Experimental study on the promotion of bone defect healing by GelMa hydrogel loaded with Puerarin composite scaffold
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摘要:
目的 通过构建搭载葛根素的甲基丙烯酰化明胶(GelMa)复合水凝胶，制备一种新型的可以促进骨缺损愈合的生物材料。 方法 扫描电子显微镜及杨氏模量测定，分析复合支架的物理表征。构建梯度浓度的葛根素+GelMa支架(20、40、80和100 μmol/L)，通过CCK-8探究各支架在各时间点对细胞增殖的影响；ALP、茜素红染色探究各支架对于成骨分化的影响，选择最适的支架。构建颅骨缺损模型，采用完全随机设计，使用随机数表法将大鼠分为空白组、GelMa组、葛根素组、葛根素+GelMa组4组，每组3只大鼠，共12只。术后8周取材，对颅骨样本进行micro-CT、Western Blotting、HE染色、Masson ' s染色、ALP免疫组化染色以评估葛根素/GelMa支架体内对于骨缺损的作用。 结果 复合支架与单纯GelMa的孔隙率及孔径大小相似；杨氏模量显示复合支架[(3.30±0.30)MPa]与单纯GelMa[(3.22±0.25)MPa]比较差异无统计学意义(t=0.359，P=0.738)；CCK-8实验显示，在80 μmol/L时葛根素对于成骨细胞增殖能力的促进作用最强(P＜0.05)；ALP、茜素红染色显示，80 μmol/L组的阳性面积百分比最大(P＜0.05)。micro-CT、Western Blotting、HE染色、Masson's染色、ALP免疫组化表明，葛根素+GelMa组的新生骨组织长入最多，其对于骨缺损的修复能力显著高于空白组(P＜0.05)。 结论 葛根素浓度为80 μmol/L的葛根素+GelMa支架具有促进成骨细胞成骨分化的能力，可以在体内有效促进骨缺损愈合，可以作为一种骨缺损修复的新型生物材料。 Abstract:Objective To prepare a new kind of biomaterial that can promote the healing of bone defect by constructing the GelMa composite hydrogel loaded with Puerarin. Methods Scanning electron microscope and Young's modulus were used to analyze the physical characteristics of the composite scaffolds. Gradient concentrations of Puerarin/GelMa scaffolds (20, 40, 80, and 100 μmol/L) were constructed, and the effects of scaffolds on cell proliferation at different time points were investigated by CCK-8. ALP and alizarin red staining were used to investigate the effect of various stents on osteogenic differentiation and select the most suitable stents. A craniotomy defect model was successfully established. Employing a completely randomized design, rats were randomly allocated into four groups by means of a random number table. These four groups were designated as the blank group, the GelMa group, the puerarin group, and the puerarin + GelMa group. Each group consisted of three rats, resulting in a total of twelve rats for the study. Samples were collected 8 weeks after surgery. micro-CT, Western Blotting, HE staining, Masson's staining, and ALP immunohistochemical staining were performed on the skull samples to evaluate the effect of Puerarin+GelMa scaffolding on bone defects in vivo. Results The porosity and pore size of the composite scaffold were similar to that of GelMa. Young's modulus showed no significant difference between the composite scaffold [(3.30±0.30) MPa] and the single GelMa [(3.22±0.25) MPa, t=0.359, P=0.738]. CCK-8 experiment showed that Puerarin had the strongest promotion effect on osteoblast proliferation at 80 μmol/L (P < 0.05). ALP and alizarin red staining showed that the positive area percentage of the 80 μmol/L group was the largest (P < 0.05). micro-CT, Western Blotting, HE staining, Masson's staining, and ALP immunohistochemistry showed that the Puerarin+GelMa group had the most new bone tissue growth, and its repair ability for bone defects was significantly higher than that of the blank group (P < 0.05). Conclusion Puerarin+GelMa scaffold with a puerarin concentration of 80 μmol/L has the ability to promote osteogenic differentiation of osteoblasts, and can effectively promote bone defect healing in vivo, which can be used as a new biomaterial for bone defect repair. -
Key words:
- Bone defect /
- Puerarin /
- Hydrogel /
- Osteanagenesis
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图 1 扫描电镜观察水凝胶支架表征(×100)
注：A为1组；B为5组。
Figure 1. Characteristics of hydrogel scaffolds observed by scanning electron microscopy (×100)
图 2 CCK-8检测各组水凝胶对细胞活性的影响
注：与1组比较，aP＜0.05。
Figure 2. The effects of hydrogels in each group on cell viability detected by CCK-8
图 3 各组水凝胶对MC3T3-E1细胞体外成骨的影响
注：A为培养7 d后ALP染色结果(×10)；B为培养14 d后茜素红染色结果(×10)。
Figure 3. Effect of hydrogel on MC3T3-E1 cells in vitro osteogenesis
图 4 术后8周micro-CT扫描结果及Western Blotting检测结果
注：A为micro-CT图片；B为各组BV/TV值比较；与空白组比较，aP＜0.05；C为Western Blotting检测各组OCN、ALP的表达。
Figure 4. The micro-CT scan results and Western Blotting detection results 8 weeks after the operation
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