A double controlled release composite system of PTH (1-34) and preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and provides a double-control-release composite system of PTH (1-34), a preparation method and application, which comprises a hydrogel support and PTH (1-34) loaded microspheres loaded on the hydrogel support; the PTH (1-34) loaded microspheres comprise a microsphere matrix and parathyroid hormone loaded on the microsphere matrix, and the microsphere matrix is composed of a slow-release polymer. Advantages: (1) the application constructs a double-control-release composite support formed by the synergistic action of PLGA, HA and CS, and combines PTH (1-34) to form a precise control-release composite system. The degradation kinetics of the composite support meets the demand of a bone regeneration time window, and the composite support has high physiological adaptability; (2) a repair system with a trinity function of "double-control-release composite support-microenvironment regulation-anatomical shape matching" is constructed, and the repair system has a remarkable curative effect on the repair of bone defects under pathological load.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a dual controlled-release composite system of PTH(1-34), its preparation method, and its application. Background Technology

[0002] Sufficient bone volume is not only fundamental for the initial stability of implants but also crucial for maintaining aesthetics and function. Currently, alveolar bone defects remain a key factor limiting implant success. Bone biomaterials can effectively induce initial cell adhesion, proliferation, differentiation, and subsequent tissue formation in both in vitro and in vivo environments. Osteoporosis has evolved into a multidimensional public health challenge. As a systemic bone metabolic disease, osteoporosis poses a challenge to dental implant treatment. Due to low bone cell density, limited migration of endogenous cells, and weak regenerative potential, alveolar bone regeneration in osteoporosis patients faces greater challenges, highlighting the necessity of targeted bone biomaterials.

[0003] Parathyroid hormone (PTH) is one of the few globally approved prescription bone-forming agents. As a core regulator of bone metabolism, this hormone promotes anabolism in bone remodeling through a three-pronged mechanism: inhibiting pre-osteoblast apoptosis, promoting their proliferation, and inducing differentiation of bone lining cells into active osteoblasts, thereby enhancing cell function and prolonging their lifespan. The PTH(1-34) fragment, by activating the PTH(1-34)R1 receptor, upregulates the Wnt / β-catenin pathway to promote osteoblast differentiation while inhibiting osteoblast apoptosis. Clinical studies have shown that intermittent low-dose PTH(1-34) administration significantly increases bone mineral density, superior to traditional anti-resorption drugs, while continuous high doses promote bone resorption. The short half-life of PTH(1-34) (approximately 5 minutes) and systemic administration-induced fluctuations in blood calcium levels limit its clinical application. Research indicates that the critical time window for PTH(1-34) administration is early intervention within one week after bone defect formation. Based on the timeline of bone healing, PTH treatment lasting 2-4 weeks achieves good therapeutic effects. Therefore, controlled release of PTH(1-34) is a key challenge in bone defect treatment, and existing PTH(1-34) therapies often fail to achieve adequate local release control. Thus, researching a product capable of sustained local delivery of PTH(1-34) during bone repair is of great significance. Summary of the Invention

[0004] The present invention aims to overcome at least one defect (deficiency) of the prior art and provide a dual controlled-release composite system for PTH(1-34), its preparation method and application, thereby achieving controlled release of PTH(1-34) drug through the composite system.

[0005] One object of the present invention is to provide a dual controlled-release complex system for PTH(1-34),

[0006] The invention includes a hydrogel scaffold and PTH(1-34) loaded microspheres on the hydrogel scaffold; the PTH(1-34) loaded microspheres include a microsphere matrix and parathyroid hormone loaded on the microsphere matrix, the microsphere matrix being composed of a sustained-release polymer.

[0007] Furthermore, the sustained-release polymer is a polylactic acid-glycolic acid copolymer, and the hydrogel scaffold is made of chitosan mixed with nano-hydroxyapatite.

[0008] In this technical solution, polylactic acid-glycolic acid copolymer (PLGA) is composed of lactic acid (LA) and glycolic acid (GA) linked by ester bonds. It has good biocompatibility and biodegradability and can transport proteins, bacterial or viral DNA and various drugs in the form of microspheres, nanoparticles and other forms.

[0009] Nano-hydroxyapatite (nHA), with its morphology and size highly similar to HA crystals in natural bone tissue, can precisely mimic the micro-nano structure of the bone matrix. This biomimetic design not only endows the 3D-printed scaffold with biocompatibility and osteoinductive activity comparable to natural bone tissue, but its unique geometric properties also ensure the uniform dispersion and suspension stability of nHA in the printing ink, significantly improving the controllability of the printing process and the uniformity of the scaffold structure.

[0010] Chitosan (CS) is a natural aminopolysaccharide with good biocompatibility and biodegradability, promoting the adhesion and proliferation of various tissue cells. The amino groups on the chitosan (CS) molecular chain stabilize the nHA dispersion system through electrostatic interactions, and its inherent flexibility effectively compensates for the brittleness of nHA; while the rigidity of nHA enhances the mechanical strength of the CS matrix. Through synergistic effects, these two components form an ideal composite material that combines hardness, compressive strength, and flexibility. This hydroxyapatite-chitosan (nHA-CS) composite system not only serves as an ideal bone regeneration scaffold due to its biomimetic properties but also becomes an excellent drug-controlled release carrier thanks to its porous structure and surface activity.

[0011] Furthermore, the nano-hydroxyapatite is in the shape of short rods with a diameter of 20–45 nm and a length of 50–170 nm.

[0012] Furthermore, the mass ratio of the nano-hydroxyapatite to chitosan is (6-8):(2-4), preferably 7:3.

[0013] Another object of the present invention is to provide a method for preparing any of the above-mentioned dual controlled-release composite systems, comprising the following steps:

[0014] S1. Preparation of PTH(1-34) loaded microspheres, including:

[0015] S11. Parathyroid hormone and polyvinyl alcohol are dissolved in water to form parathyroid hormone solution and polyvinyl alcohol solution, respectively; polylactic acid-glycolic acid copolymer is dissolved in dichloromethane to form polylactic acid-glycolic acid copolymer solution; parathyroid hormone solution is added to polylactic acid-glycolic acid copolymer solution under vortex conditions, and the mixture is formed to form colostrum;

[0016] S12. Add the colostrum to the polyvinyl alcohol solution, stir and mix to emulsify again, collect the precipitate, wash and freeze dry; obtain PTH(1-34) loaded microspheres.

[0017] S2. Preparation of chitosan ink includes: adding chitosan to an acid solution and mixing, adding a crosslinking modifier and mixing, then adding nano-hydroxyapatite powder and mixing, removing bubbles, and obtaining chitosan ink.

[0018] The preparation of the dual controlled-release complex system of S3 and PTH(1-34) includes:

[0019] S31. Disperse the PTH(1-34) microspheres obtained in step S1 into the chitosan ink obtained in step S2, mix, remove bubbles, and form a chitosan-based composite ink. After 3D printing the chitosan-based composite ink, freeze-dry it to obtain a dual controlled-release composite system of PTH(1-34).

[0020] In one or more embodiments of the present invention, PTH(1-34) is selected.

[0021] Further, in step S11, the vortex rotation speed of the polylactic acid-glycolic acid copolymer solution is 2500 rpm to 3500 rpm; the mixing is ultrasonic mixing, and the ultrasonication time is 60 to 90 s. Preferably, the vortex rotation speed is 3000 rpm, and the ultrasonication time is 60 s.

[0022] Furthermore, in step S12, the freeze-drying time is 12 to 18 hours.

[0023] Further, in step S2, the acid solution is a mixed solution of acetic acid, lactic acid, and pure water, wherein the volume ratio of acetic acid, pure water, and lactic acid is (1.5–2.5):(1.5–2.5):(0.5–2.5), preferably, the volume ratio is 2:2:1.

[0024] Further, in step S3, preferably, the air pressure for 3D printing is 80-100 kPa and the printing speed is 60-360 mm / s; more preferably, the air pressure for 3D printing is 91-110 kPa and the printing speed is 150-210 mm / s.

[0025] Another object of the present invention is to provide the application of the dual controlled-release composite system of any of the above-mentioned methods or the dual controlled-release composite system prepared by any of the above-mentioned methods in the preparation of products for repairing osteoporosis or bone defects.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) This invention integrates PLGA microspheres loaded with PTH(1-34) into an nHA-CS hydrogel system, achieving time-controlled drug release through a microsphere-gel biphasic drug release system. The controlled release depends on the synergistic effect of PLGA, HA, and CS. Lactic acid / glycolic acid generated from PLGA degradation modifies the CS molecular chain through hydrophobic association, reducing its hydrophilicity; nHA, through Ca... 2+ -PO4 3- Ion exchange buffers buffer the local acidic microenvironment; nHA nanocrystals significantly increase cross-linking density through interfacial hydrogen bonding and microspheres as physical cross-linking points, significantly delaying the de-entanglement rate of polymer chain segments. All three factors work together to slow down the hydrolysis process of polymer chains and increase degradation stability, making the composite scaffold formed by PLGA, HA and CS more suitable for complex osteogenic environments.

[0028] PLGA, HA, and CS work synergistically to form a dual-controlled-release composite scaffold, which, combined with PTH (1-34), forms a precisely controlled-release composite system. The degradation kinetics of this composite scaffold meet the bone regeneration time window requirements, manifested as follows: in the initial stage (0-7 days), CS rapidly dissolves and releases PTH (1-34), activating osteoblast differentiation; in the middle stage (7-28 days), nHA gradually dissolves to provide calcium. 2+ / PO4 3- Promotes mineralization; in the later stage (>28 days), PLGA microspheres continuously release the drug to maintain a local effective concentration. This three-stage release pattern is highly consistent with the inflammatory, repair, and remodeling phases of bone healing, and is more physiologically adaptable than a single sustained-release system.

[0029] The degradation kinetics of the composite scaffold are highly matched with the critical window period for bone repair (3-6 weeks), avoiding the mechanical support failure problem caused by excessively rapid degradation of traditional materials. This biomimetic degradation design successfully solves the "degradation-regeneration mismatch" problem of traditional materials, making the scaffold mass loss rate significantly positively correlated with the rate of new bone deposition, providing a dynamically adapted microenvironment for personalized bone repair.

[0030] (2) The present invention constructs a three-in-one repair system of “dual controlled release composite scaffold - microenvironment regulation - anatomical morphology matching”, which has a significant therapeutic effect on the repair of bone defects under pathological load repair. Attached Figure Description

[0031] Figure 1 Display: Physicochemical properties of PTH(1-34) loaded microspheres; (A) Microspheres under SEM; (B) Particle size distribution of microspheres; (C) PTH(1-34) release curves of PTH(1-34) loaded microspheres and PTH(1-34) loaded microsphere-chitosan hydrogel.

[0032] Figure 2 Display: Selection of 3D printing parameters for composite scaffolds; (A) 4X microscopic observation of linewidth under different pressures and printing speeds of PTH(1-34) microspheres-chitosan hydrogel; (B) Printing speed-linewidth graph of PTH(1-34) microspheres-chitosan hydrogel under different pressures; (C) Rheological viscosity test graph of chitosan hydrogel with different hydroxyapatite ratios.

[0033] Figure 3 Display: Construction and physicochemical property testing of PTH(1-34) microsphere-chitosan hydrogel; (A) Personalized 3D printed “CS-nHA” shaped hydrogel; (B) Electron micrograph of PTH(1-34) microsphere-chitosan hydrogel: red arrow: PTH(1-34) microsphere; blue arrow: short rod-shaped nHA; (C) Rheological test diagrams of CS hydrogel and CS-nHA hydrogel; (D) Stress-strain diagrams of CS hydrogel and CS-nHA hydrogel under tensile test; (E) Degradation rate of CS hydrogel, CS-nHA hydrogel, and S-CS-nHA hydrogel.

[0034] Figure 4 Display: Cell viability detection; (A) Live / dead staining detection of MC3T3 co-culture cells (4X microscopy); (B) Alizarin Red staining (4X microscopy); (C) CCK8 detection results; (D) ALP content detection; (E) Alizarin Red semi-quantitative analysis diagram; (F) Osteoblast gene expression levels on days 3 and 7; (G) Western blot of osteoblast protein on days 3 and 7; (H) Osteoblast protein expression levels on days 3 and 7.

[0035] Figure 5 Display: (A) CT images of the sham surgery group and the experimental group; (B) Bone mineral density analysis images of the sham surgery group and the experimental group; (C) BV / TV analysis images of the sham surgery group and the experimental group; (D) Trabecular bone thickness analysis images of the sham surgery group and the experimental group.

[0036] Figure 6 Display: In vivo osteogenic capacity detection; (A) Three-dimensional reconstruction screenshots of the skulls of three groups of rats; (B) BV analysis of the new bone formation area of ​​three groups of rats; (C) TV analysis of the new bone formation area of ​​three groups of rats; (D) BV / TV analysis of the new bone formation area of ​​three groups of rats; (E) Tb.N of the new bone formation area of ​​three groups of rats; (F) Tb.Th of the new bone formation area of ​​three groups of rats; (G) Tb.Sp of the new bone formation area of ​​three groups of rats.

[0037] Figure 7 Display: (A) HE-stained sections of rats in three groups (4×, 10× magnification); (B) Masson-stained sections of rats in three groups (4×, 10× magnification). Detailed Implementation

[0038] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the invention. To better illustrate the following embodiments, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions; it is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0039] Example 1

[0040] This embodiment provides a dual controlled-release composite system for PTH(1-34), including a hydrogel scaffold and PTH(1-34) loaded microspheres on the hydrogel scaffold; the PTH(1-34) loaded microspheres include a microsphere matrix and parathyroid hormone loaded on the microsphere matrix, the microsphere matrix being composed of sustained-release polymers.

[0041] The sustained-release polymer is a polylactic acid-glycolic acid copolymer, and the hydrogel scaffold is made of chitosan mixed with nano-hydroxyapatite.

[0042] The nano-hydroxyapatite is in the form of short rods, with a diameter of 20–45 nm and a length of 50–170 nm. The mass ratio of chitosan to nano-hydroxyapatite is 3:7.

[0043] Example 2

[0044] This embodiment provides a method for preparing the dual controlled-release composite system of Example 1, the method comprising:

[0045] S1. Preparation of PTH(1-34) loaded microspheres (using water-in-oil-in-water (w / o / w) double emulsification technology):

[0046] PTH(1-34) (Maclean's, Shanghai, China) and polyvinyl alcohol (PVA) were dissolved in deionized water (DI), and PLGA was dissolved in dichloromethane (DCM). Then, the PTH(1-34) solution was added dropwise to the PLGA solution at 3000 rpm using a magnetic stirrer (Zhengzhou Biochemical), and the mixture was sonicated for 60 seconds to form a colostrum.

[0047] The colostrum was poured into the PVA solution and stirred with a magnetic stirrer for 3 minutes to re-emulsify. Stirring was continued overnight (12 hours) at room temperature to volatilize the dichloromethane. Then, the mixture was stirred at 10,000 rpm for 4 minutes using a star centrifuge to collect the precipitate. The precipitate was collected, washed with DI, centrifuged, and repeated 4 times. After freeze-drying for 15 hours, PTH(1-34) microspheres were obtained.

[0048] S2, Preparation of chitosan ink

[0049] An appropriate amount of chitosan powder (Maclean, Shanghai, China, 6 wt%, based on the total mass of the mixed acid solution) was added to the mixed acid solution (acetic acid / pure water / lactic acid volume ratio 2:2:1) and magnetically stirred until completely dissolved to form a homogeneous chitosan sol. Citric acid (3 wt%, based on the total mass of the chitosan sol) was introduced into the chitosan sol as a crosslinking modifier, and the mixture was stirred overnight (12 h) to achieve sufficient complexation.

[0050] The short rod-shaped nano-hydroxyapatite (nHA) and chitosan were mixed in a mass ratio of 7:3. The short rod-shaped nano-hydroxyapatite (Xianfeng Nano, Jiangsu, China, diameter 20-45nm, length 50-170nm) was mixed with chitosan sol and then uniformly dispersed by high-speed shear emulsification (10,000rpm, 30min) using a star centrifuge (Thinky, Japan) to obtain the ink system.

[0051] Transfer the ink system to a planetary centrifugal mixer for two-stage processing:

[0052] ① Mixing stage: Rotation / revolution at 2200 rpm for 10 minutes to ensure system homogeneity;

[0053] ② Degassing stage: Vacuum centrifugation at 2000 rpm for 30 seconds to degas and eliminate internal microbubbles.

[0054] The chitosan ink was finally obtained.

[0055] S3, 3D printing forms a dual controlled-release composite system of PTH(1-34)

[0056] The PTH(1-34) microspheres obtained in step S1 were uniformly dispersed in the chitosan ink obtained in step S2 at a mass fraction of 10 wt% (based on the weight of the chitosan ink). A two-stage process was performed using a planetary centrifugal mixer: mixing at 2200 rpm for 10 min by rotation / revolution; followed by vacuum degassing at 2000 rpm for 30 s to form the chitosan-based composite ink.

[0057] Chitosan-based composite ink was deposited layer by layer according to a preset geometric model (40 mm long, 10 mm wide, 100 μm thick) using a bio-3D printer (envisionTEC 3D-Bioplotter). The printing pressure was 100 kPa, and the printing speed was 180 mm / s. The ink was then freeze-dried at -85°C (CHRIST freeze dryer) for 12 h. After freeze-drying, the ink was immersed in 0.5 mol / L NaOH solution (room temperature, 2 h) to neutralize residual acid groups, washed three times with ultrapure water, purged with nitrogen, and then stored in a desiccator.

[0058] Comparative Example 1

[0059] This comparative example provides a blank composite sustained-release system. The main difference between its preparation method and that of Example 2 is that step S1 is the preparation of blank microspheres, and in step S3, the PTH(1-34) microspheres are replaced with blank microspheres.

[0060] Test Example 1

[0061] Chitosan-based composite inks were prepared by setting the weight ratio of nano-hydroxyapatite to chitosan to 3:7, 4:6, 5:5, and 6:4, respectively. The chitosan-based composite inks and the chitosan-based composite ink provided in Example 2 were tested with a rheometer, and it was found that the viscosity of Example 2 was the best.

[0062] Test Example 2

[0063] The chitosan-based composite ink was transferred to a printing syringe. A 300μm needle was attached to the printing syringe. After the printing syringe was installed, nitrogen gas was turned on, and printing was performed at printing pressures of 80kPa, 90kPa, and 100kPa at speeds of 60mm / min, 120mm / s, 180mm / s, 240mm / s, 300mm / s, and 360mm / s, respectively. The width of the printing marks under the above four settings was observed under a 4× microscope.

[0064] The chitosan-based composite ink mixed with blank microspheres was used to print a special pattern CS-HA using a 3D printer. The pattern was observed at room temperature to explore whether the composite ink could adapt to bone defects with complex shapes.

[0065] Test Example 3 Characterization Test

[0066] The microstructure of the dual controlled-release composite system containing PTH(1-34) microspheres and PTH(1-34) was observed using scanning electron microscopy (SEM).

[0067] Rheological properties: The dynamic viscoelasticity of pure chitosan hydrogel and chitosan-based composite ink (nHA: chitosan = 7:3) was measured using a rheometer (Anton Paar MCR302, Austria) based on dynamic oscillation mode: frequency scan 1.0 Hz, temperature 37°C (simulating physiological environment), angular velocity 5 rad / s, storage modulus (G') and loss modulus (G”) were obtained.

[0068] Mechanical property testing: A long strip specimen (40mm×10mm) was prepared and a tensile test was performed using a universal testing machine (INSTRON E3000, UK) with a loading rate set to 10mm / min-1.

[0069] In vitro degradation analysis: Samples were immersed in PBS (pH 7.4) at 37°C until swelling equilibrium was reached. The initial mass (W0) was recorded. Samples were retrieved from the dynamic degradation system (fresh PBS replaced every 48 hours) at days 7, 14, 21, and 28, and after lyophilization, the residual mass (Wt) was measured. Degradation rate calculation formula:

[0070] Degradation rate (%) = (W0 - Wt) / W0 × 100%

[0071] Test Example 4: Drug Loading Rate and Drug Release Curve

[0072] Drug loading rate determination of PTH(1-34) microspheres: 10 mg of PTH(1-34)-loaded microspheres were accurately weighed and suspended in a mixed solvent system consisting of 1 ml of 0.9% NaCl solution and 200 μl of dichloromethane. After shaking the sample at room temperature for 1 h, the drug concentration in the supernatant was detected using a PTH(1-34) ELISA kit. The drug loading rate was calculated using the following formula: Drug loading rate (%) = (Mass of drug in microspheres / Initial drug loading mass) × 100%

[0073] Drug release curves of the PTH(1-34) microsphere-loaded system: 10 mg of drug-loaded microspheres were weighed and dispersed in 3 ml of PBS (pH 7.4), and shaken in a 37°C water bath. Samples were taken at preset time points (days 1, 2, 3, 4, 7, 10, 15, 20, 25, and 30), and the supernatant was collected by centrifugation at 1000 r / min for 5 min at 4°C. An equal volume of fresh PBS was added simultaneously to maintain system stability. The cumulative release curves were plotted after detecting the PTH(1-34) content in the samples at each time point using ELISA.

[0074] Drug release curves of the dual controlled-release composite system of PTH(1-34): A sample of the dual controlled-release composite system was immersed in 3 mL of PBS, and release experiments were conducted under the same conditions (37℃, 1000 r / min). Samples were taken at the same time sequence, and an equal volume of fresh PBS was added promptly after each collection of supernatant. The content of PTH(1-34) was determined by ELISA, and a time-cumulative release curve was plotted simultaneously.

[0075] All experiments were conducted with three parallel samples, and the data are expressed as mean ± standard deviation.

[0076] Furthermore, the PTH(1-34) microspheres described above were prepared in step 1 of the method of Example 2, and the dual controlled-release composite system of PTH(1-34) described above was provided in Example 1.

[0077] Test Example 5: Culture and Experimental Grouping of MC3T3-E1 Cells

[0078] MC3T3-E1 cells were cultured under standard conditions (37°C, 5% CO2, and 95% relative humidity) in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The medium was replaced with fresh medium every 48 hours. Cells were passaged by trypsin digestion when cell confluence reached 90%. Cells in the logarithmic growth phase (passages 3-5) were used for subsequent experiments.

[0079] The experiment was divided into a negative control group (Blank), a blank microsphere-hydrogel group (PLGA / CS-nHA), and a drug-loaded microsphere-hydrogel group (PTH(1-34)@PLGA / CS-nHA). The control group cells were routinely cultured in complete culture medium. The other two groups were cultured by immersing the materials in the culture medium and placing them in an incubator (37℃, 5% CO2 and 95% relative humidity). The cells were centrifuged at 1000 r / min for 5 min at corresponding days (1d, 3d, 5d, 7d). The supernatant was extracted and filtered through a 0.22 μm filter to obtain the corresponding extracts, which were used for cell culture.

[0080] Furthermore, the above-mentioned blank microsphere-hydrogel assembly (PLGA / CS-nHA) uses the blank composite sustained-release system provided in Comparative Example 1, and the above-mentioned drug-loaded microsphere-hydrogel assembly (PTH(1-34)@PLGA / CS-nHA) uses the dual controlled-release composite system of PTH(1-34) provided in Example 1.

[0081] Test Example 6: Cell Proliferation and Viability Assessment

[0082] 1) CCK-8 detection

[0083] Cells were loaded at 3 × 10 3Cells were seeded in 96-well plates. After 1, 3, 5, and 7 days of culture, basal medium containing 10% CCK-8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the relative cell proliferation rate was calculated.

[0084] 2) Double staining of live / dead cells

[0085] Cells were seeded as before, and treated with a calcein AM (2 μM) / propidium iodide (PI, 4 μM) double staining kit at days 1, 4, and 7: after washing with PBS, staining working solution was added, and the cells were incubated at 37°C in the dark for 30 min. Observation was performed under a fluorescence microscope (live cells showed green fluorescence, dead cells showed red fluorescence).

[0086] Test Example 7: In vitro osteogenic differentiation capacity determination

[0087] Experiments were conducted using αMEM osteogenic medium containing 10 mM β-glycerophosphate, 0.25 mM ascorbic acid, and 10 nM dexamethasone. The promoting effect of the drug-loaded microsphere-hydrogel system on osteogenic differentiation of MC3T3 cells was systematically evaluated by alkaline phosphatase (ALP) activity analysis, mineralized nodule staining, and osteogenic marker detection. The drug-loaded microsphere-hydrogel system in this test case is the dual controlled-release composite system of PTH(1-34) provided in Example 1.

[0088] 1) Alkaline phosphatase detection

[0089] MC3T3 cells (1×10⁻⁶) were seeded in 24-well plates. 5 Cells / well were collected, and after 24 hours, group interventions were performed: the control group received osteogenic medium, while the experimental groups received PLGA / CS-nHA and PTH(1-34)@PLGA / CS-nHA extracts (preparation methods are described in Test Example 5), respectively. The culture medium was changed every 48 hours. At 4, 7, and 14 days after intervention, quantitative analysis was performed using cell lysis extracts and an ALP detection kit, with absorbance measured at 405 nm using a microplate reader.

[0090] Furthermore, the above-mentioned PLGA / CS-nHA is the blank composite sustained-release system provided in Comparative Example 1, and the above-mentioned PTH(1-34)@PLGA / CS-nHA is the dual controlled-release composite system of PTH(1-34) provided in Example 1.

[0091] 2) Mineralization capacity assessment

[0092] A 6-well plate system (1×10) was adopted. 5Cells / well), intervention protocol as described in section 1). After 21 days of continuous culture, after washing with PBS, Alizarin Red S staining (0.2%, pH 4.2, room temperature for 30 minutes). After observing the formation of calcium nodules under an optical microscope, the staining complex was dissolved in 10% hexadecylpyridine chloride solution (1 mL / well). 150 μL of the solution was transferred to a 96-well plate, and absorbance was detected at 562 nm to establish a semi-quantitative analysis model for mineralization deposition.

[0093] 3) Osteogenesis-related gene testing

[0094] RT-qPCR detection system: 24-well plates were seeded with cells (1×10⁻⁶ cells / well). 4 Cells / wells were used, and the intervention protocol was the same as described in section 1). Total RNA was extracted using the TRIzol method on days 3 and 7 after intervention, and cDNA was obtained by reverse transcription. GAPDH was used as an internal control, and the expression of marker genes such as Alp, Runx2, Col1, and Ocn was detected using the SYBR Green system.

[0095] 4) Osteogenesis-related protein detection

[0096] Western blotting analysis: Cell processing was as described in section 3). After collecting total protein, SDS-PAGE electrophoresis (12% separating gel) was performed. After transfer, the membrane was blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies RUNX2 (1:1000), ALP (1:800), and COL1 (1:1500), respectively. After incubation with HRP-labeled secondary antibody (1:5000) at room temperature for 1 hour, protein expression was detected using an ECL imaging system.

[0097] Test Example 8: Animal Experiment

[0098] All animal experiments were approved by the Animal Conservation and Use Committee of Sun Yat-sen University (Approval No.: SYSU-IACUC-2024-000724) and conducted in strict accordance with the "Guidelines for Ethical Review of Laboratory Animal Welfare". A skull defect model in osteoporotic rats was used to evaluate the function of the drug-loaded microsphere-hydrogel. Ten-week-old female SD rats (n=15) were randomly divided into three groups (n=5 / group): blank control group (sham surgery), PLGA / CS-nHA group, and PTH(1-34)@PLGA / CS-nHA group. The PLGA / CS-nHA group used the blank composite sustained-release system provided in Comparative Example 1; the PTH(1-34)@PLGA / CS-nHA group used the dual controlled-release composite system of PTH(1-34) provided in Example 1.

[0099] 1) Surgical modeling process

[0100] Ovarian removal surgery (OVX): After laparotomy via a dorsal approach, both ovaries are located, the blood vessels 0.5-1cm proximal to the uterine horn are ligated, and the ovaries are removed.

[0101] Sham surgery comparison: The same incision was made to expose the ovary but the organ was preserved intact.

[0102] Model validation: Twelve weeks after surgery, some rats in the OVX group and sham-operated group were randomly sacrificed, and the femurs were taken for micro-CT scanning. The osteoporosis model was successfully confirmed by quantitative analysis of trabecular volume fraction (BV / TV) and bone mineral density (BMD).

[0103] 2) Skull defect model and intervention

[0104] Surgical Procedure: After anesthesia, the soft tissue of the skull vertex was incised to expose the parietal bone. A 5mm circular bone drill was used to symmetrically prepare a full-thickness bone defect at the junction of the sagittal and lambdoid sutures. The Blank group (no material was placed) and the experimental groups (PLGA / CS-nHA and PTH(1-34)@PLGA / CS-nHA composite systems, respectively) were filled, and the soft tissue was sutured in layers. Four weeks postoperatively, skull specimens were taken and fixed with 4% paraformaldehyde for 24 hours. The defect area was then reconstructed three-dimensionally using micro-CT (scanning resolution 10μm) to quantify the new bone volume (BV), relative new bone volume (BV / TV), number of trabeculae (Tb.N), trabecular thickness (Tb.TN), and trabecular separation (Tb.Sp). Paraffin sections were prepared 30 days after EDTA decalcification and stained with H&E and Masson trichrome.

[0105] All experiments were performed with a biological replicate count (n ≥ 3) to meet statistical power requirements. Normally distributed data are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using GraphPad Prism 9.4.1 (GraphPad Software). Levene's test was first performed to confirm homogeneity of variance, and Tukey's multiple comparisons method was used to determine statistical significance. A p-value < 0.05 was considered statistically significant.

[0106] The following is an analysis of the results of the above experiments.

[0107] I. Synthesis, printing and characterization of a dual controlled-release complex system of PTH(1-34) (PTH(1-34)@PLGA / CS-nHA)

[0108] This study prepared PTH(1-34)-loaded microspheres using a water-in-oil-in-water (w / o / w) double emulsification technique. The resulting microspheres were a white powder and stored at -20°C to maintain the drug's bioactivity. Field emission scanning electron microscopy (FE-SEM) characterization showed... Figure 1A) The microspheres have smooth surfaces, regular morphology, and a monodisperse spherical structure with uniform particle size distribution. The average particle size is 79.03 ± 8.19 μm. Figure 1 B) The microspheres exhibit good monodispersity and no significant aggregation. Quantitative analysis using enzyme-linked immunosorbent assay (ELISA) showed that the drug loading rate of the microspheres reached 68.2%, demonstrating high drug encapsulation capability.

[0109] In vitro sustained-release experiments showed that ( Figure 1 C): The burst release of the microsphere group (PTH(1-34)@PLGA) was 30.8±1.3% in the initial 24 hours, while the burst release of the PTH(1-34)@PLGA / CS-nHA group was significantly reduced to 19.4±2.1% (p<0.01), a decrease of 11.4% compared to the burst release of the PTH(1-34)@PLGA system. Both systems showed significant sustained-release characteristics, maintaining a stable linear release trend over 30 days. By the end of the 30-day experiment, the cumulative release of the PTH(1-34)@PLGA group reached 75.2±1.5%, while that of the PTH(1-34)@PLGA / CS-nHA group was 71.8±1.7%. The release curve then entered a plateau phase. The improved sustained-release performance is attributed to the physical restriction of drug diffusion by the hydrogel network. This is highly consistent with the critical time window for PTH(1-34) treatment proposed by Long et al. (initiating early intervention within 7 days after bone defect, and controlling the duration of treatment to 2-4 weeks). This system maintains an effective concentration of PTH(1-34) within 30 days, which can provide continuous stimulation for early bone repair.

[0110] The aforementioned article by Long et al. regarding the critical time window for PTH treatment is: Long, J. et al.

[0111] Dual-Cross-Linked Magnetic Hydrogel with Programmed Release of Parathyroid Hormone Promotes Bone Healing. ACS Appl. Mater. Interfaces 15, 35815-35831 (2023).

[0112] The PTH(1-34)@PLGA / CS-nHA composite scaffold constructed in this study exhibits unique dual controlled-release properties stemming from a dual barrier effect: the nHA network delays CS chain segment hydrolysis through ion exchange, while the acidic microenvironment generated by the degradation of PLGA microspheres triggers CS amino protonation, forming dynamic cross-linking sites to inhibit swelling. This pH-responsive degradation behavior intelligently matches the local inflammatory microenvironment of bone defects (early pH decrease, later pH increase), ensuring that drug release is synchronized with the tissue regeneration process. This dual controlled-release mechanism not only effectively reduces the clinical risk of fluctuations in blood calcium concentration but also enables continuous local delivery of PTH(1-34) during bone repair, providing a stable osteogenic stimulating microenvironment for bone repair.

[0113] This study revealed the dynamic mechanical response characteristics and enhancement mechanism of the CS / nHA composite system through multi-scale rheological characterization. When CS was combined with short rod-shaped nano-hydroxyapatite (nHA, diameter 20-45 nm, length 50-170 nm) at a mass ratio of 3:7, rheological tests showed that its storage modulus (G') was increased by an order of magnitude compared with the pure CS system (from 10). 2 -10 3 Pa increased to 10 3 -10Pa), the loss modulus (G”) shows a synchronous increasing trend ( Figure 3 C). This significant viscoelastic transition confirms the formation of a multi-level physical cross-linked network between the rod-shaped nHA crystals and the CS molecular chains: the high aspect ratio of nHA not only enhances the network topology through steric hindrance, but its surface hydroxyl groups also form hydrogen bonds with the amino groups of CS, jointly constructing a dynamic network system with energy dissipation capabilities. After entering the plateau phase, the storage modulus of the CS-HA hydrogel exceeds 10 kPa, indicating that it possesses moderate mechanical strength. This moderate mechanical strength can maintain the structural integrity of the bone scaffold while avoiding the cell stress shielding effect caused by excessive rigidity, thus meeting the application requirements of bone regeneration scaffolds. After uniformly dispersing PTH(1-34) microspheres in the CS-nHA matrix, the final printing ink was obtained. The scaffold obtained after freeze-drying for 12 hours showed a volume shrinkage rate of <2%, indicating good stability.

[0114] Shear rate scanning experiments further confirmed that the composite ink possesses excellent shear response characteristics. Figure 2 C): At low shear rates (0.1 s⁻¹), the apparent viscosity is as high as 4.45 × 10 mPa·s, which endows the material with solid-like behavior to prevent structural collapse; while at high shear rates (100 s⁻¹), the viscosity drops rapidly to 1.12 × 10 mPa·s, showing a significant shear thinning effect (a reduction of up to 97.5%), which provides the necessary conditions for continuous ink extrusion.

[0115] Based on system optimization using orthogonal experimental design, the optimal parameter combination of air pressure (100 kPa) and nozzle moving speed (180 mm / min) was established. This condition successfully achieved spatiotemporal matching between extrusion kinetics and deposition kinetics: the filament linewidth remained stable at 675.14 ± 11.3 μm (CV < 5%, n = 3), ensuring excellent shape fidelity. Figure 2 A, B). Microstructural analysis shows that nHA exhibits a three-dimensional interpenetrating arrangement in the CS matrix. Figure 3 (B) This biomimetic mineralization structure provides an ideal topological microenvironment for cell adhesion. Through multi-dimensional correlation analysis of rheology, process, and structure, the study resolved the inherent contradiction between "extrudability and shape retention" in hydrogel inks. The established PTH(1-34)@PLGA / CS-nHA composite system not only meets the mechanical requirements of bone tissue engineering scaffolds, but its micron-level printing precision and submicron-level topological features also create a biomimetic microenvironment for directed cell migration and bone tissue regeneration. These findings provide a reliable design paradigm for developing intelligent bone repair materials with bioactive delivery capabilities.

[0116] Clinically, because patients' bone defects are often irregular in shape, bone augmentation scaffolds need to be custom-made. Therefore, this study custom-made CS-nHA hydrogels into a special shape called "CS-nHA". Figure 3 A) This fully demonstrates that 3D printed scaffolds can accurately adapt to complex defect contours and can well meet the needs of clinical applications. Its excellent plasticity stems from the shear-thinning properties of CS-nHA ink. Figure 2 C). SEM observations showed ( Figure 3 B): The composite scaffold surface exhibits significant topological heterogeneity. Drug-loaded microspheres (79.03±8.19 μm in diameter) and short rod-shaped nHA (20-45 nm in diameter and 50-170 nm in length) are discretely and uniformly distributed in the CS matrix. No phase separation was observed. The discretely distributed nHA / microsphere composite structure on the scaffold surface simulates the multiphase characteristics of natural bone tissue (Havers system and lacunae). This spatial distribution pattern confirms that the system has a highly homogeneous, phase-separation-free structural characteristic. This topological feature may promote drug release and cell-scaffold interaction by increasing the specific surface area.26

[0117] This study successfully constructed a functionalized composite system by uniformly dispersing PTH(1-34)-loaded microspheres (68.2% drug loading) in a chitosan-short rod-shaped nano-hydroxyapatite (CS-nHA, mass ratio 3:7)-based bio-ink. The short rod-shaped nHA (50-170 nm long, 20-45 nm in diameter) formed a three-dimensional physical cross-linked network through hydrogen bonding between surface hydroxyl groups (-OH) and chitosan amino groups (-NH), as well as electrostatic interactions. This network significantly improved ink stability, with a microsphere sedimentation rate of <5% after 24 hours. Real-time monitoring of the linewidth deviation rate after printing a pre-designed scaffold showed <3% (set value vs. measured value). The volume shrinkage rate of the scaffold after freeze-drying was not significant, showing no significant difference compared to the freshly printed state, indicating that the drying process did not disrupt the microsphere-matrix interface bonding.

[0118] The CS-HA composite material constructed by introducing short rod-shaped nHA (50-170 nm in length and 20-45 nm in diameter) showed an increase in ultimate tensile stress of 96.8% (18.5±3.2→36.4±3.5 kPa) and tensile strain of 41.8% (25.1→35.6%). Figure 3 (D) This mechanical enhancement effect is attributed to the uniform dispersion of short rod-shaped nHA and the strong interfacial hydrogen bond network formed between it and the chitosan molecular chains. The high aspect ratio of the short rod-shaped nHA endows it with excellent stress transfer efficiency. Compared with spherical nHA, the rod-shaped crystals improve the mechanical properties of the composite material due to their larger specific surface area and orientation characteristics.

[0119] In vitro degradation experiments showed that ( Figure 3 E) On day 7 of degradation, the mass of all three hydrogel groups decreased, with the pure CS group showing accelerated degradation subsequently. During the 28-day degradation period, the pure CS group exhibited an exponential degradation curve due to deacetylation (degradation rate 71.16 ± 0.45%), while the CS-nHA group degraded via the Ca²⁺ ionization of nHA. 2+ -PO4 3- The buffering effect (pH stabilized at 6.8±0.3) reduced the degradation rate to 50.12±0.65%. After introducing PLGA microspheres, the degradation rate of the PTH(1-34)@PLGA / CS-nHA group further decreased to 47.87±0.71%, and its linear degradation kinetics better matched the bone regeneration time window requirements. This is attributed to a triple synergistic effect: the lactic acid / glycolic acid produced by PLGA degradation modifies the CS molecular chain through hydrophobic association, reducing its hydrophilicity; nHA, through Ca... 2+ -PO4 3-Ion exchange buffers buffer the local acidic microenvironment; nHA nanocrystals significantly increase cross-linking density through interfacial hydrogen bonding and microspheres act as physical cross-linking points, significantly delaying the rate of polymer chain de-entanglement. These three factors collectively slow down the hydrolysis process of the polymer chains, increasing degradation stability and making the composite scaffold more suitable for complex osteogenic environments. The degradation kinetics of the composite scaffold are synergistically regulated by nHA content and PLGA microsphere distribution: in the initial stage (0-7 days), CS rapidly dissolves, releasing PTH (1-34), activating osteoblast differentiation; in the middle stage (7-28 days), nHA gradually dissolves, providing Ca... 2+ / PO4 3- It promotes mineralization; in the later stage (>28 days), PLGA microspheres continuously release drugs to maintain local effective concentrations. This three-stage release pattern highly coincides with the inflammatory, repair, and remodeling phases of bone healing, making it more physiologically compatible than single sustained-release systems. Notably, the degradation kinetics of the composite scaffold are highly matched with the critical window period for bone repair (3-6 weeks), avoiding the mechanical support failure problem caused by excessively rapid degradation of traditional materials. This biomimetic degradation design successfully solves the "degradation-regeneration mismatch" problem of traditional materials, making the scaffold mass loss rate significantly positively correlated with the rate of new bone deposition, providing a dynamically adapted microenvironment for personalized bone repair.

[0120] II. Cell compatibility of the dual controlled-release complex system of PTH(1-34) (PTH(1-34)@PLGA / CS-nHA)

[0121] Cell compatibility is a prerequisite for the in vivo application of biomaterials. To evaluate the biological properties of the materials, this study used a co-culture system of MC3T3-E1 pre-osteoblasts and scaffold material extracts, and systematically analyzed the results using a dual evaluation method of CCK-8 cell proliferation assay and live / dead cell staining. Quantitative analysis showed that (n=5), cell viability in all groups showed a significant increasing trend from day 1 to day 5, with the PTH(1-34)@PLGA / CS-nHA group exhibiting the strongest proliferative effect (P<0.05). Figure 4 C), this is partly due to the fact that the burst release of PTH(1-34) in this group was only 19.4±2.1%, and partly due to the sustained-release time of PTH(1-34) reaching more than 30 days. Figure 1 C). Live / dead staining results (n=3) further confirmed the biocompatibility of the material: all experimental groups maintained high cell viability at 1, 4, and 7 days, and the survival rate of the PTH(1-34)@PLGA / CS-nHA group was significantly higher than that of the other two groups at all time points (4A), indicating that the added PTH(1-34) can significantly promote cell proliferation. The above results fully demonstrate that the composite scaffold material has good biocompatibility, and the loaded PTH(1-34) can further enhance the cell proliferation-promoting effect of the material.

[0122] III. In vitro osteogenic differentiation capacity of the dual controlled-release composite system of PTH(1-34) (PTH(1-34)@PLGA / CS-nHA)

[0123] Ideal bone repair materials need to possess the ability to precisely regulate osteogenic differentiation. Alkaline phosphatase (ALP) is an early marker of osteogenic differentiation, while matrix mineralization is a key indicator of late-stage differentiation. Based on verifying the biocompatibility of the material, this study constructed a three-tiered evaluation system of "ALP activity-ECM mineralization-gene / protein expression" to systematically analyze the in vitro osteogenic properties of PTH(1-34)@PLGA / CS-nHA.

[0124] ALP staining showed (n=3): ALP activity in the PTH(1-34)@PLGA / CS-nHA group was significantly enhanced in a time-dependent manner, increasing by approximately 3.08 times compared to the control group by day 14 (P<0.01). Figure 4 D). Rapid activation of this early osteogenic marker suggests that the material may rapidly initiate the osteogenic process via the cAMP-PKA pathway. Alizarin Red staining at day 21 showed ( Figure 4 B), PTH(1-34)@PLGA / CS-nHA group formed dense mineralized nodules, and quantitative analysis showed that the mineralized area increased by 1.85 times compared with the control group (P<0.001). Figure 4 E) indicates that the material can significantly promote ECM calcium salt deposition.

[0125] RT-qPCR detection of key osteogenic markers (n=3) revealed significant upregulation of bone-forming genes in PTH(1-34)@PLGA / CS-nHA: the early marker Alp showed significant upregulation as early as day 3 (3.99-fold, P<0.05) and persisted until day 7 (5.15-fold, P<0.01); the expression level of the transcriptional regulator Runx2 increased over time, reaching 6.74-fold on day 7 (P<0.001); the matrix protein Co11 and the late marker Opn were upregulated by 3.41-fold and 5.48-fold, respectively, at day 7 (P<0.01). Figure 4 F). Western blot results further confirmed that the protein expression levels of RUNX2, ALP, and COl1 were highly consistent with gene expression (n=3, Figure 4 GH) forms a complete gene-protein expression regulatory network.

[0126] The superior performance of the PTH(1-34)@PLGA / CS-nHA group stems from a triple synergistic mechanism: a dual-stage controlled-release system: PLGA microspheres (68.2% drug loading) and CS hydrogel synergistically regulate the release kinetics of PTH(1-34), with an early burst release (19.4±2.1%) rapidly initiating osteoogenesis, followed by a sustained release (71.8±1.7%) in the later stages (2-30 days) to maintain efficacy, and intermittent activation of PTH(1-34) 1R to promote osteoogenesis; microenvironment regulation: the morphology and size of rod-shaped nHA are similar to HA crystals in natural bone tissue, better mimicking the structure and function of natural bone tissue, and short rod-shaped nHA (length-to-diameter ratio 3.8:1) continuously releases Ca2+. 2+ / PO4 3- It promotes osteogenic gene expression in synergy with CaSR receptor. nHAP and PTH(1-34) have a synergistic effect during treatment; topological mechanical signaling: CS matrix promotes osteoogenesis by regulating cytoskeleton tension and activating corresponding mechanotransduction pathways.

[0127] IV. In vivo osteogenic capacity of the dual controlled-release complex system of PTH(1-34) (PTH(1-34)@PLGA / CS-nHA)

[0128] To investigate the inhibitory effect of the osteoporotic microenvironment on bone regeneration and to verify the repair efficacy of PTH(1-34)@PLGA / CS-nHA, this study constructed an ovariectomy (OVX)-induced osteoporosis rat model combined with a critical-sized skull defect model (5 mm in diameter). Female SD rats (n = 25) were randomly divided into an OVX group (n = 20) and a sham-operated group (Sham, n = 5). Micro-CT three-dimensional reconstruction was performed 12 weeks post-operation. Figure 5 A) Quantitative analysis showed: Bone mineral density (BMD): OVX group (0.228±0.008g / cm²). 3 Compared to the Sham group (0.457±0.009g / cm³), 3 The level decreased significantly by 49.9% (P<0.001). Figure 5 B); Bone volume fraction (BV / TV): The OVX group (8.49±0.13%) decreased by 51.9% compared with the Sham group (17.64±0.24%) (P<0.001). Figure 5 C); Trabecular bone thickness (Tb.Th): The OVX group (0.067±0.002mm) was 31.7% less than the Sham group (0.098±0.011mm) (P<0.001). Figure 5D) Bone microstructure degeneration: In the OVX group, the number of trabeculae (Tb.N) decreased by 58.3% (3.2±0.2 vs 1.4±0.1 / mm, P<0.001), and the trabecular separation (Tb.Sp) increased by 2.1 times (0.43±0.03 vs 0.91±0.05mm, P<0.001). These indicators combined confirm the successful establishment of the osteoporosis model, and the bone microstructure exhibits typical characteristics of high-turnover osteoporosis, with a 2.8-fold increase in bone resorption.

[0129] Evaluation of bone defect repair efficacy showed that after 4 weeks of intervention in a grouped osteoporosis model rat (n=5 / group), Micro-CT three-dimensional reconstruction analysis indicated significant differences between groups: the negative control group (NC) and the PLGA / CS-nHA group only showed scattered low-density images in the peripheral area (new bone volume fraction BV / TV were NC group:

[0130] The mean value of bone density in the PTH(1-34)@PLGA / CS-nHA group was 0.0973±0.0129 (0.0909±0.0031). High-density new bone tissue (BV / TV=0.2020±0.0165) was formed in the defect center area of ​​the PTH(1-34)@PLGA / CS-nHA group. The trabecular bone thickness (Tb.Th) was 0.6132±0.028 in the NC group, 0.3879±0.0149 in the PLGA / CS-nHA group, and 0.3536±0.0197 in the PTH(1-34)@PLGA / CS-nHA group. 3, mm); ④ Tb.N were: NC group: 0.2359±0.0485, PLGA / CS-nHA group: 0.2193±0.0386, PTH(1-34)@PLGA / CS-nHA group: 0.4867±0.0286, 1 / mm; ⑤ Tb.Sp were: NC group: 5.345±0.785, PLGA / CS-nHA group: 5.296±0.675, PTH(1-34)@PLGA / CS-nHA group: 3.498±0.260, mm (P<0.001, Figure 6 AG).

[0131] Osteogenic efficacy assessment confirmed that PTH(1-34)@PLGA / CS-nHA demonstrated significant therapeutic advantages in the repair of osteoporotic bone defects. Micro-CT quantitative analysis (n=5 / group) showed that PTH(1-34)@PLGA / CS-nHA was significantly superior to the NC group and the PLGA / CS-nHA group in terms of bone regeneration indicators: Bone volume fraction (BV / TV): The PTH(1-34)@PLGA / CS-nHA group reached 0.2020±0.0165, which was 2.08 times and 2.22 times higher than the NC group (0.0973±0.0129) and the PLGA / CS-nHA group (0.0909±0.0031), respectively (P<0.001). Trabecular bone thickness (Tb.Th): The trabecular bone structure in the PTH(1-34)@PLGA / CS-nHA group was more complete (0.6132±0.0283mm), which was 1.58 times and 1.73 times greater than that in the NC group (0.3879±0.0149mm) and the PLGA / CS-nHA group (0.3536±0.0197mm), respectively (P<0.001); Trabecular bone number (Tb.N): The trabecular bone network density in the PTH(1-34)@PLGA / CS-nHA group was (0.4867±0.0286mm). -1 The value was significantly higher than that of the NC group (0.2359±0.0485mm). -1 ) and PLGA / CS-nHA group (0.2193±0.0386mm) -1 The trabecular separation (Tb.Sp) of PTH(1-34)@PLGA / CS-nHA was increased by 2.06 times and 2.22 times respectively (P<0.001); the trabecular separation (Tb.Sp) was reduced by 34.56% and 33.95% respectively compared with NC group (5.345±0.785mm) and PLGA / CS-nHA group (5.296±0.675mm) (P<0.001), indicating that its microstructure connectivity was significantly enhanced.

[0132] In addition to Micro-CT scans, the study also assessed the quality of bone defect repair in each group using a hematoxylin-eosin (HE) and Masson trichrome staining system. Figure 7AB). HE staining analysis: Under low magnification (4×), the defect areas in the NC and PLGA / CS-nHA groups were mainly filled with loose connective tissue, with only a small amount of osteoid deposition visible in the peripheral areas. No mature bone tissue was observed. In the PTH(1-34)@PLGA / CS-nHA group, a continuous osteoid structure was formed in the central area. Under high magnification (10×), a significant increase in the number of osteoblasts was observed, accompanied by an active bone lining cell layer. Masson staining: The defect centers in the NC and PLGA / CS-nHA groups were mainly composed of blue collagen fibers, with only sporadic red bone matrix. In the PTH(1-34)@PLGA / CS-nHA group, a complete and continuous lamellar bone structure was observed, with a large amount of bone matrix visible in between. The histological results were highly consistent with the Micro-CT three-dimensional reconstruction data, confirming that the PTH(1-34)@PLGA / CS-nHA system significantly enhanced the regenerative function of osteoporotic bone defects by promoting osteoblast activation and matrix mineralization. In summary, this study constructed a functional scaffold system adapted for the repair of osteoporotic bone defects through multi-scale structural design and pathological microenvironment regulation.

[0133] This study observed incomplete bone regeneration in the skull defect area of ​​osteoporosis model rats, which may be caused by multiple factors: the inhibitory effect of the pathological microenvironment of osteoporosis; the successfully constructed OVX model rats exhibited typical characteristics of bone metabolism imbalance: bone mineral density (BMD = 0.228 ± 0.008 g / cm³). 3 Compared with the sham surgery group, the bone volume fraction (BV / TV = 8.49 ± 0.13%) decreased by 49.9% and 51.9% respectively (P < 0.001), and the bone microstructure deteriorated significantly (Tb.Sp increased by 2.1 times). Under this pathological condition, the osteogenic microenvironment in the defect area is poor: pro-inflammatory factors are imbalanced, stem cell differentiation is deviated, the tendency of BMSCs to differentiate into adipocytes is enhanced, and the osteogenic-adipogenic balance is disrupted. The limitations of the treatment time window: the 4-week observation period set in this experiment may not be sufficient to fully reflect the bone repair potential of the composite scaffold. The current PTH(1-34) sustained-release system (30-day cumulative release rate 71.8 ± 1.7%) has not yet covered the complete callus remodeling stage (including endochondral ossification-primary callus formation-lamellar bone remodeling), resulting in insufficient new bone volume and maturity.

[0134] V. Conclusion and Analysis

[0135] This invention utilizes a multi-scale design to construct a bone repair system with a three-in-one function of "dual controlled drug release, microenvironment regulation, and anatomical morphology matching." Compared to traditional single-component systems, this composite system exhibits significant advantages:

[0136] Dual release kinetics: PLGA microspheres (drug loading rate 68.2±3.1%) and CS / nHA hydrogel synergistically regulate PTH (1-34) release, achieving precise matching between early therapeutic concentration (burst release of 19.4±2.1% within 24 hours) and long-term maintenance concentration (cumulative release of 71.8±1.7% up to 30 days).

[0137] Microenvironment regulation: The addition of short rod-shaped nHA can better mimic the structure and function of natural bone tissue. Short rod-shaped nHA and PTH(1-34) have a synergistic effect during treatment.

[0138] Anatomical morphology matching: 3D bioprinting based on clinical CT data enables anatomical adaptation of defect areas.

[0139] In summary, this composite system improves the pathological state of the microenvironment in osteoporotic bone defects. In vivo experiments using an osteoporotic rat model confirmed its significant therapeutic effect in repairing bone defects under pathological load: Micro-CT quantitative analysis showed a 2.22-fold increase in the volume fraction of newly formed bone (BV / TV) compared to the control group (p<0.001), and histological analysis also confirmed this trend. These findings fully demonstrate the potential of this composite system, providing a new paradigm for optimizing the repair process of osteoporotic bone defects.

[0140] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A dual controlled-release complex system for PTH (1-34), characterized in that, Includes a hydrogel scaffold and PTH(1-34) microspheres loaded on the hydrogel scaffold; The PTH(1-34) loaded microspheres include a microsphere matrix and parathyroid hormone loaded on the microsphere matrix, wherein the microsphere matrix is ​​composed of a sustained-release polymer; The sustained-release polymer is a polylactic acid-glycolic acid copolymer, and the hydrogel scaffold is made of chitosan mixed with nano-hydroxyapatite. The mass ratio of nano-hydroxyapatite to chitosan is (6~8):(2~4).

2. The dual release composite system of claim 1, wherein, The nano-hydroxyapatite is in the form of short rods with a diameter of 20-45 nm and a length of 50-170 nm.

3. A method for preparing the dual controlled release composite system according to any one of claims 1 or 2, characterized by, Includes the following steps: S1. Preparation of PTH-loaded microspheres, including: S11. Parathyroid hormone and polyvinyl alcohol are dissolved in water to form parathyroid hormone solution and polyvinyl alcohol solution, respectively; polylactic acid-glycolic acid copolymer is dissolved in dichloromethane to form polylactic acid-glycolic acid copolymer solution; parathyroid hormone solution is added to polylactic acid-glycolic acid copolymer solution under vortex conditions, and the mixture is formed to form colostrum; S12. Add the colostrum to the polyvinyl alcohol solution, stir and mix to emulsify again, collect the precipitate, wash and freeze dry; obtain PTH(1-34) loaded microspheres. S2. Preparation of chitosan ink includes: adding chitosan to an acid solution and mixing, adding a crosslinking modifier and mixing, then adding nano-hydroxyapatite powder and mixing, removing bubbles, and obtaining chitosan ink; The preparation of the dual controlled-release complex system of S3 and PTH (1-34) includes: S31. Disperse the PTH (1-34) microspheres obtained in step S1 into the chitosan ink obtained in step S2, mix, remove bubbles, and form a chitosan-based composite ink. After 3D printing the chitosan-based composite ink, freeze-dry it to obtain a dual controlled-release composite system of PTH (1-34).

4. The production method according to claim 3, characterized by, In step S11, the vortex rotation speed of the polylactic acid-glycolic acid copolymer solution is 2500 rpm to 3500 rpm; the mixing is ultrasonic mixing, and the ultrasonic time is 60 to 90 seconds.

5. The preparation method according to claim 3, characterized in that, In step S12, the freeze-drying time is 12-18 hours.

6. The preparation method according to claim 3, characterized in that, In step S2, the acid solution is a mixed solution of acetic acid, lactic acid and pure water, and the volume ratio of acetic acid, pure water and lactic acid is (1.5~2.5): (1.5~2.5): (0.5~2.5).

7. The preparation method according to claim 3, characterized in that, In step S3, the air pressure for 3D printing is 91~110 kPa, and the printing speed is 150~210 mm / s.

8. The use of the dual controlled-release composite system according to any one of claims 1 or 2, or the dual controlled-release composite system prepared by any one of claims 3 to 7, in the preparation of products for repairing osteoporosis or bone defects.

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