A humanized osteoporosis mouse model and a method for constructing the same

Abstract

The application discloses a humanized osteoporosis mouse model and a construction method thereof, and belongs to the technical field of biotechnology and animal model technology.The present application is aimed at the core defects of the existing osteoporosis model, such as great species difference, insufficient humanization degree, low pathological simulation degree, unstable phenotype and the like, and the humanized osteoporosis mouse model is constructed through five core steps of experimental animal pretreatment, collagen-nano-hydroxyapatite biomimetic scaffold preparation and cell pre-planting, immune-bone metabolism double humanization system construction, gradual osteoporosis induction, model identification and verification.The present application first realizes double humanization of the immune and bone metabolism systems, greatly improves the human cell planting rate and survival time, accurately simulates the pathological process of clinical postmenopausal osteoporosis, the model phenotype is stable and uniform, and the matching degree with the clinical drug reactivity is high, so the present application can be widely used for the research and development of anti-osteoporosis drugs and the research of pathological mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and animal model technology, specifically relating to the construction technology of humanized disease animal models, and more specifically to a humanized osteoporosis mouse model and its construction method. Background Technology

[0002] Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass, bone microarchitectural deterioration, increased bone fragility, and elevated fracture risk. Postmenopausal osteoporosis (PMOP) accounts for more than 70% of clinical cases and has become a major public health issue for middle-aged and elderly women worldwide. Currently, the development of anti-osteoporosis drugs relies heavily on animal models. However, the core deficiencies in existing models directly result in a less than 30% match between preclinical efficacy evaluation results and human clinical trial results, severely hindering the progress of new drug development.

[0003] Existing animal models of osteoporosis are mainly divided into two categories, and their technical bottlenecks and limitations are as follows: The first category is the traditional rodent osteoporosis model, the most commonly used being the ovariectomized mouse (OVX) model. This model achieves acute estrogen deprivation through bilateral total ovariectomy, inducing bone loss in mice. However, it is essentially a mouse-derived bone metabolism system, which has insurmountable species differences: First, the bone remodeling cycle differs greatly between humans and mice, with humans having a cycle of 3-6 months and mice only 1-2 months, and the pathological process is completely inconsistent with bone metabolism kinetics; Second, there are significant differences between humans and mice in the spectrum of bone metabolism-related cytokines, drug target sequences, and reactivity. For example, bisphosphonates are highly effective in mouse models but have no response in some clinical populations, while more than 60% of the positive results obtained by novel targeted drugs in mouse models fail in clinical trials; Third, the traditional OVX model is an acute injury caused by a sudden drop in estrogen levels, which is completely inconsistent with the physiological and pathological process of the gradual decline in estrogen during the 3-5 years of perimenopause in clinical women, and cannot simulate the gradual imbalance of bone metabolism during perimenopause, resulting in extremely low pathological simulation.

[0004] The second category comprises existing reported humanized osteoporosis models, which attempt to address species differences through human cell transplantation. However, they still suffer from fatal technical flaws: First, insufficient humanization, failing to humanize the core pathological mechanisms. The vast majority of models only transplant human bone marrow mesenchymal stem cells (hBMSCs), while osteoclasts remain mouse-derived, failing to achieve the core regulatory system of human osteogenic-osteoclast coupling. Since the essence of osteoporosis is precisely the imbalance of osteogenic-osteoclast coupling, these models cannot simulate the pathological changes at the human cell level. A few models transplant both osteogenic and osteoclast precursor cells, but fail to construct a human immune microenvironment. Clinical studies have confirmed that immune cells (T cells, B cells, macrophages, etc.) are the core link in bone metabolism regulation, and the immune inflammation dysregulation induced by postmenopausal estrogen decline is a key driver of bone loss. Models lacking a human immune microenvironment cannot simulate the immune-bone metabolism interaction regulation pathology of clinical osteoporosis. Second, extremely low human cell colonization rates and short survival times prevent the formation of a stable human bone microenvironment. Most existing models use tail vein injection of human cells, resulting in highly non-specific cell distribution in vivo, with a bone tissue colonization rate of less than 10%. The vast majority of cells undergo apoptosis in vivo, failing to form stable human bone remodeling units. A few models using orthotopic transplantation fail to construct a biomimetic microenvironment suitable for human cell growth, resulting in cell survival times of no more than 4 weeks, which cannot meet the needs for osteoporosis induction and long-term efficacy evaluation. Thirdly, the modeling cycle is long, phenotypes are unstable, and homogeneity is poor, failing to meet the needs of large-scale drug screening. Fourthly, there is a lack of specific human osteoporosis induction protocols; existing models still use the traditional mouse-derived OVX induction method, failing to specifically induce human cells to form an osteoporotic phenotype. Often, severe bone loss occurs in mouse-derived bone tissue, but pathological changes in human cell bone metabolism are not yet observed, resulting in extremely low model effectiveness.

[0005] In summary, there is currently no humanized osteoporosis mouse model that can simultaneously achieve dual humanization of immune and bone metabolism, high colonization rate of human cells, high matching between pathological process and clinical condition, stable and uniform phenotype, and short modeling cycle. This has become a key technical bottleneck in the development of anti-osteoporosis drugs and the study of pathological mechanisms. Summary of the Invention

[0006] This invention addresses the core deficiencies of existing technologies by providing a humanized osteoporosis mouse model and its construction method that features dual humanization of immune and bone metabolism, high human cell colonization rate, high match between osteoporosis phenotype and clinical manifestations, short modeling cycle, and excellent stability and uniformity. This solves the technical problems of existing models, such as large species differences, low degree of humanization, insufficient pathological simulation, and unstable phenotypes, providing an accurate and reliable animal model for the humanization screening, efficacy evaluation, and pathological mechanism research of anti-osteoporosis drugs.

[0007] The objective of this invention can be achieved through the following technical solutions: A method for constructing a humanized osteoporosis mouse model comprises five core steps: pretreatment of immunodeficient mice, preparation of biomimetic bone scaffolds and pre-implantation of cells, construction of a dual humanized immune-bone metabolism system, progressive osteoporosis induction, and model identification and validation. The specific technical solution is as follows: 1. Animal pretreatment: SPF-grade 8-10 week old female NSG immunodeficient mice were selected. This strain of mice lacks mature T cells, B cells, and NK cells, and does not exhibit immune rejection, making them ideal hosts for human cell transplantation. After one week of acclimatization, the mice were irradiated with a low dose of 1.5-2.5 Gy of gamma rays to eliminate residual immune cells and bone marrow hematopoietic stem cells, further reducing the risk of immune rejection and providing space for hPBMCs colonization. Subsequent transplantation procedures were completed within 24 hours after irradiation.

[0008] 2. Preparation and Pre-implantation of Bionic Bone Scaffolds: A collagen-nanohydroxyapatite composite bionic scaffold was prepared. PLGA sustained-release microspheres loaded with human bone metabolism-related cytokines (M-CSF, RANKL, TNF-α) were uniformly premixed in the scaffold matrix at the optimal dosage (M-CSF 500 ng / scaffold, RANKL 1 μg / scaffold, TNF-α 200 ng / scaffold). This scaffold mimics the extracellular matrix components and porous structure of human bone tissue and can provide a suitable growth and differentiation microenvironment for hBMSCs and osteoclast precursor cells through continuous sustained release of factors. Qualified hBMSCs and human CD14+ osteoclast precursor cells were mixed at a ratio of (2-4):1, using 1×10⁻⁶ cells. 7 The cells were seeded onto the scaffold at a total cell density of cells / mL and co-cultured in vitro at 37°C and 5% CO2 for 3-5 days to allow the cells to fully adhere, proliferate, and predifferentiate within the scaffold, thus obtaining a biomimetic scaffold loaded with bone metabolism factors and containing the intended implanted cells.

[0009] 3. Construction of a dual humanized immune-bone metabolism system: After anesthetizing pretreated mice, bilateral subcapsular renal transplantation was performed. Biomimetic scaffolds with pre-selected human-derived cells were implanted into the subcapsular region of both kidneys of the mice. The subcapsular region has a very rich blood supply, which can provide sufficient nutrition and oxygen to the cells in the scaffold, greatly improving the cell survival rate and colonization rate. On the same day as the surgery, hPBMCs were injected into the tail vein of the mice to reconstruct the human immune system. Finally, a dual humanized immune-bone metabolism mouse was constructed, which fully simulates the regulatory role of the immune microenvironment on bone metabolism in the human body.

[0010] 4. Progressive Osteoporosis Induction: Bilateral ovarian artery ligation was performed on mice simultaneously with transplantation to preserve ovarian tissue and avoid the acute estrogen drop seen in traditional OVX models. From the day of surgery, tamoxifen PLGA sustained-release microspheres were subcutaneously implanted in the back. Gradual dose administration achieved progressive estrogen blockade, precisely mimicking the pathological process of progressive estrogen decline during human perimenopause. Simultaneously, the biomimetic scaffold was premixed with PLGA sustained-release microspheres loaded with human M-CSF, RANKL, and TNF-α, continuously releasing human cytokines over 8 weeks. This specifically induced the differentiation and maturation of human osteoclast precursor cells, disrupting the human osteogenic-osteoclast coupling balance and stably inducing the formation of an osteoporotic phenotype in the human bone metabolism system.

[0011] 5. Model identification and validation: After 8 weeks of induction, the model was systematically identified by detecting the human cell colonization rate, human bone metabolism markers, bone microstructure Micro-CT, histological staining, and clinical drug responsiveness. The model that meets the qualification criteria is a stable humanized osteoporosis mouse model.

[0012] Furthermore, the optimal preparation parameters for the collagen-nanohydroxyapatite composite biomimetic scaffold of the present invention are: a mass ratio of type I collagen to nanohydroxyapatite of 4:1, a scaffold porosity of 88%-90%, and an average pore size of 200-250 μm. Under these parameters, the mechanical properties of the scaffold match those of bone tissue and are most conducive to the infiltration and growth of human osteoblasts.

[0013] Furthermore, the hBMSCs described in this invention are 3rd-5th generation cells, and the flow cytometry analysis shows that the double positivity rate of CD90+ and CD105+ is ≥95%, and the negative rate of CD45- and CD34- is ≥99%; the human CD14+ osteoclast precursor cells are sorted by immunomagnetic beads, with a positive rate of ≥95%, ensuring the purity and activity of the human cells.

[0014] Furthermore, the gradual estrogen deprivation gradient dosing regimen of the present invention is as follows: tamoxifen sustained-release microspheres are administered at a dose of 0.5 mg / kg·d in the first week, 1 mg / kg·d in the second to fourth weeks, and 2 mg / kg·d in the fifth to eighth weeks. This regimen can accurately simulate the downward trend of estrogen levels in the perimenopausal period and avoid the pathological process caused by acute estrogen deprivation from deviating from clinical reality.

[0015] Furthermore, the optimal loading of the human bone metabolism-related cytokines described in this invention is: M-CSF 500ng / scaffold, RANKL 1μg / scaffold, and TNF-α 200ng / scaffold. This dosage can specifically induce human osteoclast differentiation without causing non-specific inflammatory responses, thus ensuring the specificity and stability of the osteoporosis phenotype.

[0016] Furthermore, the humanized osteoporosis mouse model constructed in this invention meets the following criteria: ≥80% colonization rate of human CD90+ osteoblasts and human CD14+ osteoclasts in the renal capsule scaffold; ≥15% proportion of human CD45+ immune cells in peripheral blood; compared with sham-operated humanized control mice of the same strain, the model mice exhibit a ≥30% decrease in tibial bone mineral density (BMD), a ≥40% decrease in bone volume fraction (BV / TV), a ≥2-fold increase in serum human bone resorption marker CTX-I levels, a ≥30% decrease in human bone formation marker PINP levels, and a ≥2.5-fold increase in the number of TRAP-positive osteoclasts on the trabecular bone surface; and a ≥85% match between the responsiveness to first-line clinical anti-osteoporosis drugs and human clinical data.

[0017] The beneficial effects of this invention are: (1) For the first time, a dual-humanized osteoporosis mouse model of immune-bone metabolism was constructed, breaking through the core bottleneck of insufficient humanization in existing models. This invention simultaneously achieved stable colonization and functional maturation of human osteoblasts, human osteoclasts, and human immune cells, and completely reconstructed the core bone metabolism regulatory network of "immune regulation-osteogenesis-osteoclast coupling" in the human body. It achieved full humanization of the pathological mechanism of osteoporosis at the cellular level, and completely solved the fatal defects of existing models, such as single-cell humanization, lack of immune microenvironment, and inability to simulate human pathological processes.

[0018] (2) The innovative "bionic scaffold pre-implantation + subcapsular renal transplantation" technical solution achieves high colonization rate and long-term survival of human cells. The collagen-nanohydroxyapatite bionic scaffold prepared by this invention accurately simulates the extracellular matrix components and porous structure of human bone, providing a suitable growth microenvironment for cells; combined with the rich blood supply under the renal capsule, the colonization rate of human cells in the scaffold reaches more than 85%, and the cell survival time exceeds 6 months, which can meet the needs of long-term drug efficacy evaluation and solve the technical problems of poor survival and low colonization rate of human cells in existing models.

[0019] (3) A protocol of "progressive estrogen deprivation + specific induction of human cytokines" was established, achieving an osteoporosis phenotype that is highly matched to clinical practice. This invention accurately simulates the physiological and pathological process of progressive estrogen decline during the perimenopausal period by combining ovarian artery ligation with gradient dose tamoxifen sustained-release administration, replacing the acute estrogen drop in the traditional OVX model; at the same time, by in-stent sustained release of human M-CSF, RANKL, and TNF-α, specific induction of human osteoclast differentiation is achieved, and a stable humanized osteoporosis phenotype can be obtained in just 8 weeks, with excellent phenotype uniformity and an individual coefficient of variation of less than 10%, which can be directly used for large-scale humanization screening of anti-osteoporosis drugs.

[0020] (4) The model’s drug responsiveness is highly matched with clinical data, which greatly improves the reliability of preclinical efficacy evaluation. The model constructed in this invention shows that its changes in bone metabolism markers, bone microstructure destruction characteristics, and responsiveness to first-line clinical anti-osteoporosis drugs (alendronate sodium and teriparatide) are highly matched with data from human clinical postmenopausal osteoporosis patients, with a matching degree of over 85%. This completely solves the problems of large species differences and low drug screening results and clinical conversion rates in traditional rodent models, providing a more accurate and reliable preclinical animal model for the development of new anti-osteoporosis drugs.

[0021] (5) The construction method of the present invention is simple to operate, has good repeatability, strong model stability, and wide applicability. All operation steps of the present invention are standardized operations, requiring no special high-end equipment, and can be completed in ordinary SPF-grade animal rooms, with 100% repeatability; after induction, the constructed model can stably maintain the osteoporosis phenotype for more than 6 months, which can not only be used for screening and efficacy evaluation of anti-osteoporosis drugs, but also for research in multiple fields such as osteoporosis pathological mechanism, bone metabolism regulation, and human bone tissue engineering, and has extremely high application value. Detailed Implementation

[0022] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0023] 1. Experimental animals: SPF-grade 8-week-old female NSG immunodeficient mice, weighing 18-22g, were housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50±5%, with a 12-hour light-dark cycle and free access to food and water. They were used for experiments after one week of acclimatization. All animal experiments were approved by the institution's animal ethics committee.

[0024] 2. Reagents and Materials: Type I collagen (mouse tail source, Sigma-Aldrich, catalog number C7661), nano-hydroxyapatite (particle size 20-50 nm), EDC, NHS (Sigma-Aldrich, catalog numbers E6383, H7377), PLGA (LA:GA=75:25, molecular weight 10000), tamoxifen (Sigma-Aldrich, catalog number T5648), human M-CSF, RANKL, TNF-α (PeproTech, catalog numbers 300-25, 310-01, 300-01A), human peripheral blood lymphocytes. Immunosorbent assay (TIA) medium (Tianjin Haoyang, catalog number LTS1077), CD14 immunomagnetic beads (Mitten, catalog number 130-050-201), hBMSCs complete culture medium (Cyagen Biosciences, catalog number MUBMX-90011), α-MEM culture medium, premium fetal bovine serum (Gibco, catalog numbers 12571063, 10099141), flow cytometry antibodies: human CD45-APC, CD90-FITC, CD105-PE, CD14-PE-Cy7 (BioLegend), human PINP, OCN, CTX-I ELISA kit (Wuhan Yilairuit), TRAP staining kit (Sigma, catalog number 387A), penicillin-streptomycin bispecific antibody (Gibco, catalog number 15140122).

[0025] Instruments and equipment: freeze dryer, cobalt-60 irradiator, gamma ray irradiator, flow cytometer, micro-CT, paraffin microtome, upright optical microscope, ELISA reader, CO2 cell culture incubator, biosafety cabinet, low-temperature high-speed centrifuge.

[0026] General experimental methods 1. Isolation, Culture, and Identification of hBMSCs: 5 mL of bone marrow aspiration fluid was collected from healthy adult volunteers, and informed consent was obtained from the volunteers. Bone marrow mononuclear cells were isolated using density gradient centrifugation and seeded into complete hBMSC culture medium. The cells were cultured at 37°C in a 5% CO2 saturated humidity incubator. The medium was changed for the first time after 48 hours to remove non-adherent cells, and then every 3 days thereafter. When the cells reached 80%-90% confluence, they were passaged using 0.25% trypsin-EDTA digestion. Cells from passage 3 were used to identify cell surface markers using flow cytometry. The results showed a CD90 and CD105 double positivity rate ≥98% and a CD45 and CD34 negative rate ≥99%, meeting the identification criteria for human mesenchymal stem cells. Cells from passages 3-5 were used for subsequent experiments.

[0027] 2. Isolation of human CD14+ monocytes and hPBMCs: 50 mL of peripheral blood was collected from healthy adult volunteers, and informed consent was obtained. The peripheral blood was diluted with an equal volume of sterile PBS and slowly stacked on top of the lymphocyte separation medium. The mixture was centrifuged horizontally at 2000 rpm for 20 min. The intermediate white membrane layer was carefully aspirated, and the cells were washed twice with sterile PBS to obtain hPBMCs. One-third of the hPBMCs were used for positive sorting using CD14 immunomagnetic beads to obtain human CD14+ monocytes. Flow cytometry identification showed a positive rate ≥96%, which were used as human osteoclast precursor cells. The remaining hPBMCs were adjusted to a cell concentration of 1 × 10^7 cells / 200 μL PBS and stored on ice for tail vein injection.

[0028] 3. Preparation of PLGA sustained-release microspheres: Drug / cytokine-loaded PLGA sustained-release microspheres were prepared using a double emulsion-solvent evaporation method. The specific steps are as follows: Tamoxifen sustained-release microspheres: PLGA was dissolved in dichloromethane to prepare an oil phase of 100 mg / mL; tamoxifen was dissolved in anhydrous ethanol as the inner aqueous phase, and added to the oil phase at a volume ratio of 5:1 (oil phase:inner aqueous phase). The mixture was ultrasonically emulsified at 300 W for 3 min under ice bath conditions to prepare the primary emulsion. The primary emulsion was added to 10 volumes of 1% PVA aqueous solution, and ultrasonically emulsified at 300 W for 5 min under ice bath conditions to prepare the secondary emulsion. The mixture was magnetically stirred at room temperature for 4 h to completely evaporate the organic solvent. The microspheres were collected by centrifugation at 12000 rpm for 10 min, washed three times with sterile PBS, freeze-dried, and stored in a sealed container at -20℃. The average particle size of the microspheres was 85±12 μm, the encapsulation efficiency was 88.2±2.1%, the in vitro sustained-release period was 8 weeks, and no significant burst release effect was observed.

[0029] Cytokine sustained-release microspheres: PLGA was dissolved in dichloromethane to prepare an oil phase of 100 mg / mL; human M-CSF, RANKL, and TNF-α were dissolved in sterile PBS containing 1% BSA as the internal aqueous phase. The remaining preparation steps were the same as for tamoxifen microspheres. The encapsulation efficiency of each cytokine microsphere was ≥85%, the in vitro sustained-release period was 8 weeks, and the cytokine activity retention rate during the sustained-release period was ≥90%.

[0030] 4. Preparation of Collagen-Nano-hydroxyapatite Composite Biomimetic Scaffold: Type I collagen was dissolved in 0.5 mol / L acetic acid solution and stirred at room temperature for 2 h to prepare a 5 mg / mL collagen solution. Nano-hydroxyapatite powder was added at a mass ratio of 4:1 (Type I collagen:nano-hydroxyapatite), and stirred continuously at room temperature for 2 h. After thorough mixing, the mixture was degassed under vacuum for 30 min to remove air bubbles. The mixture was injected into a cylindrical polytetrafluoroethylene mold (3 mm in diameter and 1 mm in height), pre-frozen at -20 °C for 4 h, and then transferred to a freeze dryer at -80 °C for 24 h to obtain a porous scaffold. The dried scaffold was immersed in a 75% ethanol solution containing 50 mmol / L EDC and 25 mmol / L NHS and crosslinked at room temperature for 24 h to improve the mechanical properties and degradation stability of the scaffold. After crosslinking, the scaffold was rinsed three times with sterile PBS for 15 min each time to remove residual crosslinking agent. The scaffold was then aliquoted, sterilized by cobalt-60 irradiation (dose 25 kGy), and aseptically stored at 4 °C for later use. The scaffold prepared by this method has a porosity of 89.2±2.1%, an average pore size of 210±35μm, and a compressive modulus of 12.5±1.3MPa, which meets the performance requirements of bone tissue engineering scaffolds.

[0031] 5. General methods for performance testing: The mice in all examples and comparative examples were tested for the following indicators after 8 weeks of induction. The sample size for each group was n=10. All tests were performed in triplicate. The results are expressed as mean ± standard deviation.

[0032] Human cell colonization rate detection: Mice were sacrificed, and scaffold tissue under the renal capsule was aseptically isolated. The tissue was washed three times with sterile PBS, minced, and then digested with 0.2% type I collagenase at 37°C for 2 hours, with pipetting every 15 minutes during digestion. After digestion, the cells were passed through a 200-mesh cell sieve and centrifuged at 1500 rpm for 5 minutes to collect a single-cell suspension. Simultaneously, 100 μL of peripheral blood from mice was collected, and erythrocyte lysis buffer was added to lyse the erythrocytes. The erythrocytes were washed twice with sterile PBS to obtain a leukocyte suspension. The cells were then added to flow cytometry antibodies: human CD45-APC, CD90-FITC, and CD14-PE-Cy7, respectively, and incubated at 4°C in the dark for 30 minutes. After washing twice with PBS and resuspending, the cells were analyzed by flow cytometry to calculate the colonization rate of human CD90+ osteoblasts and CD14+ osteoclasts within the scaffold, as well as the proportion of human CD45+ immune cells in the peripheral blood.

[0033] Serum bone metabolism marker detection: After 8 weeks of induction, 500 μL of blood was collected from the orbital vein of mice, incubated at room temperature for 2 hours, centrifuged at 3000 rpm for 15 minutes, and the supernatant serum was separated and stored at -80℃ for later use. Human PINP, OCN, and CTX-I ELISA kits were used, and the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The concentrations of human bone formation markers PINP and OCN, and human bone resorption marker CTX-I in serum were calculated based on the standard curve. For control groups without humanization treatment, the concentrations of mouse-derived PINP and CTX-I were measured.

[0034] Micro-CT examination of bone microstructure: After sacrifice, bilateral tibias were harvested, surrounding soft tissues were removed, and the bones were fixed with 4% paraformaldehyde for 24 h, washed three times with sterile PBS, and stored in 70% ethanol at 4°C. Scanning was performed using a Bruker SkyScan 1276 Micro-CT scanner with the following parameters: voltage 50 kV, current 200 μA, spatial resolution 10 μm, aluminum filter 0.5 mm, and rotation step size 0.4°. After scanning, image reconstruction was performed using NRecon software. The region 1-2 mm below the proximal growth plate of the tibia was selected as the region of interest (ROI). Core bone microstructure parameters, including bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness ([Tb.Th](Tb.Th)), and trabecular bone separation (Tb.Sp), were analyzed using CTAn software.

[0035] Histological and osteoclast staining detection: Tibial tissue and renal capsule scaffold tissue after Micro-CT scanning were collected and decalcified with 10% EDTA decalcification solution for 4 weeks, with the decalcification solution being changed every 3 days. After decalcification, the tissue was routinely dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and serially sectioned to a thickness of 5 μm. HE staining and Masson's trichrome staining were performed, and bone microstructure, collagen distribution, and cell growth were observed under an optical microscope. Tartrate-resistant acid phosphatase staining was performed using a TRAP staining kit, and the number of TRAP-positive multinucleated osteoclasts on the surface of bone trabeculae was counted under an optical microscope, expressed as the number of osteoclasts per millimeter of bone trabecular surface (N.Oc / [B.Pm](B.Pm), mm⁻¹).

[0036] Drug responsiveness verification experiment: Twelve model mice were randomly selected from each group and divided into a drug intervention group and a saline control group, with six mice in each group. The drug intervention group was administered alendronate sodium, a first-line clinical anti-osteoporosis drug, via gavage at a dose of 1 mg / kg / week for 8 consecutive weeks; the control group was administered an equal volume of sterile saline via gavage. After the drug administration was completed, the tibial bone bone mineral density (BMD) and serum creatine thrombocytopenic acid (CTX-I) levels were measured in each group. The increase rate of BMD and the decrease rate of CTX-I after drug intervention were calculated using the following formula: BMD increase rate = (BMD in the drug group - BMD in the control group) / BMD in the control group × 100% CTX-I reduction rate = (Control group CTX-I - Drug group CTX-I) / Control group CTX-I × 100% Simultaneously, the test results were compared with the efficacy data of alendronate sodium in clinical postmenopausal osteoporosis patients to calculate the matching degree between the model and clinical data. Example 1

[0037] This embodiment is the optimal implementation of the present invention, and the construction method includes the following steps: S1. Animal pretreatment: Eight-week-old female NSG mice were selected and acclimatized for one week before being irradiated with gamma rays at a dose of 2.0 Gy. Subsequent transplantation procedures were performed within 24 hours after irradiation.

[0038] S2. Pre-implantation of source cells into the biomimetic scaffold: Take the sterilized biomimetic scaffold prepared above and place it in a 24-well culture plate, one scaffold per well. Infiltrate with complete hBMSC culture medium for 2 hours, then discard excess culture medium. Take fourth-generation hBMSCs and CD14+ monocytes, mix them at a ratio of 3:1, and adjust the cell suspension concentration to 1×10⁻⁶. 7 Cells / mL were slowly added dropwise to the scaffold at a volume of 100 μL per well, and the scaffold was incubated at 37℃ with 5% CO2 for 4 hours to allow for full cell infiltration and attachment. Then, 1 mL of complete hBMSCs culture medium was added to each well, and the cells were co-cultured in vitro for 4 days, with fresh medium replaced every 2 days, to obtain the biomimetic scaffold with the intended implanted cells. After co-culture, calcein-AM / PI live / dead cell staining showed a cell viability of 93.2 ± 2.5% within the scaffold.

[0039] S3. Construction of an immune-bone metabolism dual-humanized system: Pretreated mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg / kg, fixed in a prone position on a surgical board, and the skin on the back was disinfected three times with povidone-iodine. A 0.5 cm longitudinal incision was made on both sides of the back below the costal margin, and the subcutaneous tissue and muscle were bluntly dissected to expose both kidneys. The renal capsule was gently lifted with microforceps to form a small cyst. The scaffolds for the predetermined humanized cells were implanted into the cysts under the renal capsule on both sides, one scaffold on each side. The muscle layer was sutured with microsutures, and the skin incision was sutured with silk sutures. On the same day as the operation, hPBMCs were injected into the tail vein of the mice at a dose of 1.5 × 10^7 cells / 200 μL PBS to construct immune-bone metabolism dual-humanized mice. After the operation, penicillin (200,000 U / mouse) was injected intraperitoneally for 3 consecutive days to prevent infection. The mice were fed normally, and their mental state and wound healing were observed daily.

[0040] S4. Progressive Osteoporosis Induction: One week post-transplantation, mice were anesthetized, fixed in a supine position, and the abdominal skin was disinfected three times with povidone-iodine. A 0.5cm midline incision was made in the lower abdomen to expose the abdominal cavity, and the bilateral ovaries and ovarian arteries were located. The bilateral ovarian arteries were ligated with 8-0 microsutures to preserve intact ovarian tissue and avoid acute estrogen deprivation. The abdomen was then closed layer by layer. Starting from the day of surgery, tamoxifen-loaded PLGA sustained-release microspheres were subcutaneously implanted in the back of the mice. The sustained-release period was 8 weeks, with the following dosages: 0.5 mg / kg / day in the first week, 1 mg / kg / day in the second to fourth weeks, and 2 mg / kg / day in the fifth to eighth weeks, to achieve progressive blockade of estrogen effects in vivo. Meanwhile, during the biomimetic scaffold preparation process in step S2, PLGA sustained-release microspheres loaded with human M-CSF, RANKL, and TNF-α were premixed. The loading amounts of each cytokine in each scaffold were: M-CSF 500ng, RANKL 1μg, and TNF-α 200ng, respectively. The microspheres were continuously released over 8 weeks, specifically inducing differentiation of human osteoclasts, breaking the osteogenic-osteoclast coupling balance, and inducing the formation of an osteoporotic phenotype.

[0041] S5. Model identification: After 8 weeks of induction, the mice were tested for various indicators according to the general detection methods described above to obtain a stable humanized osteoporosis mouse model. Example 2

[0042] The only difference between this embodiment and Embodiment 1 is that in step S2, the seeding ratio of hBMSCs to CD14+ monocytes is 2:1, and the total cell density remains 1×10⁻⁶. 7 The scaffold volume was measured in cells / mL, and all other steps, parameters, and reagents were exactly the same as in Example 1, thus constructing a humanized osteoporosis mouse model. Example 3

[0043] The only difference between this embodiment and Example 1 is that in step S4, the premixed cytokine sustained-release microspheres in the biomimetic scaffold have the following loading amounts in each scaffold: M-CSF 400ng, RANKL 800ng, and TNF-α 150ng. All other steps, parameters, and reagents are exactly the same as in Example 1, and a humanized osteoporosis mouse model is obtained.

[0044] Comparative Example 1 Without any irradiation pretreatment, scaffold transplantation, or human cell injection, an osteoporosis model was constructed using only traditional bilateral total ovariectomy (OVX). Specifically, 8-week-old female NSG mice were acclimatized for 1 week before undergoing bilateral total ovariectomy. They were then routinely fed for 8 weeks post-surgery to obtain the OVX osteoporosis mouse model.

[0045] Comparative Example 2 In step S2, only hBMSCs were seeded in the scaffold, without CD14+ monocytes, and the total cell density was the same as in Example 1 (1×10⁻⁶). 7 (cells / mL); In step S4, the scaffold is not loaded with human M-CSF, RANKL, or TNF-α sustained-release microspheres; the remaining steps are exactly the same as in Example 1.

[0046] Comparative Example 3 In step S2, only CD14+ mononuclear cells are seeded in the scaffold, and hBMSCs are not seeded. The total cell density is the same as in Example 1. The remaining steps are exactly the same as in Example 1.

[0047] Comparative Example 4 Without preparing a biomimetic scaffold, in step S2, hBMSCs and CD14+ monocytes were mixed at a ratio of 3:1 to prepare a cell suspension; in step S3, the cell suspension was directly injected under the renal capsule of both kidneys of mice, and the total number of cells was the same as in Example 1; the remaining steps were exactly the same as in Example 1.

[0048] Comparative Example 5 In step S4, instead of ovarian artery ligation combined with progressive tamoxifen administration, acute estrogen deprivation was performed using conventional bilateral total oophorectomy (OVX) one week after transplantation; the remaining steps were exactly the same as in Example 1.

[0049] Comparative Example 6 In step S4, the biomimetic scaffold is not loaded with human M-CSF, RANKL, or TNF-α sustained-release microspheres; the remaining steps are exactly the same as in Example 1.

[0050] Comparative Example 7 In step S2, the seeding ratio of hBMSCs to CD14+ monocytes is 1:1, and the total cell density is the same as in Example 1; the remaining steps are exactly the same as in Example 1.

[0051] Comparative Example 8 In step S3, the procedure of injecting hPBMCs via the tail vein is not performed; the remaining steps are exactly the same as in Example 1.

[0052] Comparative Example 9 In step S3, instead of performing subcapsular renal scaffold transplantation, the scaffold for which the intended cells are to be implanted is digested and ground into a single-cell suspension, mixed with hPBMCs, and then injected into mice via the tail vein. The total number of cells is the same as in Example 1. The remaining steps are exactly the same as in Example 1.

[0053] Comparative Example 10 Eight-week-old female NSG mice were irradiated with 2.0 Gy γ-rays and then injected via the tail vein with 1 × 10⁻⁶ gamma rays. 7Humanized osteoporosis model was obtained by collecting human bone marrow mononuclear cells, followed by bilateral oophorectomy 4 weeks later, and continued feeding for 8 weeks after the operation.

[0054] Comparative Example 11 Without any irradiation pretreatment, stent transplantation, or human cell injection, the animals were treated with the same ovarian artery ligation combined with progressive tamoxifen administration as in Example 1, and fed for 8 weeks; all other test indicators were exactly the same as in Example 1.

[0055] Performance testing The results of the detection of human cell colonization in each group of mice are shown in the table below.

[0056] Table 1. Results of human cell colonization in mice in each group (n=10, x±s) Example 1 87.62±3.15 85.38±2.94 22.47±2.16 Example 2 85.19±2.87 82.64±3.02 21.85±2.08 Example 3 86.34±3.01 83.71±2.88 22.03±2.11 Comparative Example 1 - - - Comparative Example 2 72.54±4.26 - 21.68±2.23 Comparative Example 3 - 61.27±5.13 21.74±2.19 Comparative Example 4 32.15±4.87 28.63±4.52 21.59±2.07 Comparative Example 5 86.87±3.22 84.92±3.05 22.16±2.12 Comparative Example 6 85.93±3.08 42.36±5.07 21.97±2.15 Comparative Example 7 78.42±3.95 76.58±3.76 22.05±2.09 Comparative Example 8 87.15±3.11 84.86±2.97 1.24±0.35 Comparative Example 9 8.76±2.13 7.54±1.98 18.62±2.31 Comparative Example 10 9.28±2.25 8.13±2.04 16.75±2.42 Comparative Example 11 - - - Table 2. Results of serum bone metabolism marker detection in mice of each group (n=10, x±s) Humanized control of sham surgery 68.74±5.26 22.35±2.17 3.26±0.35 Example 1 42.38±3.57 12.46±1.23 7.85±0.64 Example 2 44.15±3.62 13.12±1.28 7.52±0.61 Example 3 43.72±3.59 12.87±1.25 7.64±0.62 Comparative Example 1 (Mouse Source) 35.26±4.18* - 8.12±0.72* Comparative Example 2 65.27±5.13 20.84±2.09 3.54±0.38 Comparative Example 3 - - 4.12±0.42 Comparative Example 4 58.63±4.85 18.72±1.95 4.37±0.45 Comparative Example 5 51.24±4.02 15.63±1.64 6.28±0.57 Comparative Example 6 62.35±4.96 19.25±1.98 3.86±0.41 Comparative Example 7 50.17±3.98 15.24±1.58 6.15±0.55 Comparative Example 8 45.26±3.68 13.58±1.32 7.24±0.60 Comparative Example 9 66.32±5.18 21.03±2.11 3.42±0.37 Comparative Example 10 64.87±5.12 20.67±2.08 3.61±0.39 Comparative Example 11 (Mouse Source) 38.72±4.25* - 7.63±0.68* Note: * indicates results of rodent-derived biomarkers, while the rest are results of human-derived biomarkers.

[0057] Table 3. Micro-CT results of tibial bone microstructure in each group of mice (n=10, x±s) Humanized control of sham surgery 0.128±0.008 28.64±2.15 5.32±0.42 58.74±3.26 187.52±12.35 Example 1 0.082±0.005 15.72±1.23 3.15±0.28 41.26±2.57 324.76±18.42 Example 2 0.085±0.005 16.35±1.28 3.24±0.29 42.15±2.61 315.84±17.86 Example 3 0.084±0.005 16.08±1.25 3.21±0.28 41.87±2.59 319.62±18.05 Comparative Example 1 0.078±0.006 14.26±1.35 2.98±0.31 39.54±2.72 342.15±19.63 Comparative Example 2 0.115±0.007 25.37±2.02 4.86±0.39 55.23±3.14 208.43±13.57 Comparative Example 3 0.121±0.007 26.84±2.08 5.04±0.40 56.72±3.18 196.27±13.02 Comparative Example 4 0.108±0.007 23.15±1.86 4.42±0.37 52.16±3.02 227.58±15.24 Comparative Example 5 0.094±0.006 19.62±1.54 3.78±0.32 45.83±2.75 276.35±16.72 Comparative Example 6 0.118±0.007 25.93±2.05 4.95±0.39 55.84±3.15 202.76±13.28 Comparative Example 7 0.096±0.006 20.17±1.58 3.85±0.33 46.27±2.78 271.42±16.54 Comparative Example 8 0.087±0.005 16.84±1.31 3.32±0.29 42.83±2.63 308.57±17.63 Comparative Example 9 0.124±0.008 27.86±2.12 5.24±0.41 57.92±3.24 191.34±12.68 Comparative Example 10 0.122±0.007 27.15±2.09 5.17±0.41 57.35±3.21 194.68±12.87 Comparative Example 11 0.086±0.006 16.53±1.32 3.28±0.30 42.54±2.62 312.46±17.75 Table 4. Results of TRAP staining of osteoclast count in the tibia of mice in each group (n=10, x±s) Humanized control of sham surgery 2.35±0.32 - Example 1 8.74±0.65 3.72 times Example 2 8.26±0.62 3.51 times Example 3 8.42±0.63 3.58 times Comparative Example 1 9.12±0.71 3.88 times Comparative Example 2 2.64±0.35 1.12 times Comparative Example 3 3.12±0.38 1.33 times Comparative Example 4 3.57±0.41 1.52 times Comparative Example 5 6.83±0.57 2.91 times Comparative Example 6 2.87±0.36 1.22 times Comparative Example 7 6.54±0.55 2.78 times Comparative Example 8 8.25±0.63 3.51 times Comparative Example 9 2.46±0.33 1.05 times Comparative Example 10 2.58±0.34 1.10 times Comparative Example 11 8.36±0.64 3.56 times Table 5. Results of drug responsiveness testing of alendronate sodium in each model group (n=6, x±s) Example 1 28.74±2.15 62.35±3.26 89.2 Example 2 27.62±2.08 60.87±3.18 87.5 Example 3 28.15±2.12 61.54±3.22 88.4 Comparative Example 1 42.53±3.24 78.62±4.15 42.6 Comparative Example 2 5.26±0.87 8.74±1.23 12.3 Comparative Example 3 4.87±0.82 10.25±1.35 15.7 Comparative Example 4 12.54±1.52 22.36±2.08 32.5 Comparative Example 5 35.27±2.86 70.54±3.78 58.4 Comparative Example 6 6.32±0.94 9.68±1.28 13.6 Comparative Example 7 22.43±1.87 52.17±3.02 75.8 Comparative Example 8 26.85±2.06 59.74±3.15 86.2 Comparative Example 9 4.52±0.78 7.85±1.15 10.2 Comparative Example 10 5.18±0.85 8.62±1.21 11.8 Comparative Example 11 39.64±3.12 75.38±4.02 48.7 Note: In clinical practice, postmenopausal osteoporosis patients treated with alendronate sodium for 12 months showed an average increase of approximately 26% in lumbar spine BMD and an average decrease of approximately 60% in serum CTX-I. These results were used as a benchmark for calculating the matching degree.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a humanized osteoporosis mouse model, characterized in that, Includes the following steps: S1. Pretreatment of experimental animals: SPF-grade 8-10 week old female NOD-SCID IL2rg- / - (NSG) immunodeficient mice were selected, and after adaptive feeding, they were given low-dose whole-body irradiation with γ rays to eliminate the mice's own immune cells and bone marrow hematopoietic stem cells. S2. Preparation of biomimetic bone scaffold and pre-implantation of cells: A collagen-nanohydroxyapatite composite biomimetic scaffold was prepared. Polylactic acid-glycolic acid copolymer (PLGA) sustained-release microspheres loaded with human M-CSF, RANKL, and TNF-α were premixed in the scaffold matrix. Human bone marrow mesenchymal stem cells (hBMSCs) and human osteoclast precursor cells were seeded on the scaffold at a ratio of (2-4):

1. The cells were co-cultured in vitro for 3-5 days to obtain a biomimetic scaffold loaded with bone metabolism factors and pre-implanted human cells. S3. Construction of a dual humanized system: The predetermined scaffold obtained in step S2 was transplanted under the renal capsule of the mice pretreated in step S1, and human peripheral blood mononuclear cells (hPBMCs) were injected via the tail vein to construct immune-bone metabolism dual humanized mice. S4. Progressive osteoporosis induction: One week after transplantation, ovarian artery ligation combined with progressive estrogen receptor antagonist sustained-release administration was used to achieve progressive estrogen deprivation in mice. At the same time, human bone metabolism-related cytokines were continuously released in the sustained-release microspheres loaded in the scaffold to induce the human bone metabolism system to form an osteoporosis phenotype. S5. Model Identification and Validation: After 8 weeks of induction, a stable humanized osteoporosis mouse model was obtained through human cell colonization rate detection, bone metabolism marker detection, bone microstructure detection, histological examination, and drug responsiveness verification.

2. The construction method according to claim 1, characterized in that, In step S1, the dose of whole-body γ-ray irradiation is 1.5-2.5 Gy, and cell and scaffold transplantation is performed within 24 hours after irradiation.

3. The construction method according to claim 1, characterized in that, In step S2, the preparation method of the collagen-nano hydroxyapatite composite biomimetic scaffold is as follows: Type I collagen and nano hydroxyapatite are mixed in a mass ratio of (3-5):1 in 0.5 mol / L acetic acid solution, stirred evenly, and then injected into a mold. After pre-freezing at -20℃ for 4 h and freeze-drying at -80℃ for 24 h, a porous scaffold is obtained. Then, it is cross-linked with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide for 24 h, washed 3 times with sterile PBS, and sterilized by cobalt-60 irradiation to obtain a collagen-nano hydroxyapatite composite biomimetic scaffold premixed with PLGA sustained-release microspheres loaded with human M-CSF, RANKL, and TNF-α, for later use.

4. The construction method according to claim 1, characterized in that, In step S2, the human osteoclast precursor cells are human CD14+ mononuclear cells isolated from peripheral blood mononuclear cells of healthy individuals; the human bone marrow mesenchymal stem cells are 3rd-5th generation cells with a positive rate of ≥95% for CD90+, CD105+, CD45-, and CD34- as determined by flow cytometry; the total cell density after seeding is 1×10⁻⁶ cells / year. 7 cells / mL scaffold volume.

5. The construction method according to claim 1, characterized in that, In step S3, the injection dose of human peripheral blood mononuclear cells is 1 × 10⁻⁶ per mouse. 7 -2×10 7 The injection of cells was completed on the same day as the scaffold transplantation procedure.

6. The construction method according to claim 1, characterized in that, In step S4, the specific method of progressive estrogen deprivation is as follows: bilateral ovarian artery ligation is performed simultaneously with transplantation to preserve ovarian tissue. Starting from the day after the surgery, tamoxifen sustained-release microspheres, an estrogen receptor antagonist, are implanted subcutaneously in the back. The sustained-release period is 8 weeks, and the dosage is 0.5 mg / kg·d in the first week, 1 mg / kg·d in the second to fourth weeks, and 2 mg / kg·d in the fifth to eighth weeks, so as to achieve progressive blockade of the effect of estrogen in the body.

7. The construction method according to claim 1, characterized in that, In step S4, the human bone metabolism-related cytokines are human M-CSF, RANKL, and TNF-α, all loaded in polylactic acid-glycolic acid copolymer sustained-release microspheres. The PLGA sustained-release microspheres loaded with human bone metabolism-related cytokines are premixed in the biomimetic scaffold during the biomimetic scaffold preparation stage in step S2 and are a component of the scaffold matrix. The sustained-release period of the sustained-release microspheres is 8 weeks, and the loading amounts of each cytokine in the scaffold are: M-CSF 500ng / scaffold, RANKL 1μg / scaffold, and TNF-α 200ng / scaffold.

8. The construction method according to claim 1, characterized in that, In step S5, the qualification criteria for model identification are as follows: the colonization rate of human CD90+ osteoblasts and human CD14+ osteoclasts in the subcapsular scaffold is ≥80%, and the proportion of human CD45+ immune cells in peripheral blood is ≥15%; compared with the same strain of sham-operated humanized control mice, the model mice have a bone mineral density (BMD) decrease of ≥30%, a bone volume fraction (BV / TV) decrease of ≥40%, a human bone resorption marker (CTX-I) level increase of ≥2 times, a human bone formation marker (PINP) level decrease of ≥30%, and a TRAP-stained positive osteoclast number increase of ≥2.5 times.

9. The application of a humanized osteoporosis mouse model prepared by the construction method according to any one of claims 1-8 in the humanization screening and efficacy evaluation of anti-osteoporosis drugs.