What if a bone implant did not simply release a drug, but released two agents in sequence—first a low level of zinc, then quercetin—as inflammation changed over time? Clinicians continue to face challenges in repairing bone defects, especially because larger and more severe defects can require external intervention to facilitate bone regeneration and ensure overall health.

Autologous bone grafts remain the gold standard for treating large bone defects, but they are associated with donor site morbidity and graft size restrictions. Stem cells and biomaterials can instead be used to construct tissue-engineered scaffolds that mimic the structure of natural bone, providing an innovative approach to repairing bone defects.

The relationship between immune regulation and bone regeneration, especially the differentiation of bone marrow mesenchymal stem cells, is complex and has gained significant attention. Bone marrow mesenchymal stem cells play a crucial role in bone healing and can differentiate into osteoblasts, which are essential for bone formation.

Their differentiation and functionality are significantly influenced by the surrounding immune environment. Immune cells, particularly macrophages, modulate the behavior of bone marrow mesenchymal stem cells, and macrophages can assume predominantly M1, or pro-inflammatory, and M2, or anti-inflammatory, phenotypes.

M1 macrophages release Tumor Necrosis Factor-alpha and Interleukin-1 beta, which can impede bone marrow mesenchymal stem cell differentiation into bone-forming cells. Conversely, M2 macrophages encourage anti-inflammatory cytokine secretion through paracrine signaling, fostering a healing environment and steering bone marrow mesenchymal stem cells toward the osteogenic lineage.

Interleukin-10 is recognized as a crucial factor that diminishes inflammation and enhances osteogenic differentiation of bone marrow mesenchymal stem cells. The inflammatory microenvironment plays a dual role in bone healing: moderate inflammation supports healing, whereas excessive inflammation can delay or prevent it.

The NF-kappa B signaling pathway is a key regulator of the inflammatory response, and its activation is closely linked to the survival, proliferation and differentiation of bone cells. Quercetin has notable anti-inflammatory properties and may positively impact bone healing by regulating NF-kappa B activity.

The intended strategy was low anti-inflammatory activity at first, followed by quercetin release to produce strong anti-inflammatory effects and enable spatiotemporal regulation of the immune response. ZIF-8 has a large specific surface area, high porosity and stable degradation characteristics, while its porous design allows high drug-loading efficiency.

ZIF-8 is sensitive to pH changes, allowing it to release zinc ions responsively and exhibit anti-inflammatory effects. Zinc ions can be toxic at high concentrations, so maintaining a safe release concentration is crucial; GelMA hydrogels can stabilize zinc-ion release at lower concentrations.

The injectable hierarchical drug-delivery system integrated ZIF-8 and quercetin nanoparticles with GelMA hydrogel for immune modulation and bone regeneration in bone defect repair. The system enables sequential release of zinc ions and quercetin, targeting different stages of the bone immune response to achieve anti-inflammation and bone differentiation.

The experiments evaluated biocompatibility, anti-inflammatory properties and bone-regeneration efficacy in vitro and in vivo. Figure one presents the construction of the ZIF-eight/quercetin nanoparticles and their integration into a GelMA hydrogel, formed from gelatin modified with methacrylic anhydride.

After injection into a bone defect, the schematic depicts sequential release of zinc ions and quercetin, alongside changes in macrophage states, BMSC activity, and osteoblast formation. The visual matters because it links this hierarchical delivery design to immune-microenvironment regulation and bone repair.

Quercetin was embedded into ZIF-8 through one-step synthesis, using eight milligrams of quercetin while ensuring nanoparticle shape stability. Two-methylimidazole and zinc nitrate hexahydrate were dissolved in methanol, mixed, and combined with eight milligrams of quercetin before vigorous stirring for thirty minutes.

Yellow ZIF-8/Que nanoparticles were obtained by centrifugation at thirteen thousand revolutions per minute and washed three times with methanol; pure ZIF-8 was synthesized similarly without quercetin. ZIF-8/Que@GelMA was prepared by combining ZIF-8/Que nanoparticles with lyophilized GelMA.

Twenty-five milligrams of the photoinitiator LAP was dissolved in ten milliliters of PBS at fifty degrees Celsius, and zero point five grams of GelMA was added. A five percent GelMA concentration was chosen for the experiment. To simulate acidic inflammatory and neutral physiological conditions, ZIF-8/Que@GelMA was immersed in two milliliters of culture medium at pH six point zero and pH seven point four.

Samples were collected from one hour through sixty days, including one hour, three hours, six hours, twelve hours, one day, two days, three days, five days, seven days, ten days, fourteen days, thirty days, forty-five days and sixty days. Zinc ions were measured by inductively coupled plasma optical emission spectrometry, while quercetin was quantified using ultraviolet-visible absorption at three hundred seventy nanometers.

Figure three characterizes the ZIF-eight and quercetin-loaded GelMA hydrogel from formation through performance. The images show UV-crosslinking, interconnected porous structures, elemental maps confirming the composite, and adhesion and elasticity demonstrations.

Mechanical tests report Young’s modulus, swelling, and pressure–strain behavior, while the release curves show distinct zinc-ion and quercetin profiles at pH six and pH seven point four. Together, these measurements support the scaffold’s shape adaptability, tissue contact, mechanical resilience, and staged drug delivery.

UV crosslinking allowed GelMA hydrogels to be fabricated into various shapes, which is advantageous for repairing complex-shaped bone defects. The GelMA hydrogel had a porous sponge-like cross-section, while ZIF-8@GelMA and ZIF-8/Que@GelMA also had large pores and rough, convex particles corresponding to ZIF-8 nanoparticles.

ZIF-8/Que@GelMA had a higher water contact angle than GelMA alone, indicating decreased hydrophilicity, while adhesion ability remained unaffected. The zinc-ion and quercetin release curves demonstrated hierarchical release characteristics.

Initially, zinc ions showed low-concentration release from the hydrogel, whereas quercetin was almost entirely retained inside ZIF-8. As zinc-ion concentration increased, the ZIF-8/Que@GelMA metal framework began to dissolve, leading to significant quercetin release. In acidic solution at pH six, zinc ions and quercetin were released more rapidly from the ZIF-8/Que nanoparticles than in a neutral environment, demonstrating hierarchical, sustained and pH-responsive release.

Figure four evaluates how the hydrogels affect BMSC compatibility, attachment, proliferation, and migration. Live/dead images show few visibly dead cells across groups, while phalloidin staining shows attached cells with extended pseudopods; the ZIF-8 and quercetin composite displays a polygonal, well-spread morphology.

Scratch images and their quantification track closure from zero to twenty-four hours, and the CCK-8 chart reports proliferation over one to three days, providing complementary evidence for cellular response. Cell viability and proliferation of bone marrow mesenchymal stem cells were assessed with live-dead staining and the CCK-8 kit on days one, two and three.

Only a few dead cells were observed across all four groups. Bone marrow mesenchymal stem cell proliferation was highest in the ZIF-8@GelMA and ZIF-8/Que@GelMA groups, likely owing to low-concentration zinc-ion release. With quercetin release, the ZIF-8/Que@GelMA group showed greater proliferation ability than the ZIF-8@GelMA group.

Figure six links osteogenic differentiation with immune signaling in BMSCs. ALP and Alizarin Red staining, together with OCN and Runx2 fluorescence, are quantified across control, GelMA, ZIF-eight@GelMA, and ZIF-eight/Que@GelMA groups; the authors report the strongest mineralization and bone-formation markers for ZIF-eight/Que@GelMA.

In LPS-treated cells, the hydrogel lowers p65 and phosphorylated p65 while increasing IL-ten and Runx2, supporting an immunomodulatory mechanism alongside osteogenesis. Alkaline phosphatase staining and quantitative results indicated that ZIF-8/Que@GelMA produced the most substantial mineralization and bone formation among the four groups.

Alizarin Red Staining assessed mineralized bone visually, and its results aligned with alkaline phosphatase activity, validating the osteogenic effect of ZIF-8/Que@GelMA. Osteocalcin and Runx2 fluorescence intensities were most significant in the ZIF-8@GelMA and ZIF-8/Que@GelMA groups, attributed to sustained zinc-ion release.

The ZIF-8/Que@GelMA group had markedly higher fluorescence intensity than the ZIF-8@GelMA group, attributed to synergistic effects of zinc ions and quercetin. The animal procedures were approved by the Ethics Committee of Bengbu Medical University, and six-week-old male Sprague-Dawley rats were used.

The rats were divided into GelMA, ZIF-8@GelMA, ZIF-8/Que@GelMA and control groups without materials. An electric drill created a five-millimeter critical-size bone defect on both sides of the calvarium, after which hydrogel was injected and cured under ultraviolet light.

Rats were euthanized at four or eight weeks postoperatively, and harvested craniums were fixed for histological analysis. Figure seven combines micro-CT and histology to assess skull-defect repair across the control, GelMA, ZIF-eight@GelMA, and ZIF-eight/Que@GelMA groups.

The reconstructed images and bar charts report bone volume fraction, trabecular thickness, trabecular number, and bone mineral density, while H and E and Masson staining identify host bone, new bone, fibrous tissue, and vessels at four and eight weeks. Together, these measurements connect mineralized bone formation with tissue-level remodeling and show how the implanted hydrogels supported repair.

At eight weeks, micro-CT reconstruction showed that the ZIF-8/Que@GelMA group had the largest area of new bone formation within the skull defects. The control group showed only minimal new bone formation at the defect edges, and connectivity was not achieved.

ZIF-8@GelMA and ZIF-8/Que@GelMA produced the highest bone-volume-to-total-volume ratios, with similar trends for trabecular thickness, trabecular number and bone mineral density. ZIF-8/Que@GelMA had more pronounced advantages over ZIF-8@GelMA regarding bone-volume-to-total-volume and bone mineral density.

ZIF-8/Que@GelMA promoted increased bone mass and reconstruction of a high-quality, well-organized trabecular bone structure. Figure nine evaluates immune regulation in the bone defect after seven days. The fluorescence images and quantification show fewer CD86-positive M1 macrophages and more CD206-positive M2 macrophages with the ZIF-8-containing hydrogels, particularly when quercetin is included.

Immunohistochemistry similarly reports reduced IL-1 beta and TNF-alpha staining, while IL-10 staining is most intense for ZIF-8 with quercetin, supporting a shift toward a less inflammatory microenvironment. After seven days, the control group had the most pro-inflammatory M1 cells marked by CD86 and the fewest anti-inflammatory M2 cells marked by CD206.

The GelMA group reduced M1 cells compared with control, although no significant difference in M2-cell levels was observed. Both ZIF-8@GelMA and ZIF-8/Que@GelMA significantly reduced M1 cells, with no notable difference between the two treatment groups. The ZIF-8/Que@GelMA group had substantially more M2-positive cells than the other groups, indicating markedly enhanced M2 polarization after incorporating zinc ions and quercetin.

Immunofluorescence staining at four and eight weeks examined COL-1 and OCN in and outside the osteoblast cytoplasm, as well as Runx2 in the nucleus. COL-1 and Runx2 expression levels decreased from four to eight weeks in each group, suggesting that bone repair was entering a relatively mature phase.

The overall expression level of the late osteogenesis marker OCN increased from four to eight weeks. The bone immune response is a complex network involving multiple cell types and signaling pathways, but understanding of these critical connections remains preliminary.

Future research should more comprehensively and systematically explore how this system regulates interactions between immune cells and related signaling pathways. Although the animal model partially simulated the pathophysiological processes associated with bone defects, it differs from actual clinical scenarios.

Follow-up studies therefore need larger animal models that are more clinically relevant to assess treatment safety and efficacy accurately. The ZIF-8/Que@GelMA hydrogel combined sequential zinc and quercetin release with immune regulation and bone formation, while the authors caution that larger, more clinically relevant animal models are still needed.