Calcium phosphate oligomer cross-linked biomimetic nanocomposite hydrogel and preparation method and application thereof

Abstract

The application discloses a calcium phosphate oligomer cross-linked biomimetic nanocomposite hydrogel and a preparation method and application thereof, and relates to the technical field of medical materials. The application provides a self-cross-linked double-network hydrogel (GOP), wherein a Schiff base reaction main network is constructed through a Schiff base reaction between sodium alginate (OSA) oxidized by a periodate and Gel, and small-size calcium phosphate oligomers (CPO) participate in intermolecular bonding to establish a secondary network. The double-network structure solves the problem of insufficient mechanical strength of a traditional hydrogel, and the elastic modulus of the double-network hydrogel is 2-5 times higher than that of a single OSA / Gel hydrogel, which is beneficial to bearing the cyclic mechanical load of a bone defect site.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogels, their preparation methods, and applications. Background Technology

[0002] Bone defects are a common orthopedic challenge in clinical practice. Bone defects exceeding a critical size cannot heal spontaneously and require intervention with bone grafts or artificial bone substitutes. Among current clinical protocols, autologous bone grafting faces issues such as donor site complications and limited resources, while allogeneic and xenograft transplants pose risks of immunogenicity and disease transmission. Traditional synthetic bone substitutes generally lack sufficient bioactivity, making it difficult to efficiently induce bone regeneration.

[0003] Traditional gelatin and alginate hydrogels exhibit excellent biocompatibility. Their high water content and three-dimensional porous structure mimic the extracellular matrix, thus facilitating nutrient transport and metabolic waste removal.

[0004] Hydrogels have attracted much attention in bone tissue engineering due to their three-dimensional porous structure similar to the extracellular matrix and their high water content. Although traditional gel hydrogels contain cell adhesion RGD sequences, they suffer from poor mechanical strength and rapid degradation. Sodium alginate hydrogels exhibit good biocompatibility, but their in vivo degradation is uncontrollable and they lack cell adhesion sites. While current technologies attempt to combine inorganic components such as hydroxyapatite with polymer matrices, these are mostly simple physical mixtures with phase separation problems, making it difficult to simulate the organic-inorganic integrated structure of natural bone. Furthermore, their mechanical properties and osteogenic activity still fail to meet clinical needs.

[0005] Therefore, developing biomimetic hydrogel materials that combine excellent mechanical strength, controllable degradation, and high osteogenic activity is an urgent need in the field of bone tissue engineering. Summary of the Invention

[0006] The purpose of this invention is to provide calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogels, their preparation methods, and applications, in order to solve the technical problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel, comprising a Schiff base reaction main network and a CPO-mediated secondary network; The Schiff base reaction main network adopts OSA, that is, sodium periodate oxidizes sodium alginate to introduce aldehyde functional groups, which form dynamic covalent bonds with the amino group of gel through Schiff base reaction, replacing toxic chemical crosslinking agents and ensuring biocompatibility. The gel is gelatin. The CPO-mediated secondary network is introduced with 1-2 nm of CPO, namely calcium phosphate oligomer. CPO forms coordination with the carboxylate group of OSA and hydrogen bonds with the amino group of Gel to construct a dense CPO-mediated secondary network. At the same time, CPO, as an osteogenic active ingredient, provides mineralization nucleation sites.

[0008] A method for preparing a gel, comprising at least the following steps: S1: Preparation of OSA: Prepare a mixture of SA solution and sodium periodate solution. Stir the mixture magnetically at 25°C in the dark for 12 hours to obtain OSA solution. Terminate the reaction by adding ethylene glycol to the OSA solution, which also neutralizes excess periodate. Dialyze the resulting solution with deionized water for 4 days, changing the water frequently, until periodate is undetectable in the dialysate. Freeze-dry the dialyzed solution to obtain OSA. S2: To prepare CPO, calcium chloride was dissolved in anhydrous ethanol, triethylamine was added and stirred, and then mixed with phosphate ethanol solution. After centrifugation and washing, CPO dispersion was obtained. S3: To prepare a composite hydrogel, Gel solution, OSA solution and CPO dispersion were mixed and allowed to stand to form a double-network biomimetic nanocomposite hydrogel.

[0009] Furthermore, the preparation of the SA solution and sodium periodate solution mixture includes at least the following steps: Add 2 g of SA to 10 mL of anhydrous ethanol with magnetic stirring; In addition, 1.16 g of sodium periodate was dissolved in 10 mL of deionized water under light-protected conditions; Finally, the SA solution is mixed with the sodium periodate solution.

[0010] Furthermore, S2 includes at least the following steps: Add calcium chloride dihydrate to 80 mL of anhydrous ethanol. Seal the container containing calcium chloride dihydrate and anhydrous ethanol with plastic film and stir magnetically for 7-8 hours. Then add triethylamine and continue stirring magnetically for 12 hours. Add phosphoric acid to 20 mL of anhydrous ethanol and mix well to obtain a phosphoric acid ethanol solution; The phosphoric acid solution was added to the triethylamine-treated mixture, and the reaction was continued for 5 h. After the reaction was complete, the resulting mixture was centrifuged at 8600 rpm for 6 minutes. The collected solids were washed twice with anhydrous ethanol and then redispersed in 15 mL of anhydrous ethanol to obtain a CPO dispersion with a concentration of 10 mg / mL.

[0011] Furthermore, in S2, the amount of calcium chloride dihydrate is 200 mg, the amount of triethylamine is 4 mL, and the amount of phosphoric acid is 70 μL.

[0012] Furthermore, S3 includes at least the following steps: The gel was incubated at 60 °C for 12 hours and then dissolved in 420 μL of deionized water to form a homogeneous gel solution. OSA was dissolved in 420 μL PBS under vortex mixing to obtain an OSA solution; The CPO dispersion was centrifuged three times with ultrapure water, 240 μL of supernatant was removed, and the water-containing CPO precipitate was collected. The CPO precipitate was then thoroughly mixed with the OSA solution, followed by the addition of a pre-prepared gel solution under vigorous stirring. The resulting mixture was then transferred to a mold and allowed to stand for 8 hours to form a hydrogel.

[0013] Applications of calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogels: Calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogels are used in bone tissue engineering scaffolds or bone defect repair materials.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention proposes a self-crosslinking dual-network hydrogel (GOP). The Schiff base reaction main network is constructed through the Schiff base reaction between sodium alginate (OSA) oxidized by periodate and gel. At the same time, small-sized calcium phosphate oligomers (CPO) participate in intermolecular bonding to establish a secondary network. The dual-network structure solves the problem of insufficient mechanical strength of traditional hydrogels. The elastic modulus is increased by 2-5 times compared with single OSA / Gel hydrogels, which is beneficial for withstanding cyclic mechanical loads at bone defect sites. CPO, as a natural precursor of inorganic bone components, provides mineralization nucleation sites and promotes bone-like apatite deposition. At the same time, it synergistically enhances cell adhesion and osteogenic differentiation with the RGD sequence of gel. The secondary network constructed by CPO slows down the hydrolysis rate, and the degradation rate can be regulated by CPO concentration to match the bone regeneration cycle. Furthermore, because it uses natural polymer raw materials without any added toxic cross-linking agents, it is injectable and self-healing, can adaptively fill complex bone defects, and has thermosensitive solubility, making it easy to adjust or remove non-invasively after surgery. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram illustrating the preparation process and crosslinking mechanism of the GOP hydrogel of the present invention; Figure 2 This is a characterization diagram of the raw materials used in this invention. Figure 2 Figure A shows digital photographs and TEM images of CPO; Figure B shows the UV-Vis spectrum and titration analysis of OSA; Figure C shows a visual description of the hydrogel synthesis process. Figure 3 SEM images of hydrogels with different CPO contents according to the present invention; Figure 4 This is a detection image of the hydrogel of the present invention. Figure 4 A represents the FT-IR spectrum. Figure 4 B represents the XRD pattern; Figure 5 This is a performance test diagram of the hydrogel of the present invention. Figure 5 A represents the compressive stress-strain curve and the derived mechanical parameters. Figure 5 B is a schematic diagram of the hydrogel rebound test; Figure 6 This is a schematic diagram illustrating the swelling and degradation behavior of the hydrogel of the present invention. Figure 6 A is a schematic diagram of the swelling kinetics in PBS over 24 hours. Figure 6 B is a schematic diagram of the degradation curve; Figure 7 SEM images of the hydrogel of the present invention after soaking in SBF for 1 day and 3 days; Figure 8 This is a miniature CT analysis and histological staining image of rats after 8 weeks of skull defect repair according to the present invention. Figure 8 A represents a miniature CT analysis of bone regeneration in rats with skull defects at 8 weeks of age. Figure 8 B is a staining agent for hematoxylin and eosin (H&E). Figure 8 C represents Masson's trichrome staining. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0018] This invention utilizes oxidized sodium alginate (OSA), gelatin (Gel), and calcium phosphate oligomer (CPO) to successfully develop a novel dual-network hydrogel (GOP) without the need for any toxic chemical crosslinking agents. The Schiff base reaction main network is formed via a Schiff base reaction between OSA and Gel, while CPO establishes a secondary network within the polymer matrix through coordination and hydrogen bonding. This dual crosslinking strategy yields a three-dimensional self-crosslinked structure with significantly enhanced mechanical strength. Notably, the incorporation of CPO maintains excellent optical transparency and ensures uniform integration at the molecular level. Furthermore, the GOP hydrogel exhibits significant bioactivity, promoting uniform surface mineralization and demonstrating a strong potential to stimulate osteogenic growth. In vitro assays confirmed excellent cell compatibility, manifested in enhanced cell viability and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), attributed to the biocompatibility of the natural polymer components and the absence of added crosslinking agents. Importantly, implantation in a rat critical-size skull defect model validated the hydrogel's ability to support substantial bone regeneration and associated angiogenesis. In summary, the GOP hydrogel system integrates enhanced mechanical strength, excellent biocompatibility, and inherent osteogenic activity, representing a promising advanced biomaterial for bone tissue engineering and regenerative applications.

[0019] Specifically as follows: Example 1:

[0020] This embodiment specifically discloses a calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel, including a Schiff base reaction main network and a CPO-mediated secondary network; The Schiff base reaction main network adopts OSA, that is, sodium periodate oxidizes sodium alginate to introduce aldehyde functional groups, which form dynamic covalent bonds with the amino group of gel through Schiff base reaction, replacing toxic chemical cross-linking agents and ensuring biocompatibility. Introducing 1-2 nm CPO, or calcium phosphate oligomer, into the CPO-mediated secondary network allows for coordination with the carboxylate groups of OSA and hydrogen bonding with the amino groups of Gel, thus constructing a dense CPO-mediated secondary network. Simultaneously, CPO, as an osteogenic active ingredient, provides mineralization nucleation sites.

[0021] Example 2: This embodiment proposes a method for preparing a gel based on the above embodiments, which includes at least the following steps: S1: Preparation of OSA: Prepare a mixture of SA solution and sodium periodate solution. Stir the mixture magnetically at 25 °C in the dark for 12 hours to obtain OSA solution. Terminate the reaction by adding ethylene glycol to the OSA solution, which also neutralizes excess periodate. Dialyze the resulting solution to deionized water for 4 days, changing the water frequently, until periodate is undetectable in the dialysate. Freeze-dry the dialyzed solution to obtain OSA. S2: To prepare CPO, calcium chloride is dissolved in anhydrous ethanol, triethylamine is added and stirred, and then mixed with an ethanol solution of phosphoric acid. After centrifugation and washing, a CPO dispersion is obtained. S3: To prepare a composite hydrogel, Gel solution, OSA solution and CPO dispersion were mixed and allowed to stand to form a double-network biomimetic nanocomposite hydrogel.

[0022] The preparation of a mixture of SA solution and sodium periodate solution includes at least the following steps: Add 2 g of SA to 10 mL of anhydrous ethanol with magnetic stirring; In addition, 1.16 g of sodium periodate was dissolved in 10 mL of deionized water under light-protected conditions; Finally, the SA solution is mixed with the sodium periodate solution.

[0023] S2 includes at least the following steps: Add calcium chloride dihydrate to 80 mL of anhydrous ethanol. Seal the container containing calcium chloride dihydrate and anhydrous ethanol with plastic film and stir magnetically for 7-8 hours. Then add triethylamine and continue stirring magnetically for 12 hours. Add phosphoric acid to 20 mL of anhydrous ethanol and mix well to obtain a phosphoric acid ethanol solution; The phosphoric acid solution was added to the triethylamine-treated mixture, and the reaction was continued for 5 h. After the reaction was complete, the resulting mixture was centrifuged at 8600 rpm for 6 minutes. The collected solids were washed twice with anhydrous ethanol and then redispersed in 15 mL of anhydrous ethanol to obtain a CPO dispersion with a concentration of 10 mg / mL.

[0024] In S2, the dosage of calcium chloride dihydrate is 200 mg, the dosage of triethylamine is 4 mL, and the dosage of phosphoric acid is 70 μL.

[0025] The formulations of GOP hydrogels with different compositions in this embodiment are summarized in Table 1, while schematic diagrams of the preparation process and potential cross-linking mechanisms are shown in [the table]. Figure 1 The hydrogels were named GOP0, GOP3, GOP6, and GOP9.

[0026] The representative preparation process of GOP3 hydrogel is described as follows: Gel (0.075 g) was incubated at 60 °C for 12 hours and dissolved in 420 μL of deionized water to form a homogeneous solution. OSA (0.05 g) was dissolved in 420 μL of PBS under vortex mixing to obtain an OSA solution. CPO solution (400 μL, 10 mg / mL) was centrifuged three times with ultrapure water, and 240 μL of supernatant was removed, collecting the CPO precipitate containing water (160 μL). The precipitate was then thoroughly mixed with the OSA solution, and then the pre-prepared gel solution was added under vigorous stirring to adjust the final volume to 1000 μL. The resulting mixture was transferred to a mold and allowed to stand for 8 hours to form a hydrogel.

[0027] Table 1. GOP hydrogel formulations with different CPO concentrations Sample <![CDATA[Gel(g)+H2O]]> OSA(g) + PBS CPO(μL) GOP0 <![CDATA[0.075+500μLH2O]]> 0.05 + 500 μL PBS 0 GOP3 <![CDATA[0.075+420μLH2O]]> 0.05 + 420 μL PBS 160(400-240) GOP6 <![CDATA[0.075+380μLH2O]]> 0.05 + 380 μL PBS 240(800-560) GOP9 <![CDATA[0.075+340μLH2O]]> 0.05 + 340 μL PBS 320(1200-880) Example 3:

[0028] This embodiment proposes the application of calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel based on the above embodiments, and uses calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel in bone tissue engineering scaffolds or bone defect repair materials.

[0029] To further verify the effectiveness of the above embodiments, the following experiment is proposed: Characterization TEM images of CPO were acquired using a JEM-200 microscope. An ultra-diluted CPO solution was sprayed onto an ultrathin carbon film to prevent particle aggregation. Multiple images were acquired and integrated using ImageJ to improve the signal-to-noise ratio.

[0030] The synthesis of OSA, CPO, and GOP hydrogels was confirmed using Fourier transform infrared (FTIR) spectroscopy. The morphology of the hydrogels was characterized by field emission scanning electron microscopy (SEM). After freeze-drying, the hydrogel samples were immersed in liquid nitrogen and cut to expose their internal structure. All samples were sputter-coated with gold, and their cross-sectional morphology was observed under low vacuum conditions.

[0031] OSA Measurement The aldehyde content and oxidation degree (OD) of OSA were quantitatively determined using the hydroxylamine hydrochloride titration method. In short, 0.01 g of OSA was accurately weighed and dissolved in 2.5 mL of 0.05% hydroxylamine hydrochloride-methyl orange solution. After complete dissolution, the mixture was incubated for 3 hours and then titrated with 0.01 mol / L NaOH. The titration endpoint was determined by a clear color change from red to orange to yellow. The volume of NaOH consumed was recorded (denoted as ΔV). In this experiment, the hydroxylamine hydrochloride titration method was used to determine the aldehyde content and oxidation degree of SA, where the concentration of the aldehyde group can be calculated using Equations 1 and 2.

[0032] (1) (2) in, It is the number of moles of aldehyde groups on SA. CHO represents the molar concentration of NaOH, and CHO represents the concentration of aldehyde groups (mol / g). It is the mass (g) of OSA.

[0033] Meanwhile, the oxidation degree (OD) of SA can be calculated according to Formula 3:

[0034] The UV-Vis absorption spectrum of the OSA was recorded using a UV-Vis spectrophotometer.

[0035] A test solution was prepared by dissolving 0.1 g of OSA sample in 10 mL of deionized water, using deionized water as a blank control. The spectrophotometer was configured with a transmittance range of 0.0% to 100.0%T and a wavelength scan range of 200 to 800 nm. After establishing a baseline with the blank sample and adjusting for zeroing / full scale, the absorbance spectra of the SA and OSA solutions were obtained.

[0036] Mechanical properties of GOP hydrogels The mechanical properties of the composite hydrogels were evaluated under compression conditions using a universal testing machine. Four groups of GOP hydrogels with different CPO concentrations were prepared, each group containing four parallel samples. Uniaxial compression tests were performed at a constant crosshead speed of 1 mm / min until a significant stress drop was observed or the instrument's load limit was reached. The acquired data were first processed in Excel, and then stress-strain curves were plotted using Origin software. Key mechanical parameters, including maximum strain, ultimate compressive stress, and elastic modulus, were then calculated from the curves for comparative analysis.

[0037] The swelling capacity and degradation behavior of the composite hydrogels were evaluated in phosphate-buffered saline (PBS, pH 7.4). For the swelling test, four groups of GOP hydrogels with different CPO concentrations were prepared (n=5 per group). The hydrogels were freeze-dried, and their initial dry weight (W0) was recorded. The freeze-dried samples were then immersed in PBS and incubated at 37 °C. At predetermined time intervals, the hydrogels were removed, the surface moisture was blotted with absorbent paper, and the final dry weight (W1) was measured. The swelling ratio (SR) was calculated according to equation (4): SR=(W1-W0) / W0×100%(4) For the degradation test, the lyophilized hydrogel (W0) was placed in a 5 mL centrifuge tube containing 4 mL PBS and incubated in a shaker at 37°C and 120 rpm. The PBS solution was changed on days 1, 3, and 5. On day 7, the hydrogel was removed, rinsed twice with deionized water, photographed, and then lyophilized again. The final dry weight (W1) was measured, and the degradation rate DR was determined using equation (5): DR=(W0-W1) / W0×100%(5) In vitro biomineralization potential of GOP hydrogels The biomineralization potential of GOP hydrogels was evaluated by immersion in simulated body fluid (SBF), which has similar ion concentrations and pH values ​​to human blood plasma. Cylindrical GOP hydrogel samples (7 mm in diameter and 5 mm in height) were prepared, with three replicates per group immersed in 4 mL of SBF and incubated at 37 °C. The SBF solution was changed every two days. After incubation in SBF, the samples were removed, rinsed with deionized water, freeze-dried, and then characterized by SEM-EDS.

[0038] Animal surgery All animal experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of Peking University (LA2020200).

[0039] All implanted materials were immersed in 75% ethanol to ensure complete sterilization. After anesthesia induced by sodium pentobarbital, a longitudinal incision was made in the scalp of the SD rats using a sterile scalpel. The subcutaneous tissue and periosteum were then carefully lifted to expose the parietal bone. Two full-thickness circular critical-size defects (5 mm in diameter) were created on either side of the sagittal suture. The defects were filled with the appropriate test material. After an 8-week healing period, the rats were euthanized by carbon dioxide overdose. The skull containing the defects was harvested and fixed by immersion in 10% neutral buffered formalin.

[0040] Miniature CT analysis and H&E, Masson staining Skull samples were scanned using the InveonMM system miniature CT scanner, followed by parametric analysis using dedicated software. After analysis, the samples were decalcified, embedded, and sectioned in 10% EDTA solution, and finally histologically evaluated using hematoxylin and eosin (H&E) and Masson's trichrome staining.

[0041] Statistical analysis Each experiment was independently repeated three times. All numerical data are expressed as mean ± standard deviation (SD) and analyzed using SPSS Statistics 26 software (IBM, USA). Statistically significant differences between groups were verified using one-way ANOVA and post-hoc Tukey's test. *p<0.05, **p<0.01, ***p<0.001 were considered statistically significant.

[0042] Preparation and characterization of hydrogels First, the morphology and dispersibility of CPO were examined. For example... Figure 2 As shown in Figure A, CPO is uniformly dispersed in ethanol. Figure 2 A(a)). After centrifugation, the CPO precipitate maintained good integrity and showed no obvious aggregation. Figure 2 A(b) and 2A(c)). Transmission electron microscopy (TEM) further revealed that the CPO particles were monodisperse, with a size range of 1-2 nm. Figure 2 A(d)), consistent with previous reports. The small particle size and stability of CPO are attributed to triethylamine (TEA) end-capping, which enables effective intermolecular interactions with OSA and Gel during mixing. After TEA volatilization, CPO exhibits excellent dispersibility, forming a continuous, interface-free organic-inorganic composite hydrogel. This uniform integration is further confirmed by the excellent light transmittance observed in GOP hydrogels of different concentrations.

[0043] Through ultraviolet-visible spectroscopy ( Figure 2 B) The successful oxidation of SA to OSA was confirmed. Compared with the original SA, the OSA spectrum showed a new absorption peak at 240 nm, which is the characteristic peak of the aldehyde group, confirming the introduction of the aldehyde functional group through periodate oxidation. This provides the necessary chemical basis for the subsequent Schiff base reaction with Gel. The aldehyde content was quantitatively analyzed using hydroxylamine hydrochloride titration. Figure 2 B(a,b)). Upon titration with NaOH, the color of the OSA solution changed from red to yellow, indicating that the aldehyde group was consumed. Based on the volume of NaOH consumed (4.2 mL) and the formula described in Section 2.4, the aldehyde concentration and oxidation degree of the OSA were calculated to be 4.2 mmol / L and 42%, respectively.

[0044] The preparation process of GOP hydrogel is as follows: Figure 2 As shown in C. In short, mix the Gel solution with CPO ( Figure 2 C(a)), then add OSA solution ( Figure 2 C(b)). The mixture was left to stand at 37 °C for several hours to form a gel ( Figure 2C(c)). The entire process is simple, uses natural polymers, and requires no additional cross-linking agents, indicating good biocompatibility, as shown in subsequent cell experiments.

[0045] SEM images of GOP hydrogel The internal microstructure of GOP composite hydrogels with different CPO concentrations was observed by scanning electron microscopy (SEM). Figure 3 Images at 100x and 300x magnification revealed that all hydrogels exhibited a three-dimensional porous structure, similar to the topological features of the natural extracellular matrix, a typical and favorable structure for tissue engineering scaffolds. The CPO-free GOP0 hydrogel had smaller pore sizes (approximately 50-100 μm) and thinner pore walls, consistent with the microstructure of pure OSA / Gel hydrogels. In contrast, with the addition of CPO (GOP3, GOP6, and GOP9 groups), the pore size gradually increased to 150-200 μm, and the pore wall thickness also increased significantly, with this change exhibiting a gradient trend with increasing CPO concentration.

[0046] The core reason for the structural difference lies in the network regulation effect of CPO as a crosslinking agent: CPO molecules within the gel form numerous hydrogen bonds with amino groups, and these hydrogen bond interactions significantly increase the crosslinking density of the hydrogel. This allows the hydrogel to maintain a stable pore wall structure during freeze-drying, preventing the pore wall collapse characteristic of pure organic hydrogels. Simultaneously, the nanoscale size of CPO ensures its uniform dispersion, preventing pore blockage.

[0047] Chemical structure of GOP hydrogel The chemical structure of the GOP hydrogel was characterized by attenuated total reflectance infrared (ATR-IR) spectroscopy and X-ray diffraction (XRD). Figure 4 First, through the OSA spectrum at approximately 1735 cm⁻¹ -1 A new carbonyl stretching vibration peak appeared at 1600 cm⁻¹, confirming the oxidation of sodium alginate (SA) to OSA, corresponding to the introduced aldehyde group. In contrast, the SA spectrum only showed a peak at 1600 cm⁻¹. -1 The vicinity shows characteristic asymmetric stretching vibration peaks of carboxylate. Subsequently, the mixing of OSA and Gel leads to significant spectral changes, indicating network formation. A broad OH stretching region (3200-3400 cm⁻¹) is observed due to hydrogen bonding between OSA and Gel. -1 The OSA aldehyde peak broadened further. Meanwhile, the OSA aldehyde peak (1735 cm⁻¹) also broadened further. -1 The intensity of ) decreased significantly, while the Gel amino peak (1550 cm⁻¹) was significantly reduced. -1 The shift in the peak value is consistent with the Schiff base reaction that generates a dynamic imine bond. The introduction of CPO introduces additional coordination and hydrogen bonding interactions. The carboxylate peak of the OSA exhibits a red shift and attenuation, attributed to the interaction between the -COO group and Ca from CPO.2+ Coordination between them. Similarly, the amino stretching band of gel (3300cm) -1 The CPO peak broadened and intensified (10¹² cm⁻¹). -1 The displacement confirmed the hydrogen bonding between the amino groups of Gel and the phosphate groups of CPO. As shown in the infrared results, the dynamic imine bonds generated by the Schiff base reaction endow the GOP hydrogel with temperature responsiveness and self-healing capabilities. This invention develops a dual-network GOP hydrogel that integrates a thermally reversible phase transition (60°C sol-gel transition) with room-temperature self-healing capabilities. This unique combination elevates the material from a passive scaffold to a smart bone repair platform. The synergy between injectability and self-healing capabilities ensures that the material can adaptively fill and perfectly fit any complex bone defect in a minimally invasive manner, while maintaining the structural integrity within the body.

[0048] Thermosensitive solubility further provides unprecedented clinical reversibility and controllability, allowing for non-invasive adjustment or even removal of the implant post-operatively as needed.

[0049] This closed-loop functional cycle—"adaptive filling, self-repairing consolidation, and on-demand resetting"—overcomes the inherent limitations of traditional bone repair materials in terms of precise fit, long-term stability, and clinical intervention, thus providing an innovative material basis for next-generation personalized and intelligent bone regeneration strategies.

[0050] Water contact angle (WCA) results are consistent with the interactions identified by FT-IR. The GOP0 hydrogel exhibits moderate hydrophilicity (71.5°), attributed to hydrogen bonding and Schiff base crosslinking. With the addition of low CPO content (GOP3, GOP6), hydrophilicity decreases (WCA increases to 78.7° and 89.8°, respectively), as coordination and hydrogen bonding partially shield the hydrophilic groups. In contrast, the GOP9 sample with high CPO content shows enhanced hydrophilicity (WCA decreases to 63.4°), likely due to the exposure of surface phosphate groups. XRD patterns show that GOP0 (0% CPO) exhibits a broad amorphous peak at 20°–30°, characteristic of the gel / OSA organic matrix. The GOP3 hydrogel remains predominantly amorphous but shows a weak hydroxyapatite characteristic peak near 38°, confirming that CPO is uniformly integrated into the polymer network without significant phase separation—consistent with the concept of “organic-inorganic copolymerization” for forming homogeneous composites.

[0051] Mechanical properties of GOP hydrogels Compressive mechanical properties of GOP hydrogels with different CPO concentrations, such as Figure 5 As shown in Figure A. Stress-strain curve ( Figure 5A(a) indicates that with increasing CPO content, the hydrogel can withstand higher stress at lower strain. The maximum strains of GOP0, GOP3, and GOP6 are comparable ( Figure 5 A(b)), while GOP9 exhibits a significantly higher maximum strain (approximately 457 kPa). The results indicate that the maximum compressive stress ( Figure 5 A(c)) and Young's modulus ( Figure 5 A(d) all gradually increased with increasing CPO concentration. The maximum stress of the freeze-dried GOP hydrogel increased significantly with increasing CPO concentration, consistent with the trend observed in the wet state. This enhancement is attributed to network densification caused by water removal during freeze-drying, where CPO, as an inorganic filler, is tightly packed within the shrinking polymer matrix, thereby strengthening the crosslinked network.

[0052] The core reason for the improved mechanical properties is the dual-network structure constructed by CPO: GOPO forms a single network solely through the Schiff base reaction between Gel and OSA. CPO, acting as a crosslinking agent, forms multiple hydrogen bonds with the hydroxyl / carbonyl groups of OSA and the amino / hydroxyl groups of Gel, constructing a dense CPO-mediated secondary network. This structure makes the hydrogel's microstructure more compact, thereby enhancing its strength, stiffness, and toughness—all essential properties for bone repair scaffolds.

[0053] Figure 5 B shows that all hydrogel samples rapidly recovered their original shape after significant compression, demonstrating excellent elastic recovery. This property makes them suitable for withstanding cyclic mechanical loads at bone defect sites and maintaining scaffold stability during healing. Furthermore, the incorporation of CPO did not significantly affect the transparency of the hydrogels, confirming their uniform integration into the polymer matrix via intermolecular hydrogen bonds without macroscopic phase separation.

[0054] Swelling ratio and degradation of GOP hydrogel The 24-hour swelling kinetics of GOP hydrogels with different CPO concentrations are as follows: Figure 6 As shown in Figure A, all hydrogels rapidly absorbed PBS within the first 5 hours, after which the swelling rate gradually slowed down. From GOP0 to GOP6, the equilibrium swelling ratio increased with increasing CPO content, consistent with the more open porous structure observed by SEM. Figure 3 CPO improves pore connectivity through hydrogen bonding, which facilitates water permeability. However, GOP9 has a lower swelling capacity than GOP6, which is attributed to the excessively high CPO content leading to thicker pore walls and overly dense cross-linking, thus spatially limiting further expansion of the hydrogel.

[0055] After incubation in PBS for 7 days, the degradation rate of the hydrogel continued to decrease with increasing CPO content. Figure 6B): GOP0 degraded rapidly, with a mass loss of 83.44±0.06%, while GOP3, GOP6, and GOP9 experienced mass losses of 54.44±0.01%, 47.99±0.01%, and 43.04±0.02%, respectively. The core reason for the delayed degradation is that CPO acts as a cross-linking agent, forming multiple hydrogen bonds with OSA and Gel, enhancing the integrity of the network structure and reducing the damage to the molecular chains caused by hydrolysis. Notably, although high swelling is generally associated with rapid degradation, GOP hydrogels (especially GOP6) achieve a balance between improving swelling capacity and controlling the degradation rate—a property that not only ensures the porous and moist environment required for cell infiltration but also provides sustained mechanical support and biological effects for bone repair.

[0056] In vitro osteogenic potential of hydrogels The biomineralization potential of GOP hydrogels was assessed by immersing them in simulated body fluid (SBF) for 1 day and 3 days. Figure 7 OSA / Gel organic matrix (SBF) possesses ion concentrations and pH values ​​similar to human blood plasma, making it a classic in vitro model for evaluating the bone bioactivity of materials. SEM images magnified 1000x revealed a uniform and continuous mineral layer on all hydrogel surfaces, regardless of CPO content, indicating that the OSA / Gel organic matrix itself possesses intrinsic mineralization-inducing activity. EDSmapping further confirmed the presence of calcium and phosphate ions in the mineral layer, demonstrating that the deposited product is osteoapatite—the main inorganic component of bone tissue. Its formation is a crucial prerequisite for the material to support osteogenic regeneration.

[0057] As the soaking time increased from 1 day to 3 days, the mineral coverage of all hydrogel groups significantly increased, reflecting a continuous and gradual mineralization process. Notably, the mineral deposits formed by hydrogels containing CPO were denser and more uniform than those formed by hydrogels containing GPO. The core reason is that CPO, as a crosslinking agent, contains phosphate groups in its molecular structure, which can serve as nucleation sites for hydroxyapatite crystals. Simultaneously, the hydrogen bond network formed by CPO and gel stabilizes the mineral crystals after nucleation, preventing aggregation and thus significantly enhancing the biomineralization activity of the composite material. These results indicate that hydrogels possess excellent mineralization capabilities, and the incorporation of CPO further optimizes the mineralization effect, providing crucial support for osteogenic regeneration during bone repair.

[0058] In vivo osteogenic capacity assessment Based on its promising in vitro osteogenic activity, the bone repair effect of GOP hydrogel was further evaluated using a rat critical-size skull defect model (5 mm in diameter). GOP0 and GOP6 were implanted into the defect, and bone regeneration was assessed 8 weeks post-implantation. Miniature CT reconstruction and cross-sectional images were used. Figure 8A) shows that, compared with GOP0 and the control group, the GOP6 group exhibited significantly more new bone formation. The defect area implanted with GOP6 was almost completely regenerated by new bone tissue, while only limited repair was observed in the GOP0 group, and the control group showed minimal bone healing.

[0059] H&E and Masson staining ( Figure 8 (B, C) further elucidated the microstructure and composition of newly formed bone. In the GOP6 group, extensive bone regeneration accompanied by significant bone marrow formation was observed, almost bridging the defect gap by week 8. In contrast, the GOP0 group showed relatively less mature bone tissue and incomplete defect closure.

[0060] In summary: This invention characterized the composite hydrogel using scanning electron microscopy (SEM) and X-ray diffraction (XRD), and evaluated its mechanical properties, degradation behavior, and swelling capacity. The GOP hydrogel exhibited excellent biocompatibility, supported cell viability and osteogenic differentiation, and promoted vascularized healing of critical-sized skull defects in rats. The dual-network structure significantly enhanced the hydrogel's mechanical strength, modulated its degradation and swelling properties, promoted mineralization, and facilitated osteogenic processes. These results indicate that the GOP hydrogel better meets the requirements of bone tissue engineering scaffolds and has considerable potential in bone regeneration applications.

[0061] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel, characterized in that: This includes the Schiff base reaction main network and the CPO-mediated secondary network; The Schiff base reaction main network adopts OSA, that is, sodium periodate oxidizes sodium alginate to introduce aldehyde functional groups, which form dynamic covalent bonds with the amino group of gel through Schiff base reaction, replacing toxic chemical crosslinking agents and ensuring biocompatibility. The gel is gelatin. The CPO-mediated secondary network introduces 1-2 nm CPO, namely calcium phosphate oligomer, which coordinates with the carboxylate group of OSA and forms hydrogen bonds with the amino group of Gel to construct a dense CPO-mediated secondary network. At the same time, CPO, as an osteogenic active ingredient, provides mineralization nucleation sites.

2. A method for preparing a gel, used to prepare the calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel according to claim 1, characterized in that: At least the following steps are included: S1: Preparation of OSA: Prepare a mixture of SA solution and sodium periodate solution. Stir the mixture magnetically for 12 hours at 25°C in the dark to obtain OSA solution. Terminate the reaction by adding ethylene glycol to the OSA solution, which also neutralizes excess periodate. Dialyze the obtained solution with deionized water for 4 days, changing the water frequently, until periodate is undetectable in the dialysate. Freeze-dry the dialyzed solution to obtain OSA. S2: To prepare CPO, calcium chloride was dissolved in anhydrous ethanol, triethylamine was added and stirred, and then mixed with phosphate ethanol solution. After centrifugation and washing, CPO dispersion was obtained. S3: To prepare a composite hydrogel, Gel solution, OSA solution and CPO dispersion were mixed and allowed to stand to form a double-network biomimetic nanocomposite hydrogel.

3. The method for preparing the calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel according to claim 2, characterized in that: The preparation of the mixture of SA solution and sodium periodate solution includes at least the following steps: Add 2g of SA to 10mL of anhydrous ethanol with magnetic stirring; In addition, 1.16 g of sodium periodate was dissolved in 10 mL of deionized water under light-protected conditions; Finally, the SA solution is mixed with the sodium periodate solution.

4. The method for preparing the calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel according to claim 2, characterized in that: S2 includes at least the following steps: Add calcium chloride dihydrate to 80 mL of anhydrous ethanol. Seal the container containing calcium chloride dihydrate and anhydrous ethanol with plastic film and stir magnetically for 7-8 hours. Then add triethylamine and continue stirring magnetically for 12 hours. Add phosphoric acid to 20 mL of anhydrous ethanol and mix well to obtain a phosphoric acid ethanol solution; The phosphoric acid solution was added to the triethylamine-treated mixture, and the reaction was continued for 5 h. After the reaction was complete, the resulting mixture was centrifuged at 8600 rpm for 6 minutes. The collected solids were washed twice with anhydrous ethanol and then redispersed in 15 mL of anhydrous ethanol to obtain a CPO dispersion with a concentration of 10 mg / mL.

5. The method for preparing the calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel according to claim 4, characterized in that: The amount of calcium chloride dihydrate used in S2 is 200 mg, the amount of triethylamine is 4 mL, and the amount of phosphoric acid is 70 μL.

6. The method for preparing the calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel according to claim 2, characterized in that: The S3 includes at least the following steps: The gel was incubated at 60 °C for 12 hours and then dissolved in 420 μL of deionized water to form a homogeneous gel solution. OSA was dissolved in 420 μL PBS under vortex mixing to obtain an OSA solution; The CPO dispersion was centrifuged three times with ultrapure water to remove 240 μL of supernatant and collect the CPO precipitate containing water. The CPO precipitate was then thoroughly mixed with the OSA solution, followed by the addition of a pre-prepared gel solution under vigorous stirring. The resulting mixture was then transferred to a mold and allowed to stand for 8 hours to form a hydrogel.

7. The application of calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogels, characterized in that: The calcium phosphate oligomer crosslinked biomimetic nanocomposite hydrogel described in claim 1 is used in bone tissue engineering scaffolds or bone defect repair materials.

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