Abstract: BACKGROUND: Traditional topical administration of antibiotics for infected bone defects is limited by the selective proliferation of drug-resistant strains, burst drug release, and a lack of osteoinductive activity. Simple bone repair materials are ineffective in controlling the infection process. Therefore, the development of intelligent drug delivery systems with multiple biological functions has become a research hotspot in this field. 
OBJECTIVE: To construct a polyethylenimine/oxidized dextran dynamic cross-linked hydrogel-loaded quercetin composite system to achieve a temporally synergistic antibacterial and osteogenic effect and to investigate the efficacy of this composite system in treating infected bone defects.  
METHODS: (1) Oxidized dextran was prepared by sodium periodate oxidation, and quercetin nanocrystals were prepared by antisolvent precipitation. Quercetin nanocrystals were then added to a polyethylenimine solution and a Schiff base reaction was used to prepare a quercetin-loaded polyethylenimine/oxidized dextran hydrogel. The in vitro drug release from this hydrogel was characterized. (2) In vitro experiments: Rabbit bone marrow mesenchymal stem cells were seeded onto the surfaces of polyethyleneimine/oxidized dextran hydrogels and quercetin-loaded polyethyleneimine/oxidized dextran hydrogels, respectively. Cells cultured alone served as controls, and the cytocompatibility of the materials was assessed using CCK-8 assay and live-dead cell staining. After osteogenic induction, the osteogenic capacity of the materials was evaluated using alkaline phosphatase staining, Alizarin Red staining, and osteogenic gene detection. Staphylococcus aureus (or Escherichia coli, methicillin-resistant Staphylococcus aureus) was co-cultured with polyethyleneimine/oxidized dextran hydrogels and quercetin-loaded polyethyleneimine/oxidized dextran hydrogels, respectively. The bacteria cultured alone were used as the control. The antibacterial properties of the materials were evaluated by measuring the absorbance of the bacterial suspension and applying the bacterial suspension to a culture dish, respectively. (3) Animal experiment: SD rats were drilled below the greater trochanter of the left femur and bone marrow was aspirated. A 5% sodium morrhuate + Staphylococcus aureus suspension was injected into the bone marrow cavity to establish an infected femoral defect model. Four weeks after modeling, rats were randomly divided into three intervention groups: a control group (n=9) underwent only thorough debridement; a hydrogel group (n=9) and a quercetin-loaded hydrogel group (n=9) underwent thorough debridement followed by injection of a polyethyleneimine/oxidized dextran hydrogel or a quercetin-loaded polyethyleneimine/oxidized dextran hydrogel, respectively. Eight weeks after surgery, specimens were harvested for micro-CT scanning and histomorphological observation.
RESULTS AND CONCLUSION: (1) The polyethyleneimine/oxidized dextran hydrogel rapidly released the drug initially (within 3 days) and then released the drug slowly for up to 42 days. (2) CCK-8 assay and live-dead cell staining showed that the quercetin-loaded polyethyleneimine/oxidized dextran hydrogel promoted cell proliferation and exhibited good cytocompatibility compared with the polyethyleneimine/oxidized dextran hydrogel. Absorbance measurements of bacterial suspensions and culture dish coating experiments revealed that the polyethyleneimine/oxidized dextran hydrogel had no antibacterial properties, while the quercetin-loaded polyethyleneimine/oxidized dextran hydrogel significantly inhibited the growth and reproduction of Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus. Alkaline phosphatase staining, Alizarin red staining, and osteogenic gene analysis revealed that the polyethyleneimine/oxidized dextran hydrogel had no osteogenic activity, while the quercetin-loaded polyethyleneimine/oxidized dextran hydrogel exhibited excellent osteogenic activity. (3) Micro-CT scans revealed that the quercetin-loaded hydrogel group produced significantly more new bone tissue than the control and hydrogel groups. Hematoxylin-eosin and Masson staining revealed that the quercetin-loaded hydrogel group exhibited superior bone defect repair quality compared with the control and hydrogel groups. Giemsa staining revealed that numerous bacteria were observed in the control and hydrogel groups, while the quercetin-loaded hydrogel group was virtually devoid of bacteria. (4) The results showed that the quercetin-loaded polyethyleneimine/oxidized dextran hydrogel exhibited excellent antibacterial and osteogenic properties, promoting the repair of infected bone defects. 

Key words: infected bone defects, quercetin, hydrogel, drug delivery, antibacterial activity, osteogenic capacity, bone tissue engineering, sustained-release system