3D打印还原型谷胱甘肽水凝胶支架促进糖尿病骨缺损修复的效应评价和机制研究

背景

   在临床工作中，糖尿病患者骨折或骨缺损后常可见骨愈合不良，即使植入常规骨替代材料后其骨愈合效果往往也并不理想。因此，研究适合糖尿病患者的骨修复材料具有非常重要的意义。

方法

   本文提出了一种具有改善糖尿病微环境的3D打印水凝胶支架，通过抗氧化应激和调节免疫促进糖尿病环境下骨修复。所制备的支架由光固化明胶甲基丙烯酰(GelMA)和还原型谷胱甘肽(GSH)组成。GSH的羧基与GelMA的胺基通过酰胺键结合形成GelMA-g-GSH。GelMA-g-GSH水凝胶随后通过3D打印技术形成结构稳定的三维互联网状结构支架，并对其相关表征，理化性质和生物相容性初步检测，确定其最优参数。体外通过模拟糖尿病微环境，将前成骨细胞(MC3T3-E1)与支架共培养，通过 ALP 活性测定、ALP和ARS染色方法观察其成骨矿化能力，并且 RT-PCR 和 Western blot 检测各组成骨相关基因和蛋白的表达情况。小管形成实验检测支架上HUVEC 的成血管能力。RT-PCR检测各支架上 HUVEC血管生成相关基因的表达。细胞免疫荧光和RT-PCR检测巨噬细胞RAW264.7的分型极化。RNA 转录组测序探究支架对成骨的影响及分子机制。最后，我们将支架植入糖尿病小鼠颅骨缺损体内，通过 Micro-CT 和组织学染色观察各组支架的骨缺损修复效果。

结果

   该复合水凝胶支架中GSH的持续缓释，可改善糖尿病微环境，促进糖尿病骨愈合。一方面通过GSH的催化反应降低ROS的过量产生，减少氧化应激。另一方面，GSH促进巨噬细胞向M2型极化，减轻炎症反应，维持体内正常的免疫系统功能。成骨诱导实验表明，GelMA-g-GSH支架明显促进 MC3T3-E1的成骨分化，成骨相关基因和蛋白表达上调。小管形成实验表明支架培养下的 HUVEC 可以形成数量较多和形态良好的小管网络，并且 RT-PCR检测成血管相关基因表达上调。细胞免疫荧光和RT-PCR结果表明GelMA-g-GSH支架促进巨噬细胞向M2型极化。RNA-seq 分析表示GelMA-g-GSH可能通过激活 PI3K/Akt 信号通路促进 MC3T3-E1成骨分化。体内糖尿病小鼠颅骨缺损修复8周后的影像学和组织学染色结果显示，GelMA-g-GSH支架组有更多的新生骨组织长入。

结论

   利用 3D 打印技术构建的还原型谷胱甘肽水凝胶支架，通过改善糖尿病微环境具有良好的成骨和血管生成性能，可以作为修复糖尿病骨缺损的骨移植物替代材料，具有潜在的临床应用前景。

Background

       Patients with diabetes mellitus suffer from a high risk of fractures and poor bone healing ability. Surprisingly, no effective therapy is available to treat diabetic bone defect in clinic. Bone healing effect is often not ideal even after the implantation of conventional bone replacement materials. Therefore, it is of great significance to prepare bone replacement materials suitable for diabetic patients.

Methods

       In this study, 3D printed hydrogel scaffold was proposed to promote bone regeneration in the diabetic environment by anti-oxidative stress and immune regulation. The prepared scaffolds consisted of light-curing gelatin methacryloyl (GelMA) and reduced glutathione (GSH). The carboxyl group of GSH was combined with the amino group of GelMA by an amide bond to form GelMA-g-GSH. Subsequently, the GelMA-g-GSH hydrogel was formed into a three-dimensional interconnected network structure scaffold with stable structure by 3D printing technology. The related characteristics, physical and chemical properties, and biocompatibility were preliminarily tested to determine its optimal parameters. Pre-osteoblasts (MC3T3-E1) were co-cultured with the scaffolds by simulating the diabetic microenvironment in vitro. The osteogenic mineralization ability of the osteoblasts was observed by ALP activity assay, ALP staining, and ARS staining. The expression of osteogenesis related genes and proteins of MC3T3-E1 was detected by RT-PCR and Western blot. Tube formation assay was used to detect the angiogenic ability of HUVEC on the scaffolds. The expression of angiogenesis related genes in HUVEC on each scaffold was detected by RT-PCR. Cell immunofluorescence and RT-PCR were used to detect the typing and polarization of RAW264.7 macrophages. RNA transcriptome sequencing was used to detect the effect and mechanism of scaffolds on osteogenesis. Finally, the scaffolds were implanted into the calvarial defects of diabetic mice, and the bone defect repair effects of the scaffolds in each group were observed by Micro-CT and histological staining.

Results

       The sustained release of GSH in the composite hydrogel scaffold can improve the diabetic microenvironment and promote diabetic bone healing. On the one hand, it reduced the excessive production of ROS and oxidative stress through the catalytic reaction of GSH. On the other hand, GSH promoted the polarization of macrophages to M2 type, reduced inflammatory response, and maintained normal immune system function. Osteogenic induction experiments showed that GelMA-g-GSH scaffolds significantly promoted the osteogenic differentiation of MC3T3-E1, and the expression of osteogenesis-related genes and proteins was up-regulated. Tube formation assay showed that HUVEC cultured on scaffolds could form a large number of tubules with good morphology, and the gene expression of angiogenesis was up-regulated as detected by RT-PCR. Cellular immunofluorescence and RT-PCR results showed that GelMA-g-GSH scaffolds promoted the polarization of RAW264.7 to M2 type. RNA-seq analysis showed that the activation of PI3K/Akt signaling pathway promoted MC3T3-E1 osteogenic differentiation. The Micro-CT and histological staining results of the bone defects in diabetic mice at 8 weeks after repair showed that more new bone tissue grew into the GelMA-g-GSH scaffold group.

Conclusion

       The reduced glutathione hydrogel scaffold constructed by 3D printing technology has good osteogenic and angiogenic properties by improving the diabetic microenvironment. It can be used as an alternative material for bone graft to repair diabetic bone defects and has potential clinical application prospects.

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