Repair of infected bone defect with dual-ion time-sequenced release multifunctional hydrogels

doi:10.12307/2026.690

Abstract

Abstract: BACKGROUND: The development of time-sequenced functional biomaterials that can effectively inhibit bacteria early and promote bone regeneration later is key to solving the treatment challenges of infected bone defects.
OBJECTIVE: To construct a dual-ion time-sequenced release multifunctional hydrogel and evaluate its antibacterial and bone regeneration-promoting properties.
METHODS: (1) Strontium-containing mesoporous bioglass was synthesized using a surfactant template method, and sulfohyaluronic acid was prepared via metal-ligand coordination crosslinking. Sulfohyaluronic acid was dissolved in deionized water, and the strontium-containing mesoporous bioglass was added dropwise. After mixing thoroughly, silver nitrate solution was slowly added dropwise and stirred for 15 minutes to obtain a multifunctional hydrogel with a timed release of dual ions (denoted as SHA-Ag/SBG hydrogel). The micromorphology, sustained release of Sr2+ and Ag+, and swelling properties of the SHA-Ag/SBG hydrogel were characterized. (2) Staphylococcus aureus or Escherichia coli were co-cultured with SHA-Ag/SBG hydrogels, hyaluronic acid hydrogels, sulfo-hyaluronic acid hydrogels, and Ag+-loaded sulfo-hyaluronic acid hydrogels, respectively. Bacterial plating experiments were used to evaluate the antibacterial properties of the hydrogels, using bacteria cultured alone as controls. (3) Rat bone marrow mesenchymal stem cells were co-cultured with SHA-Ag/SBG hydrogels, hyaluronic acid hydrogels, sulfo-hyaluronic acid hydrogels, and Ag+-loaded sulfo-hyaluronic acid hydrogels, respectively. Cells cultured alone served as controls. After osteogenic induction, the cytocompatibility of the hydrogels was evaluated by live/dead staining, and the osteogenic properties of the hydrogels were evaluated by alkaline phosphatase staining, Alizarin red staining, and expression of osteogenesis-related genes. (4) Circular bone defects with a diameter of 2 mm penetrating the cortical bone were created on the medial side of the femoral condyle in 40 SD rats. The rats were randomly divided into four intervention groups. The normal control group (n=10) received no infection treatment. The model group (n=10) received a Staphylococcus aureus suspension injected into the bone defect to simulate infection. The control group (n=10) received a Staphylococcus aureus suspension injected into the bone defect followed by an Ag+-loaded sulfo-hyaluronic acid hydrogel. The experimental group (n=10) received a Staphylococcus aureus suspension injected into the bone defect followed by an SHA-Ag/SBG hydrogel. Samples were obtained 4 and 12 weeks after surgery and subjected to micro-CT, hematoxylin-eosin, Masson, and Giemsa staining, respectively.
RESULTS AND CONCLUSION: (1) SHA-Ag/SBG hydrogels exhibited a well-defined three-dimensional porous structure and excellent swelling properties. Ion release experiments showed that Ag+ was released rapidly in the early stages of the SHA-Ag/SBG hydrogels and gradually stabilized after 3 days. Sr2+ was released more slowly, beginning gradually after 7 days, demonstrating the dual-ion sequential release and antibacterial and bone regeneration-promoting properties. (2) SHA-Ag/SBG hydrogels and Ag+-loaded sulfo-hyaluronic acid hydrogels significantly inhibited the growth of Staphylococcus aureus and Escherichia coli, whereas hyaluronic acid hydrogels and sulfo-hyaluronic acid hydrogels showed no significant antibacterial effect. (3) Live/dead staining revealed no significant cytotoxicity in the four groups. Alkaline phosphatase and Alizarin red staining, as well as osteogenesis-related gene expression assay demonstrated that SHA-Ag/SBG hydrogels promoted the osteogenic differentiation of rat bone marrow mesenchymal stem cells. (4) Micro-CT examination showed that the SHA-Ag/SBG hydrogel effectively promoted bone regeneration in infected bone defects of rats compared with the other three groups. Hematoxylin-eosin staining, Masson staining, and Giemsa staining demonstrated that the SHA-Ag/SBG hydrogel exhibited excellent antibacterial activity and significantly promoted bone tissue recovery.

Key words: metal-ligand coordination crosslinking, hyaluronic acid, mesoporous bioactive glass, infected bone defect, antibacterial, bone regeneration, biomaterial

CLC Number:

Chen Weifei, Mei Yuandong, Ju Jihui. Repair of infected bone defect with dual-ion time-sequenced release multifunctional hydrogels[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(20): 5188-5200.

Figures/Tables 4

2.1 SHA-Ag/SBG水凝胶的制备与表征分析结果 2.1.1 含锶介孔生物玻璃的合成与表征分析根据表面模板剂法合成了含锶介孔生物玻璃，透射电镜下可见含锶介孔生物玻璃具有球形结构形态和介孔结构，粒径均一，无明显团聚现象，分散性良好(图1A)。Mapping图谱可见，硅、钙、磷和锶等元素均匀分布在微球内部(图1B)，其中Si元素占18.41%，P元素占2.12%，Ca元素占5.74%，Sr元素占3.28%。动态光散射分析显示含锶介孔生物玻璃粒径均在200 nm左右，有着良好的粒径分布(图1C)。 2.1.2 SHA-Ag与SHA-Ag/SBG水凝胶的合成与表征分析图1D显示了SHA-Ag/SBG水凝胶的制备过程，向混合均匀的硫代透明质酸溶液中加入Ag?，可见溶液逐渐从液态往固态的转变，加入含锶介孔生物玻璃后溶液由无色透明逐渐向白色转变。图1E显示了硫代透明质酸的核磁共振氢谱，在(2.6-2.8)×10-6处观察到-CH2-CH2-SH的特征峰，证明成功向透明质酸分子结构中引入了巯基(-SH)。图1F分别是 SHA-Ag、SHA-Ag/SBG水凝胶横截面的扫描电镜图像及局部放大图，两组水凝胶均具有多孔结构，显示出均匀互连的三维网状结构，其中SHA-Ag/SBG水凝胶可观察到含锶介孔生物玻璃的存在。 SHA-Ag/SBG水凝胶在10 h达到溶胀平衡后具有明显的体积变化，这有助于水凝胶在感染微环境下吸收渗出物，促进组织愈合(图1G)。图1H显示了SHA-Ag/SBG水凝胶中Sr2+、Ag+释放谱，结果显示：Ag+在早期释放速率快，Sr2+释放较Ag+释放缓慢，第7天，Ag+累计释放率达56.52%，而Sr2+累计释放率仅16.45%。从药物释放曲线可以看出，含锶介孔生物玻璃可以很好地作为离子储载库实现药物缓慢持续释放的目的，这种Ag+早期释放、Sr2+晚期释放能够很好地匹配感染性骨缺损早期抗菌、晚期成骨的需求问题。 "

2.4 SHA-Ag/SBG水凝胶促进大鼠股骨髁感染性缺损愈合能力评价结果 2.4.1 实验动物数量分析 40只SD大鼠全部进入结果分析。 2.4.2 Micro-CT检测模型组术后4，12周股骨髁骨缺损部位骨小梁最为稀疏，仅形成少量新生骨组织；实验组术后4，12周股骨髁骨缺损部位骨小梁最密集，在术后12周时形成了大量新生骨组织(图4A)。定量分析结果显示，与模型组比较，正常对照组、对照组、实验组股骨髁骨缺损部位骨密度、骨体积分数及骨小梁数量均升高，差异均有显著性意义；与正常对照组、对照组比较，实验组股骨髁骨缺损部位骨密度、骨体积分数及骨小梁数量均升高，差异均有显著性意义，见图4B。 2.4.3 Giemsa染色正常对照组骨缺损区未见细菌染色阳性信号，模型组缺损区可见大量深紫色细菌聚集，提示局部存在显著感染；对照组与实验组均显示细菌数量大幅减少，见图4C。结果表明，引入Ag?的SHA-Ag、SHA-Ag/SBG水凝胶均具有极强的抗菌能力。 2.4.4 苏木精-伊红和Masson染色苏木精-伊红和Masson染色显示，术后4周，正常对照组骨缺损区可见部分新骨形成，骨小梁数量较少，排列较为松散，同时伴有少量纤维组织填充，整体修复程度有限；模型组骨缺损区部位可见大量炎症细胞浸润，骨组织破坏明显，几乎无新骨形成，胶原沉积稀少；对照组骨缺损区域可见新生骨生成，骨小梁结构不规则，排列较松散，胶原纤维有一定程度沉积；实验组骨缺损区可见大量新骨充填，骨小梁粗大且排列规整，胶原纤维分布丰富且连续，见图4D。术后12周时，各组骨修复进一步进展，正常对照组骨缺损区新骨量略有增加，但仍未完全填充缺损区；模型组骨缺损区仍以纤维组织为主，骨组织修复基本缺失；对照组骨缺损区新生骨面积扩大，但缺损区仍存空隙，骨小梁分布不均；实验组骨缺损区表现出最显著的骨再生效果，缺损区域几乎被致密的新生骨组织完全覆盖，骨小梁增厚并呈连续结构，胶原沉积更加丰富，见图4E。结果表明，SHA-Ag/SBG水凝胶在抑制感染的同时显著促进了新骨形成，对感染性骨缺损具有良好的修复促进作用。 "

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