Method for constructing bone micro-tissue by compounding bone extracellular matrix with vascular matrix component and application of bone micro-tissue

Abstract

The embodiment of the invention discloses a method for constructing a bone micro-tissue by compounding a bone extracellular matrix with a vascular matrix component and application of the bone micro-tissue. The specific implementation mode of the method comprises the following steps: preprocessing a human femoral head cortical bone sample; performing freeze thawing treatment; preparing demineralized bone powder corresponding to the caput femoris cortical bone sample of the target person; preparing a bone source extracellular matrix microcarrier; preparing a vascular matrix component; mixing the vascular matrix component and the bone-derived extracellular matrix microcarrier in a basic culture medium to obtain a mixed suspension; transferring the mixed suspension to a rotary bioreactor; and performing dynamic culture treatment on the vascular matrix component and the bone source extracellular matrix microcarrier in the basic culture medium to obtain the bone micro-tissue. According to the implementation mode, the effect of the constructed bone micro-tissue can be improved, the secondary injury to a patient and the risk of complications are reduced, and the effect of repairing bone defects can be improved.

Description

Technical Field

[0001] The embodiments disclosed herein relate to the field of bone microtissue construction, specifically to a method and application for constructing bone microtissue by combining bone extracellular matrix with vascular matrix components. Background Technology

[0002] Bone defects are a common challenge in orthopedic clinics, especially after trauma, tumor resection, or infection, often leading to interruption of bone tissue continuity, delayed healing, or even nonunion. Currently, the main methods for repairing bone defects are autologous bone grafting, allogeneic bone grafting, or xenograft bone grafting.

[0003] However, in practice, it has been found that when using the above-mentioned bone defect repair methods, the following technical problems often arise: When autologous bone grafting is used for repair, the limited availability of donor sites increases the risk of secondary injury and complications for patients. When allogeneic bone grafting is used, there are risks such as immune rejection, disease transmission, and insufficient bone integration, resulting in poor repair efficacy of allogeneic bone for bone defects. When artificial bone materials (such as hydroxyapatite or calcium phosphate ceramic) are used to construct bone microtissues to repair bone defects, the low bioactivity of the bone microtissues makes it difficult for the degradation rate to match the bone regeneration process, and there is a lack of a natural microenvironment that promotes vascularization and cell adhesion, resulting in poor repair efficacy of the constructed bone microtissues for bone defects.

[0004] The information disclosed in this background section is only intended to enhance the understanding of the background of the inventive concept, and therefore may contain information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The summary portion of this disclosure is intended to provide a brief overview of the concepts, which will be described in detail in the detailed description portion. This summary portion is not intended to identify key or essential features of the claimed technical solutions, nor is it intended to limit the scope of the claimed technical solutions.

[0006] Some embodiments of this disclosure propose methods and applications for constructing bone microtissues by combining bone extracellular matrix with vascular matrix components, in order to solve one or more of the technical problems mentioned in the background section above.

[0007] In a first aspect, some embodiments of this disclosure provide a method for constructing bone microtissue using a combination of bone extracellular matrix and vascular matrix components. The method includes: pre-treating an acquired human femoral head cortical bone sample to obtain a pre-treated human femoral head cortical bone sample; performing a freeze-thaw treatment on the pre-treated human femoral head cortical bone sample to obtain a freeze-thawed human femoral head cortical bone sample as a target human femoral head cortical bone sample; and, in response to the construction method being a first bone microtissue construction method, performing the following first bone microtissue construction steps: preparing demineralized bone powder corresponding to the target human femoral head cortical bone sample; preparing bone-derived extracellular matrix microcarriers using a ball mill, molecular sieve, and the demineralized bone powder; preparing vascular matrix components based on a human abdominal adipose tissue sample; mixing the vascular matrix components and the bone-derived extracellular matrix microcarriers in a basal culture medium according to a target ratio to obtain a mixed suspension; transferring the mixed suspension to a rotary bioreactor and adding bone-inducing culture medium to the basal culture medium; and dynamically culturing the vascular matrix components and the bone-derived extracellular matrix microcarriers in the basal culture medium to obtain the constructed bone microtissue.

[0008] Secondly, some embodiments of this disclosure provide the application of bone microtissue in bone defect repair. The bone microtissue described above is the bone microtissue constructed as described in the first aspect above.

[0009] Thirdly, some embodiments of this disclosure provide the application of bone-derived extracellular matrix microcarriers and vascular matrix components in the construction of bone microtissues. The bone-derived extracellular matrix microcarriers are those described in the first aspect above, and the vascular matrix components are those described in the first aspect above.

[0010] The above-described embodiments of this disclosure have the following beneficial effects: the method for constructing bone microtissue using bone extracellular matrix composite vascular matrix components according to some embodiments of this disclosure can reduce the risk of secondary injury to patients and complications, and can improve the effect of bone defect repair. Specifically, the reasons for the high risk of secondary injury and complications to patients and the poor effect of bone defect repair are as follows: when autologous bone grafting is used for repair, the donor site is limited, resulting in the risk of secondary injury and complications to patients; when allogeneic bone grafting is used, there are risks such as immune rejection, disease transmission, and insufficient bone integration capacity, resulting in a poor effect of allogeneic bone in bone defect repair; when artificial bone materials (such as hydroxyapatite or calcium phosphate ceramic) are used to construct bone microtissue to repair bone defects, the bioactivity of the bone microtissue is low, making it difficult for the degradation rate to match the bone regeneration process, and there is a lack of a natural microenvironment that promotes vascularization and cell adhesion, resulting in a poor effect of using the constructed bone microtissue to repair bone defects. Based on this, some embodiments of the present disclosure describe a method for constructing bone microtissue using bone extracellular matrix combined with vascular matrix components. First, the obtained human femoral head cortical bone sample is pretreated to obtain a pretreated human femoral head cortical bone sample. This allows the obtained human femoral head cortical bone sample to be pretreated and then used to construct bone microtissue. Then, the pretreated human femoral head cortical bone sample is subjected to freeze-thaw treatment to obtain a freeze-thawed human femoral head cortical bone sample as the target human femoral head cortical bone sample. This allows for further pretreatment of the obtained human femoral head cortical bone sample. Next, in response to the construction method being a first bone microtissue construction method, the following first bone microtissue construction steps are performed: First, demineralized bone powder corresponding to the target human femoral head cortical bone sample is prepared based on the aforementioned target human femoral head cortical bone sample. This yields the prepared demineralized bone powder. Then, bone-derived extracellular matrix microcarriers are prepared using a ball mill, molecular sieve, and the aforementioned demineralized bone powder. This yields the prepared bone-derived extracellular matrix microcarriers, which are then used for further construction of bone microtissue. Next, vascular matrix components were prepared based on human abdominal adipose tissue samples. Bone microtissue could then be prepared using these vascular matrix components. Next, the aforementioned vascular matrix components and bone-derived extracellular matrix microcarriers were mixed in a basal culture medium according to a target ratio to obtain a mixed suspension. This mixture could then be further used to prepare microtissue. The mixed suspension was then transferred to a rotary bioreactor, and bone-inducing medium was added to the aforementioned basal culture medium. This allowed for dynamic culture, thereby culturing the bone microtissue. Subsequently, the vascular matrix components and bone-derived extracellular matrix microcarriers in the aforementioned basal culture medium were subjected to dynamic culture treatment to obtain cultured bone microtissue. Thus, the constructed bone microtissue was obtained.Because it uses human allogeneic bone to prepare acellular extracellular matrix microcarriers, immunogenicity can be minimized while preserving the components and structure of the natural bone matrix, which is beneficial for cell recognition and functional expression. Furthermore, because it uses vascular matrix components as seed cells, which are rich in various osteogenic and angiogenesis-related precursor cells, it can synergistically promote bone regeneration and angiogenesis. Moreover, because it employs a bioreactor for three-dimensional dynamic co-culture, simulating the mechanical and biochemical microenvironment of bone tissue growth in vivo, it improves cell survival rate, distribution uniformity, and functional maturity. Therefore, it can enhance the effectiveness of the constructed bone microtissue, thereby reducing secondary damage to patients and lowering the risk of complications, and improving the repair effect of bone defects. Attached Figure Description

[0011] The above and other features, advantages, and aspects of the embodiments of this disclosure will become more apparent from the accompanying drawings and the following detailed description. Throughout the drawings, the same or similar reference numerals denote the same or similar elements. It should be understood that the drawings are schematic, and elements are not necessarily drawn to scale.

[0012] Figure 1 This is a flowchart of some embodiments of the method and application of constructing bone microtissue by combining bone extracellular matrix with vascular matrix components according to the present disclosure; Figure 2 This is a scanning electron microscope schematic diagram of the bone-derived extracellular matrix microcarrier used in the method for constructing bone microtissue by combining bone extracellular matrix with vascular matrix components according to the present disclosure. Figure 3 This is a schematic diagram of the histological staining (HE, Masson) results of the bone-derived extracellular matrix microcarriers used in the method for constructing bone microtissues based on the bone extracellular matrix composite vascular matrix components disclosed herein. Figure 4 This is a schematic diagram of the method for constructing bone microtissue based on the bone extracellular matrix and vascular matrix components disclosed herein, and the results of histological staining (Tunel) of the bone microtissue. Figure 5 This is a schematic diagram of the method for constructing bone microtissue based on the bone extracellular matrix and vascular matrix components disclosed herein, and the results of Tublin (cytoskeleton) staining of the bone microtissue. Figure 6 This is a schematic diagram showing the results of ALP (alkaline phosphatase) staining and ARS (alizarin red) staining of bone microtissue cultured for seven days and 14 days, respectively, based on the bone extracellular matrix composite vascular matrix component construction method and application disclosed herein. Figure 7 This is a schematic diagram of the CD31 staining results of bone microtissue cultured for seven days, based on the bone extracellular matrix composite vascular matrix component construction method and application disclosed herein. Figure 8 This is an internal test image of bone microtissue constructed according to the method of constructing bone microtissue by combining bone extracellular matrix with vascular matrix components according to the present disclosure, and the application of bone microtissue. Detailed Implementation

[0013] Embodiments of this disclosure will now be described in more detail with reference to the accompanying drawings. While some embodiments of this disclosure are shown in the drawings, it should be understood that this disclosure can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this disclosure. It should be understood that the accompanying drawings and embodiments of this disclosure are for illustrative purposes only and are not intended to limit the scope of protection of this disclosure.

[0014] It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings. Unless otherwise specified, the embodiments and features described in this disclosure can be combined with each other.

[0015] It should be noted that the concepts of "first" and "second" mentioned in this disclosure are used only to distinguish different devices, page modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, page modules or units.

[0016] It should be noted that the terms "a" and "a plurality of" used in this disclosure are illustrative rather than restrictive, and those skilled in the art should understand that, unless otherwise expressly indicated in the context, they should be understood as "one or more".

[0017] The names of messages or information exchanged between multiple devices in the embodiments of this disclosure are for illustrative purposes only and are not intended to limit the scope of such messages or information.

[0018] This disclosure will now be described in detail with reference to the accompanying drawings and embodiments.

[0019] Figure 1 A flowchart 100 illustrating some embodiments of a method for constructing bone microtissues by combining bone extracellular matrix with vascular matrix components according to the present disclosure is shown. This method for constructing bone microtissues by combining bone extracellular matrix with vascular matrix components includes the following steps: Step 101: Preprocess the obtained human femoral head cortical bone sample to obtain a preprocessed human femoral head cortical bone sample.

[0020] In some embodiments, the obtained human femoral head cortical bone samples can be preprocessed to obtain preprocessed human femoral head cortical bone samples. It should be noted that the aforementioned human femoral head cortical bone samples are obtained from patients who have undergone total hip arthroplasty and whose hematological and infectious disease screening indicators are normal. The obtained human femoral head cortical bone samples are all from the femoral head portion that was surgically removed and originally planned for disposal. Written informed consent from the patient was obtained before sampling, and the samples were reviewed by the hospital's ethics committee. It should be noted that the aforementioned human femoral head cortical bone samples may not come from the same patient as the patient from whom the bone microtissue constructed based on the aforementioned human femoral head cortical bone samples was obtained.

[0021] In some optional implementations of certain embodiments, the obtained human femoral head cortical bone sample can be preprocessed using the following steps to obtain a preprocessed human femoral head cortical bone sample: The first step is to place the obtained human femoral head cortical bone sample in a sterile operating table.

[0022] The second step involves using a scalpel and periosteal elevator to remove the soft tissue, cartilage, and periosteum attached to the surface of the human femoral head cortical bone sample.

[0023] The third step is to rinse the human femoral head cortical bone sample three times with sterile saline to remove blood cells and non-bone residues.

[0024] The fourth step involves using a constant-speed, low-velocity cutter to cut the aforementioned human femoral head cortical bone sample into bone slices with a thickness of 2–3 mm along the bone grain direction, obtaining individual cortical bone slices. The rotational speed of the constant-speed, low-velocity cutter is within the range of 200–300 rpm. These cortical bone slices characterize the bone slices obtained from the cutting of the aforementioned human femoral head cortical bone sample.

[0025] The fifth step involves using sterile bone forceps to trim and cut each cortical bone fragment, and then using these trimmed cortical bone fragments as pre-treated human femoral head cortical bone samples.

[0026] In addressing the aforementioned technical problems in the process of adopting technical solutions, and considering the application scenario—the construction of bone microtissues from human femoral head cortical bone samples—the following technical problem often arises: natural human femoral head cortical bone samples contain cellular residues, have a hydrophobic surface, and their microporous structure is prone to collapse, making them unsuitable as direct scaffolds for bone microtissue construction, resulting in poor quality bone microtissues constructed from human femoral head cortical bone samples. To meet the following requirements for this application scenario: adapting to the need for constructing bone microtissues from human femoral head cortical bone samples, and addressing the urgent needs for three-dimensional construction, long-term culture, and functional maturation of bone microtissues, we have decided to adopt the following solution: Optionally, the obtained human femoral head cortical bone sample can be preprocessed using the following steps to obtain a preprocessed human femoral head cortical bone sample: The first step is to prepare thin slices of the human femoral head cortex into 0.3–0.8 mm pieces. In practice, a precision bone slicer can be used to prepare these thin slices.

[0027] The second step involves rinsing the thin bone slices repeatedly with PBS 3–5 times to remove blood, bone marrow, and soft tissue, resulting in rinsed thin bone slices. In practice, the thin bone slices are first transferred to a sterile culture dish. Then, sufficient sterile PBS (pH 7.2–7.4) is added, and the slices are gently shaken and rinsed repeatedly 3–5 times, replacing the PBS each time, until no obvious blood, bone marrow, or soft tissue is visible to the naked eye, thus obtaining rinsed thin bone slices.

[0028] The third step is to treat the rinsed thin bone fragments with 0.5% sodium deoxycholate for 15 minutes. In practice, the rinsed thin bone fragments can be immersed in the above-mentioned 0.5% sodium deoxycholate and left to stand at room temperature for 15 minutes to treat the thin bone fragments.

[0029] The fourth step involves treating the rinsed thin bone slices with 0.1% Triton X-100 for 10 minutes to obtain the target thin bone slices. These target thin bone slices characterize the rinsed thin bone slices. In practice, the thin bone slices are first thoroughly washed 2-3 times with sterile PBS to remove residual sodium deoxycholate. Then, the rinsed thin bone slices are immersed in a 0.1% Triton X-100 solution and treated at room temperature for 10 minutes to process the rinsed thin bone slices.

[0030] Fifth, digest the target bone slices with 100 U / mL DNase I at 37°C for 30 minutes to remove nucleic acid residues and obtain the digested target slices. In practice, the target bone slices can be immersed in the above 100 U / mL DNase I and then placed in a 37°C incubator for 30 minutes for digestion.

[0031] Step 6: Thoroughly wash the digested target slide with PBS and rinse twice with ultrapure water to obtain the cleaned target slide. In practice, first, thoroughly wash the digested target slide with a large amount of sterile PBS (3 to 5 times). Then, rinse the digested target slide twice with sterile ultrapure water to remove salt ions, obtaining the cleaned target slide.

[0032] The seventh step involves supercritical CO2 drying of the cleaned target slice to prevent the bone microporous structure from collapsing, resulting in a dried target slice. In practice, the cleaned target slice can be placed in a supercritical CO2 dryer, with the temperature set to 37°C, the pressure to approximately 15 MPa, and the treatment time to 20–30 minutes, to achieve the desired supercritical CO2 drying effect.

[0033] Step 8: Activate the dried target sheet using nitrogen plasma for 60–90 seconds to improve surface hydrophilicity, resulting in an activated target sheet. In practice, the dried target sheet can be laid flat in the sample chamber of a plasma treatment instrument, evacuated, and then purged with nitrogen gas for plasma treatment to improve surface hydrophilicity and obtain an activated target sheet.

[0034] Step 9: Immerse the activated target slice in a 0.5 mg / mL type I collagen solution for 30 minutes to obtain the first soaked target slice.

[0035] Step 10: Immerse the target slice after the first soaking in a 1×SBF / 5×SBF simulated body fluid and incubate at 37℃ for 24–48 hours for biomimetic mineralization to obtain the target slice after the second soaking. In practice, the target slice after the first soaking can first be immersed in a 1×SBF / 5×SBF simulated body fluid until completely submerged, and then placed in a 37℃ constant temperature incubator for 24–48 hours to obtain the target slice after the second soaking.

[0036] Step 11: The target slice after the second soaking is subjected to negative pressure loading of a mixed factor of VEGF, NGF, and BMP-2 for 2 hours to obtain a target slice after negative pressure loading treatment. In practice, firstly, the mixed factor of VEGF, NGF, and BMP-2 can be mixed to obtain a prepared mixed growth factor solution. Then, the target slice after the second soaking is placed in a negative pressure device to completely immerse it in the mixed growth factor solution. Afterward, the negative pressure of the negative pressure device is turned on to approximately -0.03 MPa for continuous loading for 2 hours, allowing the prepared mixed growth factor solution to penetrate into the pores of the bone matrix.

[0037] Step 12: Gently wash the target slice after the negative pressure loading treatment with PBS to obtain a pretreated human femoral head cortical bone sample. In practice, sterile PBS can be used to gently rinse the loaded bone slice 1–2 times to remove unbound free factors, thus obtaining a pretreated human femoral head cortical bone sample.

[0038] The above-described technical solution, as an inventive point of this disclosure, solves technical problem two: "the inability to directly serve as a scaffold for bone microtissue construction, resulting in poor performance of bone microtissue constructed from human femoral head cortical bone samples." The reasons for this inability to directly serve as a scaffold for bone microtissue construction, leading to poor performance of bone microtissue constructed from human femoral head cortical bone samples, are as follows: natural human femoral head cortical bone samples contain cellular residues, have a hydrophobic surface, and their microporous structure is prone to collapse, making them unsuitable as scaffolds for bone microtissue construction, thus resulting in poor performance of bone microtissue constructed from human femoral head cortical bone samples. By addressing these factors, it is possible to pre-treat the scaffold for bone microtissue construction, thereby improving the performance of bone microtissue constructed from human femoral head cortical bone samples. To achieve this effect, the disclosed method for constructing bone microtissue using bone-derived extracellular matrix and vascular matrix components firstly involves pre-treating human femoral head cortical bone samples through washing and decellularization to remove immunogenicity, thinning the bone sheets to ensure material exchange, using supercritical drying to maintain the microporous structure, plasma and collagen coating to enhance cell affinity, SBF mineralization to enhance osteoconductivity, and multi-factor loading to achieve simultaneous induction of neurovascular bone. This pre-treatment of human femoral head cortical bone samples effectively pre-treats the scaffold for bone microtissue construction, thereby improving the quality of bone microtissue constructed from human femoral head cortical bone samples.

[0039] Step 102: Perform freeze-thaw treatment on the pre-processed human femoral head cortical bone sample to obtain the freeze-thawed human femoral head cortical bone sample as the target human femoral head cortical bone sample.

[0040] In some embodiments, the pretreated human femoral head cortical bone sample can be subjected to freeze-thaw treatment to obtain a freeze-thawed human femoral head cortical bone sample as the target human femoral head cortical bone sample.

[0041] In some optional implementations of certain embodiments, the pre-processed human femoral head cortical bone sample can be subjected to freeze-thaw treatment via the following steps to obtain a freeze-thawed human femoral head cortical bone sample as the target human femoral head cortical bone sample: The first step involves performing the following freeze-thaw treatment steps on the pretreated human femoral head cortical bone sample, according to the preset number of freeze-thaw cycles: The first freeze-thaw treatment step involves transferring the pretreated human femoral head cortical bone sample into a pre-cooled 50ml centrifuge tube, adding an appropriate amount of liquid nitrogen, and freezing at -196℃ for 1 hour. The preset number of freeze-thaw cycles can be 5. The specific amount of liquid nitrogen added is not limited and can be set according to actual needs. It should be noted that the amount of liquid nitrogen added should be no less than 3 times the volume of the pretreated human femoral head cortical bone sample.

[0042] The second freeze-thaw treatment step involves thawing at room temperature for 30 minutes after removal. It should be noted that the pretreated human femoral head cortical bone sample needs to undergo five freeze-thaw treatment steps to fully rupture residual cells within the bone tissue and release intracellular contents.

[0043] Step 103, in response to the construction mode being the first bone microtissue construction mode, perform the following first bone microtissue construction steps: Step 1031: Based on the target human femoral head cortical bone sample, prepare demineralized bone powder corresponding to the target human femoral head cortical bone sample.

[0044] In some embodiments, demineralized bone powder corresponding to the aforementioned target human femoral head cortical bone sample can be prepared. This demineralized bone powder is characterized by removing minerals such as calcium and phosphorus, and immunogenic substances, from the target human femoral head cortical bone sample, retaining only the organic framework (mainly type I collagen), and then grinding it into a powder. The aforementioned first bone microtissue construction method characterizes the method of constructing bone microtissue. This first bone microtissue construction method characterizes the method of constructing bone microtissue without using neonatal rat DRG.

[0045] In some optional implementations of certain embodiments, demineralized bone powder corresponding to the aforementioned target human femoral head cortical bone sample can be prepared by the following steps: The first step is to place the femoral head cortical bone sample from the target human in a grinding jar pre-cooled with liquid nitrogen.

[0046] The second step involves using a high-throughput tissue homogenizer at a frequency of 30 Hz to homogenize the target human femoral head cortical bone sample three times to obtain bone powder. Each homogenization session lasts for 2 minutes. This initial homogenization of the target human femoral head cortical bone sample is then achieved.

[0047] The third step is to place the obtained bone meal on ice to cool it.

[0048] The fourth step, after initial grinding, involves passing the resulting bone powder through a stainless steel sieve with a pore size of 500 μm to remove large, insufficiently ground particles. There is no limitation on the diameter of these large, insufficiently ground particles; screening can be performed according to actual needs.

[0049] The fifth step is to collect the sieved bone meal and place it in a sterile container for later use. The specific type of sterile container is not limited here; it can be selected according to actual needs.

[0050] Step 6: Add a PBS solution containing 2% penicillin / streptomycin to the bone powder. The volume ratio of bone powder to the added PBS solution is 1:10. It should be noted that setting the volume ratio of bone powder to the added PBS solution to 1:10 ensures thorough soaking, avoids reagent waste, and enhances the antibacterial effect.

[0051] Step 7: Place the bone powder on a horizontal shaker at 4°C and shake at 80 rpm for 30 minutes. In practice, bone powder with a PBS solution containing 2% penicillin / streptomycin can be placed on a horizontal shaker at 4°C and shaken at 80 rpm for 30 minutes.

[0052] Step 8: Centrifuge at 3000 rpm for 10 minutes, discard the supernatant, and obtain defatted bone powder. In practice, a centrifuge can be used to centrifuge bone powder containing 2% penicillin / streptomycin in PBS solution after shaking treatment.

[0053] Step 9: Transfer the defatted bone meal to a 0.5N hydrochloric acid solution and gently stir magnetically for 24 hours at room temperature. The ratio of defatted bone meal to 0.5N hydrochloric acid solution is 1:15. Setting the ratio to 1:15 ensures that the liquid is concentrated and can completely submerge the bone meal, resulting in more uniform demineralization.

[0054] Step 10: Replace the hydrochloric acid solution with fresh solution every 8 hours to ensure thorough demineralization.

[0055] Step 11: After demineralization, wash the bone meal with deionized water until the pH value of the bone meal is between pH 6.5 and 7.5, and obtain the washed bone meal as demineralized bone meal.

[0056] In addressing the aforementioned technical problems in the application scenario—specifically, the construction of bone microtissue from human femoral head cortical bone samples—often encounters the following technical problem: Direct grinding and ordinary drying of human femoral head cortical bone samples to prepare demineralized bone powder results in uneven particle size, micropore collapse, and structural shrinkage, leading to poor quality bone microtissue prepared from the demineralized bone powder. To meet the specific requirements of this application scenario for preparing bone microtissue, we have decided to adopt the following solution: Optionally, demineralized bone powder corresponding to the aforementioned femoral head cortical bone sample can be prepared using the following steps: The first step is to remove surface impurities from the target human femoral head cortical bone sample. In practice, PBS can be used to quickly rinse the target human femoral head cortical bone sample to remove visible dirt.

[0057] The second step involves pre-cleaning the target human femoral head cortical bone sample with low-temperature plasma for 30 seconds under a nitrogen atmosphere to remove the surface oxide layer and trace impurities.

[0058] The third step involves using supercritical CO2 degreasing treatment on the aforementioned femoral head cortical bone samples to remove lipid impurities from their surface. In practice, the femoral head cortical bone samples can be transferred to a supercritical CO2 drying device, with the operating parameters set to a temperature of 37°C, a pressure of 15 MPa, and a treatment time of 20-30 minutes to process the samples and remove lipid impurities from their surface.

[0059] The fourth step involves treating the target human femoral head cortical bone samples with a 0.2M EDTA solution, maintaining a low-speed stirring rate at 4°C throughout the process to prevent collagen degradation. In practice, the target human femoral head cortical bone samples can be completely immersed in the EDTA solution and placed in a 4°C constant-temperature stirrer for continuous low-speed stirring for 24 hours.

[0060] The fifth step involves treating the target human femoral head cortical bone sample with a low-concentration hydrochloric acid solution. This low-concentration hydrochloric acid can be a 0.1M HCl solution.

[0061] Step 6: Dialyze the target human femoral head cortical bone sample using ultrapure water until the pH value of the sample is between pH 7.2 and 7.4. During dialysis, change the ultrapure water every 8 hours and add 0.01% antibacterial agent. In practice, the demineralized bone fragments can be transferred to a dialysis bag and immersed in a large amount of ultrapure water for dialysis. Control conditions: Change the ultrapure water every 8 hours. Add 0.01% antibacterial agent (such as sodium azide or penicillin-streptomycin) to the water to prevent microbial contamination. Continue dialysis until the pH of the bone fragments stabilizes at 7.2–7.4.

[0062] Step 7: Pre-treat the demineralized target human femoral head cortical bone sample with collagenase using 0.05% type I collagenase, followed by incubation at 37°C. In practice, the target human femoral head cortical bone sample can be immersed in the enzyme solution after dialysis and neutralization, and then incubated at 37°C for 15–20 minutes.

[0063] Step 8: Add 0.5 mg / mL fibronectin solution and soak for 20 minutes. In practice, collagenase-treated target human femoral head cortical bone samples can be transferred into this solution and soaked at room temperature for 20 minutes.

[0064] Step nine: Rinse thoroughly with ultrapure water to obtain a sample of the target femoral head cortex. In practice, the bone fragment can be rinsed repeatedly with large amounts of sterile ultrapure water 3–5 times to thoroughly remove residual enzymes, proteins, and salt ions.

[0065] Step 10: Under an ice bath environment, the rinsed target femoral head cortical bone sample is pulverized using low-power ultrasound at 40 kHz and 30 W to obtain an initial demineralized bone powder suspension. In practice, the rinsed target femoral head cortical bone sample can be placed in a sterile centrifuge tube, submerged in an appropriate amount of sterile ultrapure water, and then pulverized using low-power ultrasound under an ice bath environment.

[0066] Step 11: Filter the initial demineralized bone meal suspension through a 70μm filter to retain bone meal of uniform particle size and remove coarse impurities, obtaining a purified initial demineralized bone meal suspension. In practice, you can first filter with a 70μm filter to remove coarse bone particles. Then collect the filtrate and further filter with a 20–40μm filter to retain bone meal of the target particle size, obtaining a purified initial demineralized bone meal suspension.

[0067] Step 12: Pre-freeze the initial demineralized bone meal suspension (after impurity removal) at -80℃ for 2 hours, then gradually increase the temperature to freeze-dry, obtaining the freeze-dried initial demineralized bone meal. In practice, first, the initial demineralized bone meal suspension can be transferred to a freeze-drying bottle and pre-frozen at -80℃ for 2 hours. Then, it can be transferred to a vacuum freeze dryer and freeze-dried using a gradient temperature program: low temperature stage: maintain at around -40℃, gradually increase the temperature to room temperature to obtain the freeze-dried initial demineralized bone meal.

[0068] Step 13: The initial demineralized bone powder after freeze-drying is activated with nitrogen plasma for 60 seconds to obtain activated initial demineralized bone powder. In practice, the freeze-dried bone powder can be evenly spread thinly and dispersed on a sample tray. Then, it is placed in a plasma treatment instrument, and nitrogen gas is introduced for surface activation.

[0069] Step fourteen: Using a mixed solution of VEGF, BMP-2, and NGF, the activated initial demineralized bone meal is subjected to a slow-release encapsulation treatment of growth factors to obtain encapsulated initial demineralized bone meal. In practice, the activated initial demineralized bone meal is first immersed in the above-mentioned mixed solution of VEGF, BMP-2, and NGF to ensure thorough wetting and allow the factors to be adsorbed onto the surface of the bone meal.

[0070] Step 15: Using cryogenic spray encapsulation technology, the initially demineralized bone powder after the above encapsulation treatment becomes a carrier for the sustained release of long-acting factors, thus obtaining demineralized bone powder. In practice, the temperature parameters of the cryogenic spray encapsulation technology can be set to 4℃ and the spray pressure to 0.1MPa to process the initially demineralized bone powder after encapsulation treatment, thereby obtaining demineralized bone powder.

[0071] The above-described technical solution, as an inventive point of this disclosure, solves technical problem three: "The prepared demineralized bone powder exhibits uneven particle size, micropore collapse, and structural shrinkage, resulting in poor quality of bone microtissue prepared from the demineralized bone powder." The reasons for this uneven particle size, micropore collapse, and structural shrinkage in the prepared demineralized bone powder, leading to poor quality of bone microtissue prepared from it, are as follows: Demineralized bone powder is prepared from human femoral head cortical bone samples using direct grinding and ordinary drying methods, resulting in uneven particle size, micropore collapse, and structural shrinkage, thus leading to poor quality of bone microtissue prepared from it. Solving these factors can reduce the uneven particle size, micropore collapse, and structural shrinkage in the prepared demineralized bone powder, thereby improving the quality of bone microtissue prepared from it. To achieve this effect, the bone microtissue construction method of bone-derived extracellular matrix and vascular matrix components disclosed herein generates demineralized bone powder through the above-mentioned methods of plasma cleaning, supercritical defatting, gradient mild demineralization, pH controlled dialysis, dual modification with collagenase and fibronectin, ice bath ultrasonic pulverization, precise sieving, gradient freeze drying, plasma activation, and multi-factor low-temperature spray sustained release. This can improve the uniformity of bone particle size of the prepared demineralized bone powder and reduce micropore collapse and structural shrinkage, thereby improving the effect of bone microtissue prepared from demineralized bone powder.

[0072] Step 1032: Prepare bone-derived extracellular matrix microcarriers using a ball mill, molecular sieve, and demineralized bone powder.

[0073] In some embodiments, bone-derived extracellular matrix microcarriers can be prepared using a ball mill, molecular sieve, and the aforementioned demineralized bone powder. These bone-derived extracellular matrix microcarriers can be characterized as micron-sized scaffolds extracted from bone, retaining only the extracellular matrix (collagen, proteoglycans, growth factors, etc.), and used to "carry, nourish, and guide" cells.

[0074] In some alternative implementations of certain embodiments, bone-derived extracellular matrix microcarriers can be prepared using the following steps based on a ball mill, molecular sieve, and the aforementioned demineralized bone powder: The first step is to place the above demineralized bone powder in a freeze-drying bottle and pre-freeze it at -80℃ for 4 hours.

[0075] The second step involves transferring the material to a freeze dryer and freeze-drying it at -50°C and 0.1 mbar for 24 hours until the material is completely dry and has a porous, sponge-like structure, thus obtaining the freeze-dried bone matrix. In practice, the demineralized bone powder is transferred to a freeze dryer and freeze-dried at -50°C and 0.1 mbar for 24 hours until the material is completely dry and has a porous, sponge-like structure, thus obtaining the freeze-dried bone matrix. This freeze-dried bone matrix can characterize the process of using freeze-drying technology to create a dry, porous, and fluffy solid material from demineralized and degreased bone matrix.

[0076] The third step involves using a ball mill to perform a second fine grinding of the freeze-dried bone matrix, based on preset grinding time, preset grinding speed, preset cooling interval time, and preset grinding cycles, to obtain the ground freeze-dried bone matrix. Specifically, the preset grinding speed is 300 rpm, the preset grinding time is 2 minutes, and the preset grinding cycles are 5. The preset cooling interval time represents the cooling time between each two grinding cycles and can be 5 minutes.

[0077] The fourth step involves passing the ground freeze-dried bone matrix through molecular sieves with pore sizes of 100 μm and 60 μm to collect bone-derived extracellular matrix microcarriers with a size between 10 and 60 μm. These bone-derived extracellular matrix microcarriers are irregular porous particles with a surface exhibiting a natural bone microstructure.

[0078] Step 1033: Prepare vascular matrix components based on human abdominal adipose tissue samples.

[0079] In some embodiments, vascular matrix components can be prepared from human abdominal adipose tissue samples. These vascular matrix components can be a "multifunctional hybrid army of cells" extracted from fat, possessing a full range of capabilities including angiogenesis, osteogenics, nerve nourishment, and immune regulation, and can be a cell source for constructing bone microtissues.

[0080] In some alternative implementations of certain embodiments, the vascular matrix components can be prepared from a human abdominal adipose tissue sample via the following steps: The first step is to obtain abdominal adipose tissue from a human body. The volume of the obtained abdominal adipose tissue ranges from 50 to 100 ml. It should be noted that the patient from whom the abdominal adipose tissue was obtained is the same patient from whom the constructed bone micro-tissue was used. Written informed consent from the patient was obtained before the tissue was collected, and the procedure was reviewed by the hospital's ethics committee.

[0081] The second step involves using a digestive solution containing collagenase to shake and digest the aforementioned human abdominal adipose tissue in a constant temperature water bath at 37°C, thereby obtaining shake-digested human abdominal adipose tissue. The shaking and digestion time is between 40 and 60 minutes, and the digestive solution is type I collagenase with a concentration of 1 to 2 mg / ml.

[0082] The third step involves centrifuging the shaken and digested abdominal adipose tissue according to a preset centrifugal force and a preset centrifugal time, resulting in centrifuged abdominal adipose tissue. The preset centrifugal force is between 300 and 400 g, and the preset centrifugal time is 10 minutes. In practice, the shaken and digested abdominal adipose tissue can be placed in a centrifuge and centrifuged according to the preset centrifugal force and time.

[0083] The fourth step is to remove the upper layer of oil and liquid, and collect the bottom layer of cell sediment.

[0084] The fifth step involves treating the precipitate with erythrocyte lysis buffer to further purify the vascular matrix components, yielding purified vascular matrix components. The specific concentration of the erythrocyte lysis buffer is not limited here and can be set according to actual needs. In practice, the erythrocyte lysis buffer can be mixed with the aforementioned bottom cell precipitate to further purify the vascular matrix components.

[0085] Step 6: Adjust the concentration of the purified vascular matrix components to 1×10⁻⁶. 6 ~5×10 6 The adjusted vascular matrix composition was obtained by measuring cells / ml and used as the vascular matrix composition for preparation.

[0086] Step 1034: According to the target ratio, the vascular matrix components and bone-derived extracellular matrix microcarriers are mixed in the basal culture medium to obtain a mixed suspension.

[0087] In some embodiments, the vascular matrix component and the bone-derived extracellular matrix microcarriers can be mixed in a basal culture medium according to a target ratio to obtain a mixed suspension. The mixed suspension characterizes the mixture obtained by mixing the vascular matrix component and the bone-derived extracellular matrix microcarriers in the basal culture medium. The target ratio characterizes the ratio between the vascular matrix component and the bone-derived extracellular matrix microcarriers. The target ratio can be 2 × 10⁻⁶. 4 Vascular matrix component: 1 mg bone-derived extracellular matrix microcarriers. The type of basal culture medium is not limited here and can be set according to actual needs. For example, the basal culture can be serum-free human mesenchymal stem cell culture medium (MSC-T4).

[0088] Step 1035: Transfer the mixed suspension to a rotary bioreactor and add bone-inducing medium to the basal medium.

[0089] In some embodiments, the mixed suspension can be transferred to a rotary bioreactor, and bone-inducing medium can be added to the aforementioned basal medium. The bone-inducing medium can be osteogenic induction medium (Soleb G 1492, China). The specific type of rotary bioreactor is not limited here and can be selected according to actual needs.

[0090] Step 1036: Dynamically culture the vascular matrix components and bone-derived extracellular matrix microcarriers in the basal culture medium to obtain the constructed bone microtissue.

[0091] In some embodiments, the vascular matrix components in the above-mentioned basic culture medium and the above-mentioned bone-derived extracellular matrix microcarriers can be dynamically cultured to obtain the constructed bone microtissue.

[0092] In some optional implementations of certain embodiments, the vascular matrix components in the above-mentioned basal culture medium and the above-mentioned bone-derived extracellular matrix microcarriers can be dynamically cultured through the following steps to obtain the constructed bone microtissue: The first step, in response to determining that the aforementioned vascular matrix component and the aforementioned bone-derived extracellular matrix microcarriers are in the initial stage included in the preset culture phase, sets the rotation speed of the culture shaker to 8–12 rpm and the culture angle to 5°–10° to promote cell adhesion and initial distribution. The aforementioned initial stage refers to days one through three of culture. The aforementioned preset culture phase can characterize the pre-defined stages for culturing the aforementioned vascular matrix component and the aforementioned bone-derived extracellular matrix microcarriers. The aforementioned preset culture phase can include an initial stage, a middle stage, and a late stage. The aforementioned culture shaker can be an inclined thermostatic shaker. The culture conditions for each stage included in the aforementioned preset culture phase are 37°C and 5% CO2, with half of the culture medium replaced every 2–3 days. The aforementioned culture angle can characterize the angle between the tabletop of the aforementioned inclined thermostatic shaker and the horizontal plane.

[0093] The second step involves determining that the aforementioned vascular matrix components and the aforementioned bone-derived extracellular matrix microcarriers are in the intermediate stage included in the aforementioned preset culture phase, setting the rotation speed of the culture shaker to 15–20 rpm and the culture angle to 15°–20°. The aforementioned intermediate stage refers to the fourth to tenth day of culture.

[0094] Thirdly, in response to determining that the aforementioned vascular matrix components and the aforementioned bone-derived extracellular matrix microcarriers are in the later stage included in the aforementioned preset culture phase, the rotation speed of the culture shaker is set to 25–30 rpm, and the culture angle is set to 25°–30°. The aforementioned later stage refers to days 11 to 21 of culture.

[0095] In addressing the technical problems mentioned above, and considering the application scenario, the following technical problem often arises when constructing bone microtissues: Simply creating pure bone microtissues without considering nerve supply and vascular networks results in extremely low physiological simulation and poor quality of the constructed bone microtissues; constructing bone microtissues by first creating bone and then seeding cells results in blood vessels and nerves being separated from the bone matrix by only two layers, failing to form a functional network and further contributing to poor quality. To meet the specific requirements of this application scenario—namely, the need to construct bone microtissues with neurovascular structures—we have decided to adopt the following solution: Optionally, in response to the construction method being the second bone microtissue construction method, the following second bone microtissue construction steps are performed: The first step, the first pre-defined stage, involves isolating and culturing human abdominal adipose tissue fibroblasts (SVFs) from the abdominal fat. This first pre-defined stage is from day 0 to 3. In practice, the human abdominal adipose tissue SVFs are first minced into small tissue pieces of approximately 1 mm³. Then, 0.1% type I collagenase digestion solution is added, and the mixture is incubated at 37°C with shaking for 45–60 min. Next, an equal volume of complete culture medium is added to terminate the digestion, and the mixture is filtered through 100 μm and 70 μm cell sieves. Then, the mixture is centrifuged at 1200 rpm for 5 min, the supernatant is discarded, and the vascular matrix component (SVF) precipitate is obtained. Finally, the SVF is resuspended and seeded into culture dishes, and cultured using low-glucose DMEM + 10% FBS as per standard procedures. Days 0–3 are the SVF adhesion and expansion stage, and cells are cultured until they reach 70%–80% confluence. The above-mentioned second method for constructing bone microtissue can characterize the method of constructing bone microtissue. This second method can characterize the method of constructing bone microtissue using neonatal rat DRGs.

[0096] The second step involves adding VEGF at 10 ng / mL and ascorbic acid at 50 μg / mL to obtain pre-constructed vascular primordia. In practice, the following can be added to the SVF culture medium on day 3: VEGF: 10 ng / mL, ascorbic acid: 50 μg / mL, and cultured for another 24–48 hours to induce SVF differentiation towards the vascular endothelium, forming early vascular primordia structures.

[0097] The third step, in the second pre-designed stage, involves taking a newborn rat DRG, microsurgically dissecting it, and removing the capsule. In practice, first, a newborn SD rat (1–3 days old) can be taken, disinfected with 75% alcohol, and then dissected. Next, the spine is exposed, and the dorsal root ganglion (DRG) is carefully dissected. Then, under a stereomicroscope, the epineurium, capsule, and connective tissue are dissected using micro-forceps, leaving only the ganglion itself.

[0098] The fourth step involves digesting the tissue with collagenase D for 20 minutes, followed by gentle pipetting to obtain neurospheres. In practice, the cleaned DRG is first transferred to a 0.1% collagenase D solution. Then, it is incubated at 37°C for 20 minutes. Afterward, the tissue is gently pipetted several times to disperse the nerve tissue into clusters of nerve cells (neurospheres). Finally, it is gently centrifuged to remove any residual enzyme solution.

[0099] The fifth step involves pretreating the obtained neurospheres in a nerve culture medium containing NGF and GDNF for 24 hours to promote neurite growth. In practice, neurospheres are first inoculated into culture dishes pre-coated with poly-L-lysine / laminusoids. Then, a dedicated nerve basal medium is added, along with NGF and GDNF. Finally, the mixture is pretreated at 37°C in a 5% CO2 incubator for 24 hours to promote outward neurite growth.

[0100] Step 6: In the third pre-defined stage, pretreated SVF cells (3 days old) are mixed with osteogenic induction solution. In practice, firstly, SVF cells pretreated for 3 days are taken, digested with trypsin, centrifuged, and resuspended. Then, the cells are thoroughly mixed with the osteogenic induction solution. The osteogenic induction solution can be a mixture of high-glucose DMEM, 10% FBS, dexamethasone, sodium β-glycerophosphate, and ascorbic acid.

[0101] Step 7: Add β-TCP nanoparticles (10% w / v) to enhance bone mineralization. In practice, a 10% (w / v) suspension of β-tricalcium phosphate (β-TCP) nanoparticles can be prepared. Then, add it to the cell suspension in the above ratio and gently mix. The prepared 10% (w / v) β-tricalcium phosphate (β-TCP) nanoparticle suspension includes a 1:9 ratio of 10% (w / v) β-TCP stock solution to cell suspension.

[0102] Step 8: Add type I collagen gel to form a mixed sol.

[0103] Step 9: While the gel is semi-solidified, implant DRG nerve fragments (1–2 fragments / microtissue). In practice, first, the mixed sol can be placed in a 37°C environment until it begins to thicken and semi-solidify. Then, the pre-treated DRG nerve fragments can be added using a micromanipulator, with the density controlled at 1–2 nerve fragments / single microtissue. Finally, gently mix to ensure the nerve fragments are evenly distributed within the collagen gel.

[0104] Step 10: Crosslinking at 37℃ to form a gel, creating a neural-matrix composite microtissue. In practice, first, the sol containing neural fragments and cells is transferred into a micromold or culture well. Then, it is placed in a 37℃, 5% CO2 incubator for crosslinking for 20–30 minutes. Finally, the collagen completely solidifies, forming a three-dimensional neural-matrix composite microtissue.

[0105] Step 11: In the fourth pre-set stage, add NGF 50 ng / mL, GDNF 20 ng / mL, and VEGF 10 ng / mL. This fourth pre-set stage is from day 3 to 7. In practice, first add NGF: 50 ng / mL, GDNF: 20 ng / mL, and VEGF: 10 ng / mL to the culture medium. Finally, simultaneously promote nerve growth, angiogenesis, and stromal cell differentiation.

[0106] Step 12: In the fifth pre-set stage, standard osteogenic induction solution is used, where the fifth pre-set stage is from day 7 to 14. In practice, the solution is first replaced with standard osteogenic induction solution to continuously induce osteogenic differentiation. Then, the solution is changed every 2 days.

[0107] Step 13: Add NGF to maintain at 20 ng / mL, and add TGF-β1 at 2 ng / mL. In practice, on the basis of osteogenic induction, you can continue to add: NGF: 20 ng / mL (to maintain nerve survival and growth) and TGF-β1: 2 ng / mL (to promote extracellular matrix deposition and tissue remodeling) to improve the integrity of micro-tissue structure.

[0108] Step fourteen, in the sixth preset stage, high-mineralization osteogenic fluid. The sixth preset stage is from day 14 to 21. In practice, firstly, a high-mineralization osteogenic induction fluid (increasing the concentration of sodium β-glycerophosphate) can be used. Then, calcium salt deposition is accelerated, significantly improving the level of microtissue mineralization.

[0109] Step 15: Stop exogenous neurotrophic factors and rely on micro-tissue auto-secretion.

[0110] Step sixteen: Hypoxic 3% O2 ​​culture to obtain vascular matrix components. In practice, first, the oxygen concentration in the incubator is adjusted to 3% O2 ​​(hypoxic environment). Then, culture continues until the experimental endpoint. Subsequently, hypoxia significantly promotes angiogenesis, cell survival, and tissue maturation. Finally, vascular matrix components are obtained.

[0111] The above-described technical solution, as an inventive point of this disclosure, solves technical problem four: "resulting in extremely low physiological simulation, leading to poor quality of the constructed bone microtissue, and creating a 'two-layer skin' between blood vessels and nerves and the bone matrix, preventing the formation of a functional network, thus resulting in poor quality of the constructed bone microtissue." The reasons for this extremely low physiological simulation, poor quality of the constructed bone microtissue, and the creation of a "two-layer skin" between blood vessels and nerves and the bone matrix, preventing the formation of a functional network, are as follows: When constructing bone microtissue, the following technical problem four often occurs: Only simple bone microtissue is constructed without considering nerve innervation and vascular networks, resulting in extremely low physiological simulation and poor quality of the constructed bone microtissue; Using a method of constructing bone microtissue by first creating bone and then seeding cells results in a "two-layer skin" between blood vessels and nerves and the bone matrix, preventing the formation of a functional network, thus resulting in poor quality of the constructed bone microtissue. If these factors are resolved, the physiological simulation can be improved, the quality of the constructed bone microtissue can be enhanced, the "two-layer skin" situation between blood vessels and nerves and the bone matrix can be reduced, and the quality of the constructed bone microtissue can be improved. To achieve this effect, the bone microtissue construction method of bone-derived extracellular matrix combined with vascular matrix components disclosed in this paper first systematically solves a series of core defects of existing bone microtissues through key designs such as SVF prevascularization, DRG nerve pretreatment, time-sequential multi-factor induction, β-TCP enhanced mineralization, in situ collagen embedding of nerves, and end-stage hypoxia culture. These defects include functional singularity (no nerves or blood vessels), poor integration of nerves, blood vessels and bone, central necrosis, insufficient mineralization, weak mechanical properties, short-lived growth factor effects, simple cell composition, non-physiological oxygen environment, and difficulty in maintaining long-term stability. This can improve the physiological simulation, enhance the effect of the constructed bone microtissue, reduce the "two-layer skin" situation of blood vessels and nerves and bone matrix, and improve the effect of the constructed bone microtissue.

[0112]

experiment

[0113] Experiment 2: HE staining was used to identify the structure of DBM microcarriers and observe the decellularization effect. Masson's trichrome staining is a dye that specifically stains collagen. Figure 3As shown, staining the DBM microcarriers allows for a direct visualization of the distribution and retention of collagen in the extracellular matrix. Histological analysis revealed a clear trabecular network structure in the decellularized bone matrix under hematoxylin-eosin staining, but no cell nuclei were observed in the original lacunae, indicating effective removal of cellular components. Masson's trichrome staining confirmed that collagen fibers (stained blue) were well-preserved in the decellularized bone matrix, with a clear arrangement.

[0114] Experiment 3: Frozen sections of the constructed bone microtissue were prepared, and strong permeabilization immunostaining buffer (Beyotime P0097, China) was added and incubated at room temperature for 5 min. An appropriate amount of TUNEL assay solution was prepared and thoroughly mixed. Note: The prepared TUNEL assay solution must be used immediately and cannot be frozen. Each sample used 50 μl of assay solution, including 5 μl of TdT enzyme and 45 μl of fluorescent labeling solution. The total liquid volume can be reduced to 20 μl depending on the sample size. The samples were washed twice with PBS, dried, and circled with an immunohistochemistry pen. 50 μl of TUNEL assay solution was added to the sample, and the samples were incubated at 37°C in the dark for 60 min. An anti-evaporation membrane was used to prevent evaporation and to ensure the TUNEL assay solution evenly covered the sample. The samples were washed twice with PBS, and the cell nuclei were counterstained with DAPI for 10 min. The samples were washed three times with PBS, and then the slides were washed with distilled water to remove any remaining impurities. The slides were then mounted with an anti-fluorescence quenching mounting medium and observed using a multicolor fluorescence quantitative analyzer to capture panoramic images (e.g., [image not provided]). Figure 4 As shown, only a small number of cells underwent apoptosis. The percentage of positive cells was quantitatively analyzed using ImageJ. Each group was repeated three times at each time point.

[0115] Experiment 4: Cell tissues were first fixed with 4% paraformaldehyde at room temperature for at least 30 minutes. Then, a 15% sucrose solution prepared with double-distilled water was used to dehydrate and precipitate the bone microtissue for 30 minutes. The tissues were then embedded in OCT gel and frozen. 5μm sections were prepared using a cryostat (Leica, Germany) at -25℃. Poly-L-lysine-coated slides (Boster Biological, China) were used as slides, which were then stored at -20℃. Before staining, the slides were warmed to room temperature for 30 minutes, then washed twice with distilled water for 5 minutes each time. The samples were incubated for 5 minutes at room temperature with rapid antigen retrieval solution for frozen sections (Beyotime P0090, China). Note that the original rapid antigen retrieval solution for frozen sections needs to be diluted five times its volume with double-distilled water. The samples were then washed three times with immunostaining washing solution (Beyotime P0106, China), for 5 minutes each time. After drying the sections, use an immunohistochemistry pen (Biosharp, China) to draw circles around the tissue. Add 5% BSA blocking solution and incubate at 37°C for 30 min. Cover with an anti-evaporation membrane to prevent the liquid from drying out. Shake dry; do not wash. Add 1:200 diluted Tublin primary antibody and incubate at 37°C for 2 hours or 4°C overnight. Cover with an anti-evaporation membrane to prevent the liquid from drying out. Wash with immunostaining wash solution, 5 times for 5 min each. Add HRP-labeled secondary antibody and incubate at 37°C for 30 min. Cover with an anti-evaporation membrane to prevent the liquid from drying out. Wash with immunostaining wash solution, 5 times for 5 min each. Dilute concentrated fluorescent dye 1:200 with TSAbuffer and add the corresponding TSA fluorescent dye reaction solution to the circle. Incubate at room temperature for 1-15 min in the dark. Wash with immunostaining wash solution, 5 times for 5 min each. Add DAPI staining solution and incubate at room temperature for 7 min. Wash with immunostaining wash solution, 3 times for 5 min each. Before mounting, wash the slides with double-distilled water for 5 minutes to ensure they are clean and free of excess reagent or salt residue. After drying the slides, mount them with an anti-fluorescence attenuation mounting medium. Observe and take panoramic images using a multicolor fluorescence quantitative analyzer. The photographs are as follows: Figure 5 ,like Figure 5 As shown, the bone microtissue stained with Tublin (cytoskeleton) after seven days of culture showed good cell adhesion and natural morphology.

[0116] Experiment 5: (1) Alkaline phosphatase (ALP) staining: Frozen sections of the constructed bone microtissue were prepared, and ALP staining was performed using the BCIP / NBT alkaline phosphatase staining kit (Beyotime C3206, China). The BCIP / NBT staining working solution was prepared, including 3 ml of alkaline phosphatase staining buffer, 10 μl of BCIP solution (300X), and 20 μl of NBT solution (150X). Other dosages were adjusted accordingly. The slides were dried, and an appropriate amount of BCIP / NBT staining working solution was added to ensure full coverage of the sample. The slides were incubated at room temperature in the dark for 5-30 minutes until the desired color depth was achieved. The BCIP / NBT staining working solution was removed, and the slides were washed 1-2 times with distilled water. After dehydration, the slides were mounted with neutral resin and histologically imaged using a panoramic confocal digital slide scanner (Damai, China). Each group was replicated 3 times at each time point.

[0117] (2) Alizarin Red Staining: Frozen sections of the constructed bone microtissue were prepared, and then alizarin red staining solution was added to fully cover the specimens. Staining was performed at room temperature for 10 min. Excess staining solution was removed by washing three times with PBS, followed by dehydration. After mounting with neutral resin, the samples were observed and images acquired using a panoramic confocal digital slide scanner (Damai, China). Three replicates were performed for each time point in each group. Figure 6 As shown, bone microtissue stained with ALP (alkaline phosphatase) after seven days of culture and bone microtissue stained with ARS (alizarin red) after 14 days of culture showed significant osteogenic expression in both early and late stages.

[0118] Experiment 6: Cell tissues were first fixed with 4% paraformaldehyde at room temperature for at least 30 minutes. Then, a 15% sucrose solution prepared with double-distilled water was used to dehydrate and precipitate the bone microtissue for 30 minutes. The tissues were then embedded in OCT gel and frozen. 5μm sections were prepared using a cryostat (Leica, Germany) at -25℃. Poly-L-lysine-coated slides (Boster Biological, China) were used as slides, which were then stored at -20℃. Before staining, the slides were warmed to room temperature for 30 minutes, then washed twice with distilled water for 5 minutes each time. The samples were incubated for 5 minutes at room temperature with rapid antigen retrieval solution for frozen sections (Beyotime P0090, China). Note that the original rapid antigen retrieval solution for frozen sections needs to be diluted five times its volume with double-distilled water. The samples were then washed three times with immunostaining washing solution (Beyotime P0106, China), for 5 minutes each time. After drying the sections, use an immunohistochemistry pen (Biosharp, China) to draw circles around the tissue. Add 5% BSA blocking solution and incubate at 37°C for 30 min. Cover with an anti-evaporation membrane to prevent the liquid from drying out. Shake dry; do not wash. Add 1:200 diluted CD31 primary antibody and incubate at 37°C for 2 hours or 4°C overnight. Cover with an anti-evaporation membrane to prevent the liquid from drying out. Wash with immunostaining wash solution, 5 times for 5 min each time. Add HRP-labeled secondary antibody and incubate at 37°C for 30 min. Cover with an anti-evaporation membrane to prevent the liquid from drying out. Wash with immunostaining wash solution, 5 times for 5 min each time. Dilute concentrated fluorescent dye 1:200 with TSAbuffer and add the corresponding TSA fluorescent dye reaction solution to the circle. Incubate at room temperature for 1-15 min in the dark. Wash with immunostaining wash solution, 5 times for 5 min each time. Add DAPI staining solution and incubate at room temperature for 7 min. Wash with immunostaining wash solution, 3 times for 5 min each time. Before mounting, wash the slides with double-distilled water for 5 minutes to ensure they are clean and free of excess reagent or salt residue. After sectioning and drying, mount the slides with an anti-fluorescence attenuation mounting medium and observe and capture panoramic images using a multicolor fluorescence quantitative analyzer. Figure 7 As shown, Figure 7 CD31 staining of bone microtissue cultured for seven days can characterize the vascular markers, which show a reticular distribution and significant expression.

[0119] This disclosure also provides an application of bone microtissue in bone defect repair. The bone microtissue described above is as follows: Figure 1 The bone micro-tissue constructed in the process.

[0120] This disclosure also provides the application of bone-derived extracellular matrix microcarriers and vascular matrix components in the construction of bone microtissues. The bone-derived extracellular matrix microcarriers are as described above. Figure 1 The bone-derived extracellular matrix microcarriers described herein, wherein the aforementioned vascular matrix components are as described above. Figure 1 The vascular matrix components described in [the text].

[0121] The above-described embodiments of this disclosure have the following beneficial effects: the method for constructing bone microtissue using bone extracellular matrix composite vascular matrix components according to some embodiments of this disclosure can reduce the risk of secondary injury to patients and complications, and can improve the effect of bone defect repair. Specifically, the reasons for the high risk of secondary injury and complications to patients and the poor effect of bone defect repair are as follows: when autologous bone grafting is used for repair, the donor site is limited, resulting in the risk of secondary injury and complications to patients; when allogeneic bone grafting is used, there are risks such as immune rejection, disease transmission, and insufficient bone integration capacity, resulting in a poor effect of allogeneic bone in bone defect repair; when artificial bone materials (such as hydroxyapatite or calcium phosphate ceramic) are used to construct bone microtissue to repair bone defects, the bioactivity of the bone microtissue is low, making it difficult for the degradation rate to match the bone regeneration process, and there is a lack of a natural microenvironment that promotes vascularization and cell adhesion, resulting in a poor effect of using the constructed bone microtissue to repair bone defects. Based on this, some embodiments of the present disclosure describe a method for constructing bone microtissue using bone extracellular matrix combined with vascular matrix components. First, the obtained human femoral head cortical bone sample is pretreated to obtain a pretreated human femoral head cortical bone sample. This allows the obtained human femoral head cortical bone sample to be pretreated and then used to construct bone microtissue. Then, the pretreated human femoral head cortical bone sample is subjected to freeze-thaw treatment to obtain a freeze-thawed human femoral head cortical bone sample as the target human femoral head cortical bone sample. This allows for further pretreatment of the obtained human femoral head cortical bone sample. Next, in response to the construction method being a first bone microtissue construction method, the following first bone microtissue construction steps are performed: First, demineralized bone powder corresponding to the target human femoral head cortical bone sample is prepared based on the aforementioned target human femoral head cortical bone sample. This yields the prepared demineralized bone powder. Then, bone-derived extracellular matrix microcarriers are prepared using a ball mill, molecular sieve, and the aforementioned demineralized bone powder. This yields the prepared bone-derived extracellular matrix microcarriers, which are then used for further construction of bone microtissue. Next, vascular matrix components were prepared based on human abdominal adipose tissue samples. Bone microtissue could then be prepared using these vascular matrix components. Next, the aforementioned vascular matrix components and bone-derived extracellular matrix microcarriers were mixed in a basal culture medium according to a target ratio. This mixing process further prepared the microtissue. The mixed suspension was then transferred to a rotary bioreactor, and bone-inducing medium was added to the basal culture medium. This allowed for dynamic culture, thereby culturing the bone microtissue. Next, the vascular matrix components and bone-derived extracellular matrix microcarriers in the basal culture medium were dynamically cultured according to preset culture parameters to obtain cultured bone microtissue. Thus, the constructed bone microtissue was obtained.Because it uses human allogeneic bone to prepare acellular extracellular matrix microcarriers, immunogenicity can be minimized while preserving the components and structure of the natural bone matrix, which is beneficial for cell recognition and functional expression. Furthermore, because it uses vascular matrix components as seed cells, which are rich in various osteogenic and angiogenesis-related precursor cells, it can synergistically promote bone regeneration and angiogenesis. Moreover, because it employs a bioreactor for three-dimensional dynamic co-culture, simulating the mechanical and biochemical microenvironment of bone tissue growth in vivo, it improves cell survival rate, distribution uniformity, and functional maturity. Therefore, it can enhance the effectiveness of the constructed bone microtissue, thereby reducing secondary damage to patients and lowering the risk of complications, and improving the repair effect of bone defects.

[0122] The above description is merely a selection of preferred embodiments of this disclosure and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in the embodiments of this disclosure is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in the embodiments of this disclosure.

Claims

1. A method for constructing bone microtissue by combining bone extracellular matrix with vascular matrix components, comprising: The obtained human femoral head cortical bone samples were preprocessed to obtain preprocessed human femoral head cortical bone samples. Pre-treated human femoral head cortical bone samples were subjected to freeze-thaw treatment to obtain freeze-thawed human femoral head cortical bone samples as target human femoral head cortical bone samples. In response to the construction method being the first bone microtissue construction method, the following first bone microtissue construction steps are performed: Based on the target human femoral head cortical bone sample, demineralized bone powder corresponding to the target human femoral head cortical bone sample was prepared; Bone-derived extracellular matrix microcarriers were prepared using a ball mill, molecular sieve, and the demineralized bone powder. Vascular matrix components were prepared based on human abdominal adipose tissue samples. According to the target ratio, the vascular matrix component and the bone-derived extracellular matrix microcarrier are mixed in a basal culture medium to obtain a mixed suspension; The mixed suspension was transferred to a rotary bioreactor, and bone-inducing medium was added to the basal medium; The vascular matrix components in the basal culture medium and the bone-derived extracellular matrix microcarriers were dynamically cultured to obtain the constructed bone microtissue.

2. The method according to claim 1, wherein, The preprocessing of the obtained human femoral head cortical bone sample to obtain a preprocessed human femoral head cortical bone sample includes: The obtained human femoral head cortical bone sample was placed in a sterile operating table; The soft tissue, cartilage, and periosteum attached to the surface of the human femoral head cortical bone sample were removed using a scalpel and periosteal elevator. The human femoral head cortical bone sample was rinsed three times with sterile saline to remove blood cells and non-bone residues. The human femoral head cortical bone sample was cut into bone slices with a thickness of 2–3 mm along the bone texture direction using a constant speed low speed cutting machine to obtain individual cortical bone slices. Each cortical bone fragment was trimmed and cut using sterile bone forceps, and the trimmed cortical bone fragments were used as pre-processed human femoral head cortical bone samples.

3. The method according to claim 1, wherein, The step of performing freeze-thaw treatment on the pretreated human femoral head cortical bone sample to obtain a freeze-thawed human femoral head cortical bone sample as the target human femoral head cortical bone sample includes: Based on a preset number of freeze-thaw cycles, the following freeze-thaw treatment steps were performed on the pretreated human femoral head cortical bone sample: The pretreated human femoral head cortical bone sample was transferred into a pre-cooled 50ml centrifuge tube, an appropriate amount of liquid nitrogen was added, and the sample was frozen at -196℃ for 1 hour. After removing from the freezer, thaw at room temperature for 30 minutes.

4. The method according to claim 1, wherein, The step of preparing demineralized bone powder corresponding to the target human femoral head cortical bone sample includes: The target human femoral head cortical bone sample was placed in a grinding jar pre-cooled with liquid nitrogen; The target human femoral head cortical bone sample was ground three times at a frequency of 30 Hz using a high-throughput tissue grinder to obtain bone powder. Each grinding session lasted for 2 minutes to achieve preliminary grinding of the target human femoral head cortical bone sample. The obtained bone meal was placed on ice to cool; After initial grinding, the resulting bone powder is passed through a stainless steel sieve with a pore size of 500μm to remove large particles that are not fully ground. Collect the sieved bone meal and place it in a sterile container for later use; Add a PBS solution containing 2% penicillin / streptomycin to the bone meal; Place on a horizontal shaker at 4℃ and shake at 80 rpm for 30 minutes; Then centrifuge at 3000 rpm for 10 minutes, discard the supernatant, and obtain defatted bone meal; The defatted bone meal was transferred to a 0.5N hydrochloric acid solution and gently stirred magnetically at room temperature for 24 hours. The hydrochloric acid solution was replaced with fresh solution every 8 hours to ensure complete demineralization. After demineralization, the bone meal is washed with deionized water until the pH value of the bone meal is between pH 6.5 and 7.5, and the washed bone meal is used as demineralized bone meal.

5. The method according to claim 1, wherein, The preparation of bone-derived extracellular matrix microcarriers using a ball mill, molecular sieve, and the demineralized bone powder includes: The demineralized bone powder was placed in a freeze-drying bottle and pre-frozen at -80°C for 4 hours. The material was transferred to a freeze dryer and freeze-dried at -50°C and 0.1 mbar for 24 hours until it was completely dry and became porous and spongy, thus obtaining a freeze-dried bone matrix. According to the preset grinding time, preset grinding speed, preset cooling interval time and preset grinding number, a ball mill is used to perform secondary fine grinding on the freeze-dried bone matrix to obtain the ground freeze-dried bone matrix. The preset grinding speed is 300 rpm, the preset grinding time is 2 minutes and the preset grinding number is 5 times. The ground freeze-dried bone matrix was sequentially passed through molecular sieves with pore sizes of 60 μm and 10 μm to collect bone-derived extracellular matrix microcarriers with a size between 10 and 60 μm. The bone-derived extracellular matrix microcarriers were irregular porous particles with a surface exhibiting natural bone microstructure.

6. The method according to claim 1, wherein, The preparation of vascular matrix components based on human abdominal adipose tissue samples includes: Human abdominal adipose tissue was obtained, wherein the volume of the obtained human abdominal adipose tissue was in the range of 50~100ml; The human abdominal adipose tissue was subjected to a shake digestion process using a digestive solution containing collagenase in a constant temperature water bath at 37°C to obtain shake digested human abdominal adipose tissue. The shake digestion process lasted for 40 to 60 minutes. The digestive solution was type I collagenase with a concentration of 1 to 2 mg / ml. According to the preset centrifugal force and preset centrifugal time, the human abdominal adipose tissue after oscillation digestion is centrifuged to obtain centrifuged human abdominal adipose tissue. The preset centrifugal force is in the range of 300~400g and the preset centrifugal time is 10 minutes. Remove the upper layer of oil and liquid, and collect the bottom layer of cell sediment; The precipitate was treated with erythrocyte lysis buffer to further purify the vascular matrix components, resulting in purified vascular matrix components. The concentration of the purified vascular matrix components was adjusted to 1×10⁻⁶. 6 ~5×10 6 The adjusted vascular matrix composition was obtained by measuring cells / ml and used as the vascular matrix composition for preparation.

7. The method according to claim 1, wherein, The dynamic culture treatment of the vascular matrix components in the basal culture medium and the bone-derived extracellular matrix microcarriers to obtain cultured bone microtissue includes: In response to determining that the vascular matrix component and the bone-derived extracellular matrix microcarrier are in the initial stage of a preset culture stage, the rotation speed of the culture shaker is set to 8–12 rpm, and the culture angle is set to 5°–10° to promote cell adhesion and initial distribution. In response to determining that the vascular matrix component and the bone-derived extracellular matrix microcarrier are in the intermediate stage included in the preset culture stage, the rotation speed of the culture shaker is set to 15–20 rpm and the culture angle is set to 15°–20°. In response to determining that the vascular matrix component and the bone-derived extracellular matrix microcarrier are in the later stage of the preset culture stage, the rotation speed of the culture shaker is set to 25–30 rpm, and the culture angle is set to 25°–30°.

8. The application of a type of bone microtissue in bone defect repair, wherein, The bone microtissue is the bone microtissue constructed as described in any one of claims 1-7.

9. The application of a bone-derived extracellular matrix microcarrier and vascular matrix component in the construction of bone microtissues, wherein, The bone-derived extracellular matrix microcarrier is the bone-derived extracellular matrix microcarrier as described in any one of claims 1-7, and the vascular matrix component is the vascular matrix component as described in any one of claims 1-7.

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