A method for preparing human acellular dental alveolar bone matrix microparticles

Abstract

The present application relates to the technical field of acellular matrix repair materials, and discloses a preparation method of human acellular alveolar bone matrix microparticles, comprising the following steps: step S1: obtaining fresh and clean human alveolar bone from a clinic; and step S2: processing the obtained human alveolar bone and performing two cleaning operations on the human alveolar bone. According to the preparation method, after the human alveolar bone obtained from the clinic is cleaned, the cells, blood stains and exogenous DNA in the alveolar bone are successfully removed by using an ultrasonic cleaner and a common deionized water treatment liquid on the market without relying on a nuclease, the immunogenicity of the material is effectively reduced, the biocompatibility and safety are significantly improved, the natural structure and collagen integrity of the alveolar bone are retained, the preparation process is simplified, and the use of chemical reagents is reduced, so that the acellular alveolar bone matrix microparticles which are safe, non-toxic and suitable for scientific research and clinical application are prepared.

Description

Technical Field

[0001] This invention relates to the field of decellularized matrix repair materials technology, specifically a method for preparing human decellularized alveolar bone matrix microparticles. Background Technology

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

[0003] Repairing periodontal bone defects in patients with periodontal disease is a major challenge for periodontists and orthodontists. Current treatment methods include guided tissue regeneration, growth factor injection, stem cell transplantation, and autologous / artificial bone grafting. Since periodontal bone tissue regeneration is a complex process involving the synergistic effects of multiple bioactive factors, the preparation of decellularized bone materials presents technical challenges. Although methods such as NaCl-SD and Dispase-Triton combined with nuclease treatment have been explored to remove cellular components, these methods are limited by reagent cytotoxicity, antigen residue, and difficulty in washing, which can affect the recellularization of decellularized bone materials after implantation. In addition, researchers have also tried multiple decellularization methods such as repeated freeze-thaw cycles, mechanical vibration, and biological agents (including enzymes and non-enzymes), but their practical application still has limitations. Therefore, we propose a method for preparing decellularized alveolar bone matrix microparticles. Summary of the Invention

[0004] This invention provides a method for preparing human decellularized alveolar bone matrix microparticles, which has the advantages of simple process, convenient operation, low equipment cost, short preparation cycle, easy transportation and storage, and greater convenience for clinical application, thus solving the problems mentioned in the background art.

[0005] This invention provides the following technical solution: a method for preparing human decellularized alveolar bone matrix microparticles, comprising the following steps:

[0006] Step S1: Obtain fresh, clean human alveolar bone from the clinic;

[0007] Step S2: Process the obtained human alveolar bone and perform two cleaning operations on it;

[0008] Step S3: Immerse in decellularization solution for 10 minutes;

[0009] Step S4: Thoroughly clean the human alveolar bone once to obtain human decellularized alveolar bone matrix, and record the surface characteristics of the human decellularized alveolar bone matrix at the same time;

[0010] Step S5: Sublime drying of human decellularized alveolar bone matrix for 24 hours;

[0011] Step S6: Use a medium-throughput tissue grinder and simultaneously introduce nitrogen gas to rapidly grind the decellularized alveolar bone matrix of human teeth to obtain decellularized alveolar bone matrix microparticles.

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

[0013] Step S8: After sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, resuspend the decellularized alveolar bone matrix microparticles, 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 alveolar bone to be taken from adolescents, specifically referring to the alveolar bone around the tooth roots of orthognathic surgery patients aged 18-25 years that has no clinical application value. After taking the alveolar bone, the necrotic periodontal ligament and soft tissue need to be removed, and then it is repeatedly rinsed with phosphate buffer solution. Before the next stage begins, the fresh alveolar bone is soaked in deionized water.

[0015] As a preferred technical solution of the present invention, step S2 specifically includes: taking out the human alveolar bone matrix from 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.

[0016] As a preferred technical solution of the present invention, step S3 specifically includes: taking a 17% ethylenediaminetetraacetic acid solution, immersing the cleaned human alveolar bone matrix in the solution for 10 minutes, and then thoroughly rinsing 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 human alveolar bone matrix into small pieces and performing decellularization treatment to obtain decellularized alveolar bone matrix; then observing the decellularized alveolar bone matrix using scanning electron microscopy to evaluate the decellularization effect; the remaining human alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours; after sublimation, it is ground using a medium-throughput tissue homogenizer at a speed of 1200 rpm for 5 minutes, repeated 3-6 times if necessary, to obtain decellularized alveolar bone matrix microparticles; subsequently, it is sterilized using a high-temperature autoclave for 12 hours; 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 at a temperature of 37°C for three days; and filtered through a 70 μm filter. The samples were stored at -80℃ until needed, and the surface characteristics of the decellularized alveolar bone matrix microparticles were observed. The specific observation procedure 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 to the dehydrated samples, and they were placed in a fume hood to dry at room temperature for 8-10 hours. Subsequently, gold was plated on the dried sample surface 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. Then, according to the manufacturer's instructions, the chemical composition of human decellularized alveolar bone matrix was analyzed using X-ray scanning electron microscopy. Finally, according to the manufacturer's instructions, the chemical characteristics of the decellularized alveolar bone matrix microparticles were analyzed using Fourier transform infrared spectroscopy.

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

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

[0020] 1. The method for preparing decellularized alveolar bone matrix microparticles involves cleaning clinically obtained human alveolar bone and then using an ultrasonic cleaner and commercially available deionized water treatment solution to successfully remove cells, blood stains, and exogenous DNA from the alveolar bone without relying on nucleases. This effectively reduces the immunogenicity of the material and significantly improves its biocompatibility and safety. This method not only preserves the natural structure and collagen integrity of the alveolar bone but also simplifies the preparation process and reduces the use of chemical reagents, thereby producing safe, 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 microparticles involves adding an environmental temperature-sensitive gelling agent to create a shell, thereby producing human decellularized alveolar bone matrix microparticles. This eliminates the need for manual selection or weighing of the required amount of restorative material during use, and also avoids the loss of powdered restorative material. This allows dentists to directly use single or multiple human decellularized alveolar bone matrix microparticles during alveolar tooth restoration, ensuring a smooth restoration process and accelerating the dentist's treatment efficiency. Attached Figure Description

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

[0023] Figure 2 This is a schematic diagram of the preparation process of hDABMPs of the present invention;

[0024] Figure 3 This is a schematic diagram of hAB and hDABM at high and low magnifications according to the present invention;

[0025] Figure 4 This is a schematic diagram of the XPS analysis results of hDABM in this invention;

[0026] Figure 5 This is a schematic diagram of the FTIR detection results of hydroxyapatite (HAP) and hDABMPs in this invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Please see Figures 1-5 A method for preparing human decellularized alveolar bone matrix microparticles includes the following steps:

[0029] Step S1: Obtain fresh, clean human alveolar bone from the clinic;

[0030] Step S2: Process the obtained human alveolar bone and perform two cleaning operations on it;

[0031] Step S3: Immerse in decellularization solution for 10 minutes;

[0032] Step S4: Thoroughly clean the human alveolar bone once to obtain human decellularized alveolar bone matrix, and record the surface characteristics of the human decellularized alveolar bone matrix at the same time;

[0033] Step S5: Sublime drying of human decellularized alveolar bone matrix for 24 hours;

[0034] Step S6: Use a medium-throughput tissue grinder and simultaneously introduce nitrogen gas to rapidly grind the decellularized alveolar bone matrix of human teeth to obtain decellularized alveolar bone matrix microparticles.

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

[0036] Step S8: After sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, resuspend the decellularized alveolar bone matrix microparticles, 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 alveolar bone to be taken from adolescents, specifically referring to alveolar bone around the tooth roots of orthognathic surgery patients aged 18-25 years that has no clinical application value. After taking the alveolar bone, necrotic periodontal ligament and soft tissue need to be removed, and then it is repeatedly rinsed with phosphate buffer solution. Before the next stage begins, the fresh alveolar bone is soaked in deionized water.

[0038] In a preferred embodiment, step S2 specifically includes: removing the human alveolar bone matrix from deionized water and then immersing it in 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 includes: taking a 17% ethylenediaminetetraacetic acid solution, immersing the cleaned human alveolar bone matrix in the solution for 10 minutes, and then thoroughly rinsing it with sterile water.

[0040] In a preferred embodiment: Steps S4-S7 specifically include: using a handheld cutter to cut human alveolar bone matrix into small pieces and performing decellularization treatment to obtain decellularized alveolar bone matrix; then observing the decellularized alveolar bone matrix using scanning electron microscopy to evaluate the decellularization effect; the remaining human alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours; after sublimation, it is ground using a medium-throughput tissue homogenizer at 1200 rpm for 5 minutes, repeated 3-6 times if necessary, to obtain decellularized alveolar bone matrix microparticles; subsequently, it is sterilized using a high-temperature autoclave for 12 hours; then 50 g 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 incubated in an incubator at 37°C for three days; the sample is then filtered using a 70 μm filter. The samples were stored at -80℃ until needed. The surface characteristics of the decellularized alveolar bone matrix microparticles were observed. The specific observation procedure was as follows: 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 to the dehydrated samples, and they were dried at room temperature in a fume hood for 8-10 hours. Subsequently, gold was deposited on the dried sample surface using an ion sputtering device. The morphology of human alveolar bone and human decellularized alveolar bone matrix was observed and photographed under a scanning electron microscope. Following the manufacturer's instructions, the chemical composition of the human decellularized alveolar bone matrix was analyzed using X-ray scanning electron microscopy. Finally, according to the manufacturer's instructions, the chemical characteristics of the decellularized alveolar bone matrix microparticles were analyzed using Fourier transform infrared spectroscopy.

[0041] In a preferred embodiment, step S8 specifically includes: resuspending 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.

[0042] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

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

Claims

1. A method for preparing human decellularized alveolar bone matrix microparticles, characterized in that, Includes the following steps: Step S1: Obtain fresh, clean human alveolar bone from the clinic; Step S2: Process the obtained human alveolar bone and perform two cleaning operations on it; Step S3: Immerse in decellularization solution for 10 minutes; Step S4: Thoroughly clean the human alveolar bone once to obtain human decellularized alveolar bone matrix, and record the surface characteristics of the human decellularized alveolar bone matrix at the same time; Step S5: Sublime drying of human decellularized alveolar bone matrix for 24 hours; Step S6: Use a medium-throughput tissue grinder and simultaneously introduce nitrogen gas to rapidly grind the decellularized alveolar bone matrix of human teeth to obtain decellularized alveolar bone matrix microparticles. Step S7: Record the surface characteristics of the obtained decellularized alveolar bone matrix microparticles, and place the remaining decellularized alveolar bone matrix microparticles into a sterilization device for sterilization. Step S8: After sterilization with sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, resuspend the decellularized alveolar bone matrix microparticles, 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 decellularized alveolar bone matrix microparticles according to claim 1, characterized in that: Step S1 specifically includes: taking alveolar bone from the tooth roots of adolescent orthognathic surgery patients aged 18-25 years who have no clinical application value; after taking the alveolar bone, removing necrotic periodontal ligament and soft tissue, rinsing it repeatedly with phosphate buffer solution, and then soaking the fresh alveolar bone in deionized water before starting the next stage.

3. The method for preparing human decellularized alveolar bone matrix microparticles according to claim 1, characterized in that: Step S2 specifically includes: removing the human alveolar bone matrix from the deionized water and then immersing it in 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 decellularized alveolar bone matrix microparticles according to claim 1, characterized in that: Step S3 specifically includes: taking a 17% ethylenediaminetetraacetic acid solution, immersing the cleaned human alveolar bone matrix in the solution for 10 minutes, and then thoroughly rinsing it with sterile water.

5. The method for preparing human decellularized alveolar bone matrix microparticles according to claim 1, characterized in that: Steps S4-S7 specifically include: using a handheld cutter to cut human alveolar bone matrix into small pieces and decellularizing them to obtain decellularized alveolar bone matrix; then observing the decellularized alveolar bone matrix using scanning electron microscopy to evaluate the decellularization effect; the remaining human alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours; after sublimation, it is ground using a medium-throughput tissue homogenizer at 1200 rpm for 5 minutes, repeated 3-6 times to obtain decellularized alveolar bone matrix microparticles; subsequently, it is sterilized in an autoclave for 12 hours; then 50 grams of decellularized alveolar bone matrix microparticles are resuspended in 100 ml of solution containing 100 U / ml penicillin and 100 ml of... The samples were incubated in sterile phosphate buffer at a concentration of mg / ml streptomycin for three days at 37°C. The samples were then filtered through a 70μm filter and stored at -80°C until needed. The surface characteristics of the decellularized alveolar bone matrix microparticles were observed. The specific observation procedure was as follows: Human decellularized alveolar bone matrix and human alveolar bone matrix samples were dehydrated in gradient ethanol solutions with concentrations of 60%, 75%, 85%, 95%, and 100%, respectively, for 10 minutes each. Hexamethyldisilane was then added to the dehydrated samples, and the samples were dried at room temperature in a fume hood for 8-10 hours. Subsequently, gold was deposited on the dried sample surface using an ion sputtering device. The morphology of human alveolar bone and human decellularized alveolar bone matrix was observed and photographed under a scanning electron microscope. Following the manufacturer's instructions, the chemical composition of the human decellularized alveolar bone matrix was analyzed using X-ray scanning electron microscopy. Finally, according to the manufacturer's instructions, the chemical characteristics of the decellularized alveolar bone matrix microparticles were analyzed using Fourier transform infrared spectroscopy.

6. The method for preparing human decellularized alveolar bone matrix microparticles according to claim 1, characterized in that: Step S8 specifically includes: resuspending 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.

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