3D-Printed Vancomycin-Loaded ZIF-8@GMP Scaffolds with Sustained Antibacterial, Anti-Inflammatory, and Osteoinductive Properties for Bone Tissue Engineering

Abstract

Gelatin-based hydrogels have garnered significant attention in bone tissue engineering due to their excellent biocompatibility and ease of processing. However, their inherent limitations, such as poor mechanical strength and weak anti-inflammatory and antibacterial properties, restrict their broader application. To overcome these challenges, we developed a multifunctional 3D-printed scaffold by in situ growth of zeolitic imidazolate framework-8 (ZIF-8) nanoparticles within a creatine phosphate and methacrylic anhydride-modified gelatin matrix (GMP, methacrylation degree: 52.00%, creatine phosphate grafting rate: 17.89%), followed by vancomycin (VAN) loading. The resulting VAN/ZIF-8@GMP scaffolds exhibited prominent sustained drug release (78.89% VAN released at 96 h, vs 94.42% for direct blending scaffolds), enhanced antibacterial activity with 100% inhibition rate against both Escherichia coli (MIC = 32 μg/mL) and Staphylococcus aureus (MIC = 2 μg/mL), and improved mechanical properties (compressive strength: 192 kPa). The incorporation of ZIF-8 with pH-responsive Zn²⁺ release endows the scaffolds with anti-inflammatory effects (reducing protein denaturation by ∼26% at 72 h) and osteogenic stimulation. In vitro assays confirm their better antibacterial performance against Gram-positive bacteria, support MC3T3-E1 cell proliferation, and promote mineralization with significantly increased alkaline phosphatase (ALP) activity during 21 days of osteogenic induction. This multifunctional scaffold provides a promising strategy for infection prevention and bone regeneration in orthopedic applications.

1. Introduction

Gelatin has the advantages of good biocompatibility, wide sources, easy availability, and good plasticity, , and has played an indispensable role as a major bioactive substance in the application of medical materials in the past few decades. However, its disadvantages cannot be ignored, such as poor anti-inflammatory performance, potential bacterial infection problems, uncontrollable degradation rate, and poor mechanical strength, which limit the application of gelatin materials in the field of bone tissue engineering. Therefore, the development of 3D-printed gelatin bone tissue engineering scaffold materials with better antibacterial activity, anti-inflammatory performance, and osteoinductive performance is of great significance.

Li et al. constructed a dual-cross-linked system of bone tissue engineering scaffolds by cross-linking methacrylated gelatin and methacrylated gellan gum through photo-cross-linking and ionic cross-linking, significantly improving the mechanical properties of gelatin scaffolds. Montazerian et al. polymerized dopamine and conjugated it with methacrylated gelatin, and after dual-cross-linking treatment, the tensile strength and toughness of in situ polydopamine-modified methacrylated gelatin were increased by 5.7 times and 3.3 times, respectively, compared with GelMA control, further enhancing the mechanical properties of gelatin scaffolds. However, merely improving the mechanical properties is not sufficient for the clinical application of scaffolds. Therefore, we promote the coupling effect of osteogenesis and angiogenesis and prevent drug burst release. Cheng et al. encapsulated deferoxamine (DFO), a drug that induces the recruitment of osteogenesis-related cells and angiogenesis activity, in liposomes to effectively maintain the biological activity of DFO and control the local drug concentration. Xu et al. further improved the osteogenesis and angiogenesis performance of the scaffold by incorporating black phosphorus nanosheets (BP) and DFO into the methacrylated gelatin scaffold. BP can control the release of phosphate ions, and DFO as an iron chelator can promote angiogenesis by activating key angiogenic genes such as vascular endothelial growth factor (VEGF), significantly enhancing the osteoinductive ability and mechanical properties of the gelatin scaffold.

Metal–organic frameworks (MOFs) are coordination compounds formed by metals and organic ligands with 3D porous structures. The adaptability, high porosity, simple functionalization, and stimulus responsiveness of MOFs have promoted their potential applications in biomedicine, including bioimaging, anticancer, antibacterial, biosensing, and biocatalysis. ZIF-8 is a subclass of MOFs with high thermal and hydrothermal stability. In the field of MOFs, ZIF-8 nanoparticles are stable under physiological conditions and decompose under acidic conditions, possessing excellent intrinsic properties such as high drug loading capacity and simple synthesis methods, making it one of the most promising materials in the MOFs field. − The zinc ions produced after the decomposition of ZIF-8 have anti-inflammatory and antibacterial activities and also have the ability to stimulate bone formation and mineralization. Therefore, in situ growth of ZIF-8 in modified gelatin can endow the scaffold with good antibacterial ability, osteogenic ability, and mechanical properties.

Vancomycin is a tricyclic glycopeptide and is recognized as an excellent antibacterial drug due to its resistance to Gram-positive pathogens including Staphylococcus aureus. The incorporation of VAN into various drug delivery systems has antibacterial effects on the growth of Gram-positive bacteria. , Its toxicity is dose-dependent and has no adverse effects on osteogenic differentiation of stem cells. Hu et al. found that compared with natamycin and norfloxacin, VAN can better promote cell osteogenic differentiation and has a higher cell survival rate. Since directly loading drugs into the scaffold would cause a burst release of the drugs at the initial stage of degradation, and the inflammatory site of bone tissue requires that the 3D-printed bone tissue engineering scaffold has a long-term release performance of antibacterial substances to ensure that the postoperative trauma site is not infected during bone healing, this paper proposes to grow ZIF-8 in situ in gelatin material (GMP) modified by creatine phosphate and methacrylic anhydride and load VAN with ZIF-8 to prepare 3D-printed VAN/ZIF-8@GMP bone tissue engineering scaffolds with excellent drug sustained release performance, antibacterial and anti-inflammatory activity, mechanical properties, and osteogenesis promotion functions. Compared with vancomycin (VAN) alone, the VAN/ZIF-8@GMP scaffold exhibits comprehensive and superior performance in orthopedic infection prevention and bone regeneration with the following core advantages: First, VAN alone is prone to rapid burst release and short-term metabolism in vivo, failing to maintain effective antibacterial concentrations throughout the bone healing cycle. In contrast, the VAN/ZIF-8@GMP scaffold achieves controlled release through the encapsulation of VAN by ZIF-8 nanoparticles. More importantly, under acidic conditions (pH 5.37, mimicking inflammatory microenvironments), ZIF-8 decomposes rapidly to accelerate VAN release, enabling targeted antibacterial effects at infection sites; this ″on-demand release″ feature is unattainable with free VAN, which diffuses nonspecifically without environmental responsiveness. Second, VAN alone is primarily effective against Gram-positive bacteria (e.g., Staphylococcus aureus) but has limited inhibitory effects against Gram-negative bacteria (e.g., Escherichia coli). The VAN/ZIF-8@GMP scaffold integrates the antibacterial activity of VAN with the Zn²⁺released from ZIF-8 degradation: Zn²⁺not only enhances the antibacterial effect on Gram-positive bacteria but also significantly inhibits Gram-negative bacteria by disrupting bacterial membrane integrity. Third, Free VAN is a soluble drug without structural integrity, unable to provide mechanical stabilization for bone defects. The VAN/ZIF-8@GMP scaffold is sufficient to withstand the mechanical stress during bone regeneration. This structural-functional integration ensures the scaffold serves as both a drug delivery system and a bone regeneration templatean advantage that free VAN cannot match.

2. Experimental Section

2.1. Preparation of 3D-Printed Multifunctional VAN/ZIF-8@GMP Scaffold

5 g of gelatin was mixed with deionized water to make a 5% (w/v) gelatin solution at 40 °C. At a magnetic stirrer with a speed of 500 rpm, 3 mL of methacrylic anhydride (MA) was added to the above gelatin solution at a frequency of 0.5 mL/min. Seal the beaker with a transparent film and react for 2 h. The gelatin modified by methacrylic anhydride, namely, GelMA, was obtained. Dissolve 0.2 g of creatine phosphate in 50 mL of MES buffer solution with pH 5.0, and add 0.18 g of 1-ethyl-(3-(dimethylamino)­propyl) carbonyl diimide hydrochloride (EDC) and 0.10 g of N-hydroxysuccinimide (NHS). React at room temperature for 20 min until a uniform solution is formed. Then, slowly add the above-mentioned creatine phosphate solution drop by drop to GelMA, react for 24 h, change the water every 12 h, and undergo dialysis for 5 days. The dialyzed solution was freeze-dried at −20 °C to obtain creatine phosphate-modified methacrylate gelatin (GMP). The schematic diagram of the synthesis is shown in Figure A.

1.

(A) Schematic illustration for the synthesis procedure of creatine phosphate-modified methacrylate gelatin (GMP); (B) schematic illustration of the fabrication process of the VAN/ZIF-8@GMP scaffold: 2 g of GMP was dissolved in 4 mL of zinc nitrate solution with a concentration of 5 mg/mL at 40 °C, then 1 mg/mL vancomycin­(VAN), and 100 mg/mL 2-methylimidazole were added and react for 3 days, and the samples were 3D-printed to obtain the VAN/ZIF-8@GMP scaffolds.

Take 2 g of the GMP freeze-dried sample prepared by the above method and dissolve it in 4 mL of zinc nitrate solution with a concentration of 5 mg/mL at 40 °C. After stirring until the sample becomes a uniform solution, add 1% (w/v) of I-2959 photoinitiator and 1 mg/mL VAN, add 2-methylimidazole solid powder to make its concentration 100 mg/mL, and react for 3 days. The samples were printed as scaffolds at 35 °C using the Cellink 3D bioprinting device and then photo-cross-linked for 10 min under ultraviolet light to obtain the VAN/ZIF-8@GMP scaffold. The synthetic route is shown in Figure B.

2.2. Performance Test of Scaffolds

2.2.1. In Vitro Drug Release Behavior Experiment

The drug-loaded scaffold samples were respectively immersed in 10 mL of phosphate-buffered normal saline (PBS) with pH 7.41 and then placed in a 37 °C constant temperature water bath shaker with an oscillation speed of 100 rpm to simulate the normal tissue physiological environment in vivo. At a fixed time point, 500 μL of the release medium was removed from the release system. Meanwhile, an equal amount of PBS was replenished to the system from which the release medium was removed. The OD values at 280 nm of drug release at each time point were calculated by an enzyme-linked immunosorbent assay (ELISA) reader. The standard absorbance–concentration curve was established based on the standard PBS solution of the drug to determine the drug concentration released by the stent, and the in vitro drug release behavior of the drug-loaded stent was studied. Each group of stents was measured in parallel three times, and the average values were taken.

2.2.2. In Vitro Ion Release Behavior Experiment

To study the in vitro release behavior of zinc ions, the scaffold samples were immersed in 10 mL of PBS culture medium with pH 5.37 and pH = 7.41. They were shaken at 100 rpm for 3 days in a 37 °C constant temperature water bath shaker to simulate the inflammatory and normal tissue physiological environment in vivo. At specific time points (3, 6, 9, 12, 24, 48, 72 h, 120 h), 500 μL of the supernatant was extracted and the same volume of fresh PBS was added to maintain the original volume. The concentration of zinc ions in the supernatant was analyzed by an inductively coupled plasma atomic emission spectrometer, and the cumulative release percentage based on the amount of zinc ions added was calculated.

2.2.3. In Vitro Degradation Behavior

Before the experiment, the scaffold was fabricated, and its mass was measured and recorded as m ₁. The scaffold samples were respectively immersed in PBS with pH = 7.41, and then placed in a 37 °C constant temperature water bath shaker with an oscillation speed of 100 rpm to simulate the normal tissue physiological environment in vivo. At a fixed time point, the bracket sample was taken out, washed three times with deionized water, and dried in a vacuum drying oven for 24 h. Then, it was freeze-dried in a vacuum freeze-dryer until a constant weight was reached. The degradation curve of the scaffold with weight loss varying over time was plotted to explore the in vitro degradation behavior of the scaffold.

2.2.4. In Vitro Anti-Inflammatory Activity

The in vitro anti-inflammatory activity was studied by inhibiting albumin denaturation. The aqueous solution of 1% bovine serum albumin (BSA) was adjusted to pH 6.5 with glacial acetic acid. The 100 mg drug-loaded scaffold sample was suspended in 10 mL PBS, incubated at 37 °C, and the supernatant was collected for 24 and 48 h. Add 1 mL of the supernatant of the drug-loaded scaffold sample to 1 mL of BSA solution. Then, the solution was heated to 70 °C for 20 min, followed by cooling to room temperature. The turbidity of the solution was measured at 660 nm using a microplate reader. Each group of stents was measured in parallel three times, and their average values were taken. PBS solution (excluding stents) was used as the control group. The calculation formula for the percentage of protein denaturation inhibition is as follows

$$\mathrm{Inhibition\; percentage}=(A_{\mathrm{c}}-A_{\mathrm{s}})/A_{\mathrm{c}}\times 100\%$$

where A _c is the absorbance of BSA and A _s is the difference in absorbance between BSA and the scaffold sample.

2.2.5. In vitro Antibacterial Activity

The in vitro antibacterial activity of the scaffolds was evaluated by a plate counting method. The bacterial suspensions of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were uniformly dispersed in a sterile liquid medium, cocultured with the scaffold in a 37 °C constant temperature shaking box for 24 h, and further diluted. Taking the scaffolds without the drug as the blank experimental group, the bacterial concentration was approximately 10⁹ CFU mL^–1. Subsequently, 0.1 mL of the inoculated bacterial suspension was evenly spread on the bacterial culture dishes covered with nutrient agar and cultivated in a constant temperature shaking incubator at 37 °C for 24 h. The solid culture medium was photographed, the number of colonies was counted, and the antibacterial ratio was calculated. To determine the minimum inhibitory concentration (MIC), the micro dilution method was used to dilute the drug-loaded scaffold extract to a gradient concentration of 256–0.25 μg/mL, and the bacterial solution was diluted 100 times for later use. Add 100 μL of bacterial solution to each well, incubate at 37 °C for 24 h, and observe the minimum drug concentration without bacterial growth as MIC. The experiment used the extraction solution of the GMP scaffold as the blank control group.

2.2.6. In Vitro Biocompatibility Assessment

Mouse preosteoblasts (MC3T3-E1) were selected for the experiment. MC3T3-E1 cells were cultured in 10 mL of 89% basal medium (DMEM) containing 10% fetal bovine serum and 1% penicillin–streptomycin solution and then cultured in an incubator at 37 °C and 5% CO₂. Change the cell growth medium every 3 days.

When the cell density in the cell culture dish reaches 80%, the cells are digested with a 0.25% trypsin/EDTA solution, collected by centrifugation, and evenly distributed in 1 mL of the growth medium. Subsequently, the cells are resuspended in several cell culture dishes for subsequent cell proliferation experiments. The freeze-dried scaffolds (1.5 mm × 1.5 mm × 0.5 mm) were transferred to 96-well plates, disinfected under ultraviolet irradiation for 8 h, and then 100 μL of cell suspension containing 4 × 10³ cells was inoculated in each well. After culturing for 1, 2, 3, 5, and 7 days in a CO₂ incubator (5%, 37 °C), 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well. Under the same conditions, after incubation for 2 h, 100 μL of the solution from each well was transferred to a new 96-well plate, and the optical density (OD) value at 450 nm was detected using a microplate reader. The number of proliferating cells is proportional to the OD value. Each sample was tested three times, and the average value was calculated.

2.2.7. Live/Dead Cell Staining Assay

Fluorescence staining was performed using the live/dead cell staining assay (Calcein-AM/PI Double staining assay) to explore the cytocompatibility of the stents to be tested in each group. As a control, use a blank as the control group. The MC3T3-E1 cells were seeded on the scaffolds with a density of 1.0 × 10⁴ cells/well. After the cells were cultured for 1, 3, and 5 days, respectively, the culture medium was removed and the scaffolds containing the cells were gently rinsed with PBS three times. According to the assay instructions, 200 μL of live/dead cell staining solution was added to each scaffold sample. After incubation at room temperature for 30 min, fluorescence imaging of the cells on the scaffold was performed using an inverted fluorescence microscope. In fluorescence images, living cells and dead cells are stained green and red, respectively.

2.2.8. Osteogenic Performance Experiment

In order to determine the osteogenic performance of the scaffold, a certain amount of scaffolds were immersed in the osteogenic differentiation medium to prepare the sample extract. The cell growth medium in the 48-well culture plate containing MC3T3-E1 cells, which was cultured in a 37 °C, 5% CO₂ incubator for 24 h, was replaced with the osteogenic differentiation medium supplemented with ascorbic acid and β-glycerophosphate, and the sample extract was added. Change the culture medium every 3.5 days and add an in vitro extract solution of the sample.

2.2.9. ALP Staining and ALP Activity Detection

To evaluate the osteogenic differentiation of stent-induced MC3T3-E1 cells, ALP staining was performed using the BCIP/NBT alkaline phosphatase substrate kit 7 days after osteogenic induction. The steps are as follows: Add 20% fixative to each well to fix the cells for 30 min. After complete washing with DPBS, the cells were incubated with BCIP solution, NBT solution, and alkaline phosphatase chromogenic buffer at room temperature for 30 min. After being stained, the cells were observed with an inverted microscope (Nikon Eclipse TE2000-U, Japan). The supernatant was collected after 7, 14, and 21 days of osteogenic induction, and the concentration of ALP was detected using an alkaline phosphatase kit. Transfer the sample supernatant to a 96-well plate, and add 50 μL of p-nitrophenylphosphoric acid substrate solution (pNPP) to each well. After incubating in a 5%, 37 °C CO₂ incubator for 30 min, the reaction was terminated by adding the stop solution. The absorbance of the sample at 405 nm was detected by a microplate reader. Calculate the alkaline phosphatase activity of the sample according to the standard curve. Three parallel experiments were conducted for statistical analysis.

2.2.10. Calcium Deposition Experiment

The ability of the stents to induce the formation of mineralized nodules was evaluated by alizarin red S (ARS) staining. After coculturing MC3T3-E1 cells with scaffolds in bone-induced medium for 7 days, they were washed twice with PBS and fixed with 20% fixative for 20 min. Subsequently, the cells were stained with ARS solution for 30 min, and the mineralized nodules were observed under an inverted microscope.

2.2.11. Statistical Analysis

All data were expressed as mean ± standard deviation (SD). The results were analyzed using GraphPad Prism 9. One-way analysis of variance (ANOVA) with Tukey test was used to determine the statistical differences of the experimental data. Represented by *p value <0.05, **p value <0.01, ***p value <0.001, and ****p value <0.0001, ns indicates that there is no significant difference in the data.

2.3. Characterization

The surface morphology of the samples was observed using scanning electron microscopy (SEM, Regulus 8100). The functional groups of the samples were tested by Fourier transform infrared spectrometry (FTIR, Nicolet 5700). The structure of the sample was determined by a nuclear magnetic resonance spectrometer (Bruker 400 MHz). The crystal structure of the samples was analyzed by using the Philips X’Pert MPD X-ray powder diffractometer (XRD). The elemental composition of the sample was characterized by an X-ray energy spectrometer (ESCALAB 250) analysis method. The rheological properties were tested by a DHR-2 rheometer of TA Discovery Company. The porosity of the scaffolds was determined by the weighing method. The compressive mechanical properties of the samples were tested using the 5KPlus universal testing machine of LLOYD Company with a size of 10 × 10 × 5 mm under ambient conditions. The strain was set at 80%, the load was 1 KN, and the loading rate was controlled at 0.5 mm·min^–1. The maximum compressive strength and Young’s modulus of the samples were calculated based on the strain–stress curve.

3. Results and Discussion

3.1. Components and Rheological Properties of GMP

Figure A shows the ¹H NMR spectra of gelatin and GMP. The vinyl proton peaks introduced by methacrylic anhydride are indicated by blue shading (approximately at 5.42 and 5.66 ppm), and the lysine methylene signals are shown by purple shading. After grafting with creatine phosphate, the proton peak area of the glycine methylene in gelatin changes, as indicated by the pink shading, indicating the successful grafting of creatine phosphate.

2.

(A) ¹H NMR results for Gelatin and GMP to determine the grafting rates of methacrylic anhydride and creatine phosphate in GMP; (B) FTIR spectra of the GMP scaffold and Gelatin scaffold; (C) XRD patterns of ZIF-8@GMP and ZIF-8

The degree of methacrylation of gelatin was determined by the lysine methylene proton peak area: Degree of methacrylation = (1 – (lysine methylene area of GMP)/(lysine methylene area of gelatin)) × 100%. The grafting rate of creatine phosphate was determined by the following glycine methylene proton peak area: Grafting rate of creatine phosphate = (glycine methylene area of gelatin - glycine methylene area of GMP)/glycine methylene area of gelatin. The grafting rates of methacrylic anhydride and creatine phosphate in GMP were found to be 52.00% and 17.89%, respectively.

FTIR was used to further identify the chemical functional groups in the GMP scaffold and confirm the successful synthesis of GMP, as shown in Figure B. GMP exhibits characteristic peaks of the PO₄ ^3– group at 1032 cm^–1, 528 cm^–1, and 419 cm^–1 compared to Gel, confirming the successful grafting of creatine phosphate.

Figure C shows the XRD patterns of the prepared ZIF-8@GMP sample and the ZIF-8 sample. It can be clearly seen that the ZIF-8@GMP sample has sharp and significant diffraction peaks corresponding to the (311), (222), (321), (410), (422), (500), and (622) crystal planes of ZIF-8, respectively, indicating the successful preparation of the ZIF-8@GMP sample.

The rheological properties of GMP were experimentally investigated to study its printability. Figure A,C show the shear rate-viscosity curves of Gel and GMP, respectively. As shown in the figures, at 35 °C, the viscosities of both hydrogel materials decrease with increasing shear rate, showing a typical ″shear thinning″ phenomenon. Figure B,D show the effects of strain on the storage modulus (G′) and loss modulus (G″) of Gel and GMP ink at 35 °C, respectively. As shown in the figures, the G′ and G″ curves of GMP printing ink experience a plateau period as the strain increases, followed by a rapid decrease and intersection. Compared with Gel, GMP undergoes a gel–sol transition at a strain of 100%, and the G′ and G″ of the modified GMP significantly increase. The G′ and G″ curves also indicate that the GMP hydrogel has low viscosity under large shear forces, ensuring that the GMP printing ink can be successfully extruded through extrusion 3D printing, while the rapid increase in viscosity when the shear force decreases ensures the formability of the GMP printing ink after printing.

3.

Rheological characterization of the pre-UV (uncross-linked) inks. (A) Variation of Gel viscosity with shear rate at 35 °C. (B) Effect of strain on Gel storage modulus (G′) and loss modulus (G″). (C) Variation of GMP viscosity with shear rate at 35 °C. (D) Effect of strain on GMP storage modulus (G′) and loss modulus (G″)

3.2. Morphological Structure of the VAN/ZIF-8@GMP Scaffold

The macroscopic morphologies of the GMP, ZIF-8@GMP, and VAN/ZIF-8@GMP scaffolds prepared by low-temperature 3D printing are shown in Figure A–D. From the macroscopic morphology images, it can be observed that the scaffold’s grid-like structures are interconnected, with good shape fidelity and high printing resolution, and the printed scaffolds do not collapse due to gravity.

4.

Macroscopic morphology of modified gelatin scaffold: (A) GMP, (B) ZIF-8@GMP, (C) VAN/ZIF-8@GMP; (D) Cross-section image of VAN/ZIF-8@GMP. SEM images of the (E) GMP scaffold, (F) ZIF-8@GMP scaffold, and (G) VAN/ZIF-8@GMP scaffold; EDS elemental mapping images of the (H) ZIF-8@GMP scaffold and (I) VAN/ZIF-8@GMP scaffold.

An ideal bone tissue engineering scaffold should have a highly interconnected porous structure with interconnected pores similar to the natural extracellular matrix, which is more conducive to cell adhesion, proliferation, migration, growth, and the transport of nutrients and metabolic products. Therefore, the microstructure and internal fine structure of GMP scaffolds, ZIF-8@GMP scaffolds, and VAN/ZIF-8@GMP scaffolds were observed by scanning electron microscopy. As shown in Figure E–G, it can be seen that the pore structure inside the scaffolds gradually increases with the modification of the scaffolds. The ZIF-8 loaded in the modified scaffolds is uniformly distributed in the cross-section of the scaffolds. Subsequently, energy dispersive X-ray spectroscopy was performed on the two ZIF-8-loaded scaffolds, and the results are shown in Figure H,I. It can be concluded from the results that the ZIF-8@GMP scaffold contains C, N, and Zn elements, while the VAN/ZIF-8@GMP scaffold contains C, N, Zn, and Cl elements. Both ZIF-8-modified scaffolds contain Zn ions, and the VAN/ZIF-8@GMP scaffold also contains Cl ions from VAN. Moreover, both ions are uniformly distributed within the detection range, which indirectly indicates that ZIF-8 is uniformly distributed in the scaffold matrix.

3.3. Mechanical Properties of VAN/ZIF-8@GMP Scaffolds

The mechanical properties of Gel scaffolds, GMP scaffolds, ZIF-8@GMP scaffolds, and VAN/ZIF-8@GMP scaffolds were tested using a universal testing machine, with Gel scaffolds as the control group to verify the optimization of the mechanical properties of Gel scaffolds through the introduction of double bonds for photo-cross-linking and in situ growth of ZIF-8. As shown in Figure , the stress–strain curves of each sample group exhibit similar behavior, with stress gradually increasing as compression increases. After the first modification of Gel scaffolds with methacrylic anhydride and creatine phosphate, the compressive strength of GMP scaffolds increased by 12 kPa compared to that of Gel scaffolds. Subsequently, based on this GMP scaffold, further scaffold modification was carried out by incorporating metal–organic frameworks, i.e., in situ growth of ZIF-8. After the incorporation of ZIF-8, the mechanical properties of ZIF-8@GMP scaffolds and VAN/ZIF-8@GMP scaffolds were further improved compared to GMP scaffolds, with compressive strengths of 167 and 192 kPa, respectively. It can be seen that each modification step positively correlates with improvement of the mechanical properties of the scaffolds. The trend of Young’s modulus changes in the three modified scaffolds is consistent with the results of compressive strength.

5.

Mechanical properties of the Gel scaffold, GMP scaffold, ZIF-8@GMP scaffold, and VAN/ZIF-8@GMP scaffold. (A) Representative stress–strain curves. (B) Compressive strength of the scaffolds. (C) Young’s modulus of the scaffolds. All data represent the mean ± s.d. (n = 3) and statistical significance was assessed by one-way ANOVA with Tukey’s test. *p value <0.05, **p value <0.01, and ***p value <0.001.

3.4. In Vitro Release and Degradation Properties

To compare the effect of ZIF-8 metal–organic frameworks on the in vitro release performance of VAN, (V+G) scaffolds were prepared by directly blending VAN with hydrogels, and both (V+G) scaffolds and VAN/ZIF-8@GMP scaffolds (VZG) were immersed in 10 mL of PBS with pH = 7.41 to simulate the vancomycin release curve under normal physiological conditions of tissues in vivo. As shown in Figure A, it can be seen that both scaffolds have a relatively obvious burst release in the early 6 h of drug release, and then as time increases, the cumulative drug release curves show a relatively gentle sustained release state. By 96 h, (V+G) scaffolds have released 94.42% (cumulative release amount 7.56 mg/cm³), while VZG has released 78.89% (cumulative release amount 6.31 mg/cm³), and there is still a trend of continued sustained release. The early burst release of the drug is believed to be due to the release of VAN not encapsulated in the scaffolds in PBS, followed by the release of VAN encapsulated in ZIF-8 as the scaffolds degrade. Finally, the release of drugs encapsulated in ZIF-8 occurs through the degradation of ZIF-8. Therefore, VZG scaffolds have better sustained release effects than (V+G) scaffolds, proving the successful construction of the ZIF-8 sustained release drug delivery system.

6.

(A) Cumulative release profiles of VAN from the VAN + GMP blended scaffold and VAN/ZIF-8@GMP scaffold; (B) cumulative release profiles of Zn²⁺ from the VAN/ZIF-8@GMP scaffold; (C) in vitro degradation behavior of the GMP scaffold, ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold in PBS at 37 °C; (D) porosity of the scaffolds.

ZIF-8 nanoparticles are stable under physiological conditions but decompose under acidic conditions. The zinc ions produced after the decomposition of ZIF-8 have anti-inflammatory and antibacterial activities and also have the ability to stimulate new bone formation and mineralization. Therefore, if Zn²⁺ can be released slowly in the early stage of bone formation, it can greatly promote the ability of new bone formation without causing toxicity. As shown in Figure B, the scaffolds were respectively immersed in 10 mL of the PBS culture medium with pH = 5.37 and pH = 7.41 to simulate the ion release behavior in the inflammatory and normal tissue physiological environments in vivo. In the initial release period of the first 3 h, there was a significant burst release of Zn²⁺ in the VAN/ZIF-8@GMP scaffolds under both acidic and neutral conditions, which was attributed to the breaking of the coordination bond between Zn²⁺ and imidazole, leading to the decomposition of ZIF-8. However, in the initial release period, the ion release percentage of the VAN/ZIF-8@GMP scaffold under acidic conditions was much higher than that under neutral conditions, with a difference of approximately 52.56%. It can be seen that in the initial release period, almost all Zn²⁺ in the VAN/ZIF-8@GMP scaffold under acidic conditions was released; while under neutral conditions, the VAN/ZIF-8@GMP scaffold only released 49.47% (cumulative release amount 6.80 mg/cm³) at 12 h and continued to release until 120 h, with an ion release percentage of 73.39% (cumulative release amount 10.10 mg/cm³) at that time, indicating that there is still potential for continuous release and the release period can meet the requirements of the new bone formation period, achieving the purpose of stimulating new bone formation and mineralization. The release of VAN in the scaffold at pH 5.37 is shown in Figure A, and the release rate of VAN is significantly higher than that of pH 7.41, indicating that the decomposition of ZIF-8 under weak acidity releases the VAN encapsulated in it.

Figure C shows the in vitro degradation behavior curves of GMP scaffolds, ZIF-8@GMP scaffolds, and VAN/ZIF-8@GMP scaffolds in PBS at 37 °C. It can be seen that by the 28th day, the ZIF-8@GMP scaffolds and VAN/ZIF-8@GMP scaffolds were almost completely degraded, and the GMP scaffold was degraded to 85.79%, indicating that all three materials are excellent biodegradable materials with good degradation properties. By observation of the degradation curves of the scaffolds prepared from these three materials, it can be seen that the degradation rate of the VAN/ZIF-8@GMP scaffold is significantly higher than that of the other two scaffolds, with the GMP scaffold having the slowest degradation rate. The porosity test of the stent is shown in Figure D, VAN/ZIF-8@GMP. The highest porosity reached 80.6%, while GMP had the lowest porosity. This indicates that the incorporation of ZIF-8 increases the number of pores in the microstructure of the scaffold, thereby accelerating the degradation rate of the scaffold, which is consistent with the SEM results in Figure .

3.5. In Vitro Anti-Inflammatory Activity and Antibacterial Performance

Taking the GMP scaffold as the control group, the inhibitory effect on protein denaturation was studied on the ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold to evaluate the anti-inflammatory activity of the scaffolds. The results are shown in Figure A. It can be seen that the GMP scaffold has almost no inhibitory effect on protein denaturation, while the ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold have inhibition rates of 26.23% and 25.97% at 72 h, respectively, with almost the same inhibition rate. This indicates that Zn²⁺ in the modified scaffolds plays a role in inhibiting protein denaturation, proving the anti-inflammatory effect of Zn²⁺. At the same time, it can be seen that the anti-inflammatory activity of the ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold gradually increases with time, which is consistent with the trend of the cumulative release curve of Zn²⁺ in Figure B, indirectly proving that the anti-inflammatory effect increases with the increase in the cumulative release of Zn²⁺.

7.

(A) Anti-BSA denaturation studies of the GMP scaffold, ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold; (B) antibacterial activities of the GMP scaffold, ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold against E. coli and S. aureus. All data represent the mean ± s.d. (n = 3) and statistical significance was assessed by one-way ANOVA with Tukey’s test. *p value <0.05, **p value <0.01, ***p value <0.001, and ****p value <0.001.

Vancomycin (VAN) is a tricyclic glycopeptide and is recognized as an excellent antibacterial drug due to its dose-dependent toxicity and no adverse effects on the osteogenic differentiation of stem cells. The in vitro antibacterial activity of GMP scaffolds, ZIF-8@GMP scaffolds, and VAN/ZIF-8@GMP scaffolds was evaluated by the plate count method. Figure B shows the macroscopic and quantitative analysis images of the antibacterial effects of three types of scaffolds on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). From the macroscopic images, it can be observed that the GMP scaffold has almost no antibacterial effect and the number of bacteria in the macroscopic images is almost the same as that in the blank group. However, with the incorporation of ZIF-8, the number of bacteria in the culture dish significantly decreases, demonstrating that Zn²⁺ produced by the degradation of ZIF-8 has certain antibacterial properties. The addition of VAN further enhances the antibacterial effect of the modified scaffolds on E. coli and S. aureus. The quantitative analysis graph more intuitively presents the antibacterial effects of the three scaffolds, and the results are consistent with the macroscopic images. For E. coli, the antibacterial rate of the ZIF-8@GMP scaffold is 42.92%, and that of the VAN/ZIF-8@GMP scaffold is 100%. For S. aureus, the antibacterial rate of the ZIF-8@GMP scaffold is 67.37% and that of the VAN/ZIF-8@GMP scaffold is 100%. This indicates that the addition of VAN endows the scaffold material with better antibacterial performance. According to the dilution method, for S. aureus, the MIC value of the scaffold extract is 2ug/mL, and for E. coli, the MIC value of the scaffold extract is 32ug/mL.

3.6. In Vitro Biocompatibility and Osteogenic Performance

The biocompatibility of the modified scaffolds was evaluated through cytotoxicity and cell proliferation experiments. MC3T3-E1 cells were seeded on GMP scaffolds, ZIF-8@GMP scaffolds, and VAN/ZIF-8@GMP scaffolds for 1, 3, 5, and 7 days, and cell proliferation was assessed by using the CCK-8 assay. As shown in Figure A, cells on all three scaffolds showed a growth trend. With the extension of culture time, the number of cells in each group significantly increased and there were significant differences in cell density between each measurement point. This indicates that MC3T3-E1 cells can rapidly proliferate on each type of scaffold. Among them, the cell density on the GMP scaffold was higher than that on the other two scaffolds at 1, 3, and 5 days. At 7 days, there was no significant difference in cell density among the three modified scaffolds, indicating that all three modified scaffolds have good biocompatibility and no obvious cytotoxicity and can promote cell proliferation.

8.

(A) Proliferation of MC3T3-E1 cells on the GMP scaffold, ZIF-8@GMP scaffold, and VAN/ZIF-8@GMP scaffold after 1, 3, 5, and 7 days of culture in vitro; (B) The live­(green)/dead­(red) fluorescence images of MC3T3-E1 cell cocultured with the GMP scaffold, ZIF-8@GMP scaffold, and VAN/ZIF-8@GMP scaffold after 1, 3, and 5 days of culture in vitro. All data represent the mean ± s.d. (n = 3) and statistical significance was assessed by one-way ANOVA with Tukey’s test. *p value <0.05, **p value <0.01, ***p value <0.001, and ****p value <0.001.

Figure B shows the fluorescence microscopic images of cells on GMP scaffolds, ZIF-8@GMP scaffolds, and VAN/ZIF-8@GMP scaffolds after 1, 3, and 5 days of culture, stained with live/dead staining. The extensive green fluorescence coverage indicates that all of the cells in the samples have high viability. On the first day, all samples showed high cell viability, and most cells in all scaffolds remained round and did not cover the entire cell culture dish. After 3 days of culture, cells in all groups showed an elongated morphology and covered most of the cell culture dish. The cells on the GMP scaffold elongated more than those in the other groups, almost the same as that of the blank group. Notably, by the seventh day, almost all cells on the GMP scaffold, ZIF-8@GMP scaffold, and VAN/ZIF-8@GMP scaffold presented a slender morphology and covered almost the entire cell culture dish, indicating that all scaffolds have good cell compatibility and strong cell proliferation ability.

ALP staining, ALP activity, and alizarin red S staining were performed on MC3T3-E1 cells to evaluate the osteogenic performance of the scaffolds. The ALP activity of cells was observed after coculturing the scaffolds and MC3T3-E1 cells for 7, 14, and 21 days, as shown in Figure A. On the seventh day, the ALP activity of the ZIF-8@GMP scaffold with ZIF-8 incorporated and the VAN/ZIF-8@GMP scaffold was higher than that of the other groups, while the ALP activity of the GMP group was higher than that of the Gel group and the blank group, with significant differences. However, there was no significant difference between the ZIF-8@GMP scaffold and the VAN/ZIF-8@GMP scaffold groups. Moreover, as the osteogenic differentiation culture time increased, the ALP content gradually increased, and this trend was similar in all samples. This is consistent with the ALP staining results shown in Figure B.

9.

(A) Quantitative analysis of ALP activity after osteogenic induction for 7, 14, and 21 days; (B) ALP staining and (C) ARS staining of MC3T3-E1 cell cultures with extract-treated of the GMP scaffold, ZIF-8@GMP scaffold, and VAN/ZIF-8@GMP scaffold for 7 days. All scale bars are 100 μm.

The alizarin red S staining results of MC3T3-E1 cells on the scaffold samples on the 14th day are shown in Figure C. As can be seen from the figure, compared with other groups, the GMP scaffold, ZIF-8@GMP scaffold, and VAN/ZIF-8@GMP scaffold presented larger areas of deep red regions. Among these samples, the ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold presented the largest and most intense red regions. This indicates that the grafted modification of creatine phosphate on the Gel scaffold has a positive promoting effect on osteogenesis, which is attributed to the osteogenic effect of the phosphate group in creatine phosphate. Subsequently, after in situ growth of ZIF-8 on the GMP scaffold, the Zn2+ released from the decomposition of ZIF-8 played a role in early osteogenesis, up-regulating ALP expression and further promoting osteogenesis, thereby making the osteogenic effects of the ZIF-8@GMP scaffold and VAN/ZIF-8@GMP scaffold higher than that of the GMP scaffold. Therefore, creatine phosphate and Zn²⁺ synergistically promote the formation of new bone.

4. Conclusions

In this study, we successfully fabricated a multifunctional VAN/ZIF-8@GMP scaffold using low-temperature 3D printing technology. The scaffold demonstrated excellent printability, shape fidelity, and a well-interconnected porous structure suitable for bone tissue engineering. Incorporation of ZIF-8 and vancomycin endowed the scaffold with robust antibacterial and anti-inflammatory properties, as well as a pH-responsive sustained release capability. Moreover, the scaffold showed enhanced mechanical strength and promoted osteogenic differentiation of preosteoblasts, verified by ALP activity and calcium deposition assays. Collectively, the VAN/ZIF-8@GMP scaffold represents a promising candidate for bone defect repair, offering comprehensive functionality, including infection control, inflammation modulation, and bone regeneration support.

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J.Z., H.W., and Z.L. contributed equally. J.Z.: Methodology, writingoriginal draft preparation. H.W.: Conceptualization. Z.L.: Data curation. S.Z.: Methodology. H.Z.: Investigation. T.Z.: Validation. J.L.: Writingreviewing and editing. X.X.: Supervision

The authors declare no competing financial interest.