited self-repair ability, these tissues are easily damaged by inflammation, trauma and systemic diseases, so periodontal disease has become the main cause of tooth loss in adults.

[0003] Restoration of periodontal bone defects in patients with periodontal disease is a major challenge faced by periodontists and orthodontists. Current treatment methods include guided tissue regeneration, growth factor injection, stem cell transplantation therapy, and autologous / artificial bone transplantation. Since periodontal bone tissue regeneration is a complex process involving the synergistic action of multiple bioactive factors, the current technical challenges for the preparation of decellularized bone materials in related technologies, although the NaCl-SD method and the Dispase-Triton method combined with the nuclease treatment method have been explored to remove cellular components, they are limited by the cytotoxicity of the reagents, antigen residues, and difficulty in cleaning, which will affect the recellularization of the decellularized bone materials after implantation in the body. In addition, researchers have also tried multiple decellularization methods such as repeated freezing and thawing, mechanical vibration, and biological agents (including enzymes and non-enzymes), but there are still limitations in practical applications. For this reason, we proposed a method for preparing decellularized alveolar bone matrix particles. Summary of the invention

[0004] The present invention provides a method for preparing human acellular alveolar bone matrix particles, which has the advantages of simple process, convenient operation, low equipment cost, short preparation cycle, easy transportation and storage, and higher convenience in clinical application, thus solving the problems raised by the above-mentioned background technology.

[0005] The present invention provides the following technical solution: a method for preparing human acellular alveolar bone matrix particles, comprising the following steps:

[0006] Step S1: obtaining fresh and clean human alveolar bone from the clinic;

[0007] Step S2: processing the obtained human alveolar bone and performing a cleaning operation on it twice;

[0008] Step S3: soaking in the decellularization solution for 10 min;

[0009] Step S4: thoroughly cleaning the human alveolar bone to obtain a human decellularized alveolar bone matrix, and recording the surface characteristics of the human decellularized alveolar bone matrix;

[0010] Step S5: performing a sublimation drying process on the human decellularized alveolar bone matrix, usually for 24 hours;

[0011] Step S6: using a medium throughput tissue grinder and introducing nitrogen to rapidly grind the human decellularized alveolar bone matrix to obtain decellularized alveolar bone matrix microparticles;

[0012] Step S7: recording the surface characteristics of the obtained decellularized alveolar bone matrix microparticles, and placing the remaining decellularized alveolar bone matrix microparticles into a sterilization device for sterilization;

[0013] Step S8: Resuspend the decellularized alveolar bone matrix microparticles after sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, filter the sample with a 70 μm filter, and transport or store it at a temperature of -80°C.

[0014] As a preferred technical solution of the present invention: step S1 specifically includes: the population from which human alveolar bones are taken is adolescents, specifically referring to human alveolar bones around the tooth roots of orthognathic surgery patients aged 18-25 and having no clinical application value; after taking the human alveolar bones, the necrotic periodontal membrane and soft tissue need to be removed, and then they are repeatedly rinsed with phosphate buffered saline solution, and then the fresh human alveolar bones are immersed in deionized water before the next stage begins.

[0015] As a preferred technical solution of the present invention: Step S2 specifically includes: taking out the human alveolar bone matrix in deionized water, and then putting it into an ultrasonic cleaner, and cleaning the human alveolar bone matrix twice at a frequency of once every 30 minutes.

[0016] As a preferred technical solution of the present invention: Step S3 specifically comprises: taking a 17% ethylenediaminetetraacetic acid solution, then soaking the cleaned human alveolar bone matrix in the solution for 10 minutes, and then thoroughly washing it with sterile water.

[0017] As a preferred technical solution of the present invention: Steps S4-S7 specifically include: using a handheld cutter to cut the human alveolar bone matrix into small pieces and perform decellularization treatment to obtain decellularized alveolar bone matrix, then observing the decellularized alveolar bone matrix through a scanning electron microscope to evaluate the decellularization effect, and the remaining human alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours, and after sublimation, a medium-throughput tissue grinder is used to grind at a speed of 1200 rpm for 5 minutes, and repeated 3-6 times if necessary to obtain decellularized alveolar bone matrix microparticles, and then sterilized in a high-temperature autoclave for 12 hours, and then 50 grams of decellularized alveolar bone matrix microparticles are resuspended in a sterile phosphate buffer (100 ml) containing 100 U / ml penicillin and 100 mg / ml streptomycin, and cultured in an incubator set at a temperature environment of 37°C for three days, and filtered with a 70 μm filter. The samples were stored at -80°C until needed, and the surface characteristics of the decellularized alveolar bone matrix microparticles were observed. The specific observation process was as follows: the obtained human decellularized alveolar bone matrix and human alveolar bone matrix samples were dehydrated in gradient ethanol solutions (60%, 75%, 85%, 95% and 100%, each soaked for 10 minutes), hexamethyldisilane was added dropwise on the dehydrated samples, and they were placed in a fume hood and dried at room temperature for 8-10 hours. Subsequently, gold was plated on the surface of the dried samples using an ion sputtering device, and the morphology of human alveolar bone and human decellularized alveolar bone matrix was observed and photographed under a scanning electron microscope. The chemical composition of the human decellularized alveolar bone matrix was analyzed using an X-ray scanning electron microscopy spectrometer according to the manufacturer's instructions, and then the chemical properties of the decellularized alveolar bone matrix microparticles were analyzed using a Fourier transform infrared spectrometer according to the manufacturer's instructions.

[0018] As a preferred technical solution of the present invention: Step S8 specifically includes: resuspending the decellularized alveolar bone matrix microparticles after sterilization with sterile PBS containing 100U / mL penicillin and 100mg / mL streptomycin, filtering the sample with a 70μm filter, and transporting or storing it at a temperature environment of -80°C.

[0019] The present invention has the following beneficial effects:

[0020] 1. The method for preparing human decellularized alveolar bone matrix microparticles, after cleaning the clinically obtained human alveolar bone, uses an ultrasonic cleaner and a deionized water treatment solution commonly available on the market to successfully remove cells, blood stains and exogenous DNA in the alveolar bone without relying on nucleases, effectively reducing the immunogenicity of the material and significantly improving the biocompatibility and safety. This method not only retains the natural structure and collagen integrity of the alveolar bone, but also simplifies the preparation process and reduces the use of chemical reagents, thereby preparing safe and non-toxic decellularized alveolar bone matrix microparticles suitable for scientific research and clinical applications.

[0021] 2. The method for preparing human decellularized alveolar bone matrix particles is to prepare human decellularized alveolar bone matrix particles by adding a shell prepared by an environmental temperature-sensitive gelling agent. When taking the particles, there is no need to manually select or weigh the amount of the required repair material, and the powdered repair material can be prevented from falling and increasing the material loss. Therefore, when performing alveolar tooth repair on patients, dentists can directly use single or multiple human decellularized alveolar bone matrix particles, thereby ensuring the smoothness of the entire repair process and accelerating the dentist's treatment efficiency for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the preparation process of the present invention;

[0023] Figure 2 Schematic diagram of the preparation process of hDABMPs of the present invention;

[0024] Figure 3 Schematic diagram of hAB and hDABM of the present invention at high and low magnifications;

[0025] Figure 4 The present invention is a schematic diagram of the XPS analysis results of hDABM;

[0026] Figure 5 It is a schematic diagram of the FTIR detection results of hydroxyapatite (HAP) and hDABMPs of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] See also Figure 1-Figure 5 A method for preparing human acellular alveolar bone matrix particles comprises the following steps:

[0029] Step S1: obtaining fresh and clean human alveolar bone from the clinic;

[0030] Step S2: processing the obtained human alveolar bone and performing a cleaning operation on it twice;

[0031] Step S3: soaking in the decellularization solution for 10 min;

[0032] Step S4: thoroughly cleaning the human alveolar bone to obtain a human decellularized alveolar bone matrix, and recording the surface characteristics of the human decellularized alveolar bone matrix;

[0033] Step S5: performing a sublimation drying process on the human decellularized alveolar bone matrix, usually for 24 hours;

[0034] Step S6: using a medium throughput tissue grinder and introducing nitrogen to rapidly grind the human decellularized alveolar bone matrix to obtain decellularized alveolar bone matrix microparticles;

[0035] Step S7: recording the surface characteristics of the obtained decellularized alveolar bone matrix microparticles, and placing the remaining decellularized alveolar bone matrix microparticles into a sterilization device for sterilization;

[0036] Step S8: Resuspend the decellularized alveolar bone matrix microparticles after sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, filter the sample with a 70 μm filter, and transport or store it at a temperature of -80°C.

[0037] In a preferred embodiment: Step S1 specifically includes: the population from which human alveolar bones are taken is adolescents, specifically human alveolar bones around the tooth roots of orthognathic surgery patients aged 18-25 and having no clinical application value; after taking the human alveolar bones, the necrotic periodontal membrane and soft tissue need to be removed, and then they are repeatedly rinsed with phosphate buffered saline solution, and then the fresh human alveolar bones are soaked in deionized water before the next stage begins.

[0038] In a preferred embodiment: Step S2 specifically includes: taking out the human alveolar bone matrix in deionized water, and then putting it into an ultrasonic cleaner to clean the human alveolar bone matrix twice at a frequency of once every 30 minutes.

[0039] In a preferred embodiment: step S3 specifically comprises: taking a 17% ethylenediaminetetraacetic acid solution, then soaking the cleaned human alveolar bone matrix in the solution for 10 minutes, and then thoroughly washing it with sterile water.

[0040] In a preferred embodiment: Steps S4-S7 specifically include: using a handheld cutter to cut the human alveolar bone matrix into small pieces and perform a decellularization treatment to obtain a decellularized alveolar bone matrix, then observing the decellularized alveolar bone matrix by scanning electron microscopy to evaluate the decellularization effect, and the remaining human alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours, and after sublimation, a medium-throughput tissue grinder is used to grind at a speed of 1200 rpm for 5 minutes, and repeated 3-6 times if necessary to obtain decellularized alveolar bone matrix microparticles, and then sterilized in a high-temperature autoclave for 12 hours, and then 50 grams of decellularized alveolar bone matrix microparticles are resuspended in a sterile phosphate buffer (100 ml) containing 100 U / ml penicillin and 100 mg / ml streptomycin, and cultured in an incubator set at a temperature environment of 37°C for three days, and the sample is filtered with a 70 μm filter , and stored at -80°C until needed, and the surface characteristics of decellularized alveolar bone matrix microparticles were observed. The specific observation process was as follows: the obtained human decellularized alveolar bone matrix and human alveolar bone matrix samples were dehydrated in gradient ethanol solutions (60%, 75%, 85%, 95% and 100%, each soaked for 10 minutes), hexamethyldisilane was dropped on the dehydrated samples, and placed in a fume hood for drying at room temperature for 8-10 hours, and then, the dried sample surface was gold-plated using an ion sputtering device, and the morphology of human alveolar bone and human decellularized alveolar bone matrix was observed and photographed under a scanning electron microscope, and then the chemical composition of human decellularized alveolar bone matrix was analyzed using an X-ray scanning electron microscopy spectrometer according to the manufacturer's instructions, and then the chemical properties of decellularized alveolar bone matrix microparticles were analyzed using a Fourier transform infrared spectrometer according to the manufacturer's instructions.

[0041] In a preferred embodiment: Step S8 specifically includes: sterilizing with sterile PBS containing 100U / mL penicillin and 100mg / mL streptomycin and then resuspending the decellularized alveolar bone matrix microparticles, filtering the sample with a 70μm filter, and transporting or storing it at a temperature environment of -80°C.

[0042] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0043] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing human acellular alveolar bone matrix particles, characterized in that: The following steps are involved: Step S1: obtaining fresh and clean human alveolar bone from the clinic; Step S2: processing the obtained human alveolar bone and performing a cleaning operation on it twice; Step S3: soaking in the decellularization solution for 10 min; Step S4: thoroughly cleaning the human alveolar bone to obtain a human decellularized alveolar bone matrix, and recording the surface characteristics of the human decellularized alveolar bone matrix; Step S5: performing a sublimation drying process on the human decellularized alveolar bone matrix, usually for 24 hours; Step S6: using a medium throughput tissue grinder and introducing nitrogen to rapidly grind the human decellularized alveolar bone matrix to obtain decellularized alveolar bone matrix microparticles; Step S7: recording the surface characteristics of the obtained decellularized alveolar bone matrix microparticles, and placing the remaining decellularized alveolar bone matrix microparticles into a sterilization device for sterilization; Step S8: Resuspend the decellularized alveolar bone matrix microparticles after sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, filter the sample with a 70 μm filter, and transport or store it at a temperature of -80°C.

2. The method for preparing human acellular alveolar bone matrix particles according to claim 1, characterized in that: Step S1 specifically includes: the population from which human alveolar bones are taken is adolescents, specifically referring to human alveolar bones around the tooth roots of orthognathic surgery patients aged 18-25 and having no clinical application value; after taking the human alveolar bones, the necrotic periodontal membrane and soft tissue need to be removed, and then they are repeatedly rinsed with phosphate buffered saline solution, and then the fresh human alveolar bones are soaked in deionized water before the next stage begins.

3. The method for preparing human acellular alveolar bone matrix particles according to claim 1, characterized in that: Step S2 specifically includes: taking out the human alveolar bone matrix in deionized water, and then putting it into an ultrasonic cleaner to clean the human alveolar bone matrix twice at a frequency of once every 30 minutes.

4. The method for preparing human acellular alveolar bone matrix particles according to claim 1, characterized in that: Step S3 specifically includes: taking a 17% ethylenediaminetetraacetic acid solution, then soaking the cleaned human alveolar bone matrix in the solution for 10 minutes, and then thoroughly washing it with sterile water.

5. The method for preparing human acellular alveolar bone matrix particles according to claim 1, characterized in that: Steps S4-S7 specifically include: using a handheld cutter to cut the human alveolar bone matrix into small pieces and perform decellularization to obtain decellularized alveolar bone matrix, then observing the decellularized alveolar bone matrix under a scanning electron microscope to evaluate the decellularization effect, and the remaining human alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours, and after sublimation, it is ground at a speed of 1200 rpm for 5 minutes using a medium-throughput tissue grinder, and repeated 3-6 times if necessary to obtain decellularized alveolar bone matrix microparticles, and then sterilized in a high-temperature autoclave for 12 hours, and then 50 grams of decellularized alveolar bone matrix microparticles are resuspended in sterile phosphate buffer (100 ml) containing 100 U / ml penicillin and 100 mg / ml streptomycin, and cultured in an incubator set to a temperature environment of 37°C for three days, and the sample is filtered with a 70 μm filter and incubated at -80 The samples were stored at 400 °C until needed, and the surface characteristics of the decellularized alveolar bone matrix microparticles were observed. The specific observation process was as follows: the obtained human decellularized alveolar bone matrix and human alveolar bone matrix samples were dehydrated in gradient ethanol solutions (60%, 75%, 85%, 95% and 100%, each soaked for 10 minutes), hexamethyldisilane was added dropwise on the dehydrated samples, and the samples were placed in a fume hood and dried at room temperature for 8-10 hours. Subsequently, the dried sample surfaces were gold plated using an ion sputtering device, and the morphologies of human alveolar bone and human decellularized alveolar bone matrix were observed and photographed under a scanning electron microscope. The chemical composition of the human decellularized alveolar bone matrix was analyzed using an X-ray scanning electron microscopy spectrometer according to the manufacturer's instructions, and the chemical properties of the decellularized alveolar bone matrix microparticles were analyzed using a Fourier transform infrared spectrometer according to the manufacturer's instructions.

6. The method for preparing human acellular alveolar bone matrix particles according to claim 1, characterized in that: Step S8 specifically includes: resuspending the decellularized alveolar bone matrix microparticles after sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, filtering the sample with a 70 μm filter, and transporting or storing it at a temperature of -80°C.

Images