微地貌氟磷灰石-聚己内酯纳米纤维三维支架对牙周膜干细胞增殖、矿化及分化的影响

    Effect of micro-geomorphic fluorapatite-polycaprolactone nanofiber three-dimensional scaffolds on the proliferation, mineralization and differentiation of periodontal ligament stem cells

    • 摘要:
      目的: 分析微地貌氟磷灰石(FA)-聚己内酯(PCL)纳米纤维三维支架对牙周膜干细胞增殖、矿化及分化的影响。
      方法: 构建大鼠模型分为3组,其中阴性对照组植入胶原凝胶,观察组植入微地貌FA-PCL纳米纤维三维支架,空白对照组不做任何处理,对比牙周膜改建相关因子。取正畸拔除的年轻恒牙牙周组织,原代培养并分离筛选出牙周膜干细胞;分别接种于FA-PCL纳米纤维三维支架和PCL纳米纤维三维支架,再将细胞随机分为A ~ F组:A组,加入终质量浓度为10 ng/mL的牙龈卟啉单胞菌脂多糖(PG-LPS) + FA-PCL组;B组,加入10 ng/mL的PG-LPS + PCL组;C组,加入10 μg/mL的PG-LPS + FA-PCL组;D组,加入10 μg/mL的PG-LPS + PCL组;E组,纯FA-PCL组;F组,纯PCL组。观察细胞增殖、分化、矿化活性的变化。
      结果: 空白对照组、阴性对照组及观察组骨小梁数目(Tb.N)、组织矿物质密度(TMD)、骨体积分数(BV/TV)、骨矿物质密度(BMD)、骨小梁厚度(Tb.Th)、骨小梁分离度(Tb.Sp)水平差异有统计学意义,其中观察组Tb.N、TMD、BV/TV、BMD、Tb.Th水平最高,Tb.Sp水平最低(P < 0.05 ~ P < 0.01);空白对照组、阴性对照组及观察组大鼠组织HE染色病理学评分、白介素(IL)-6、IL-1b水平差异无统计学意义(P > 0.05);而观察组单核细胞趋化蛋白-1水平最低(P < 0.05)。 A组和B组细胞在24 h和48 h时增殖能力均高于其余各组(P < 0.05),但A组和B组间细胞增殖能力差异无统计学意义(P > 0.05);Runx2、Spp1级碱性磷酸酶表达水平E组细胞均最高,A组和C组均高于B、D及F组,A组细胞中各指标水平高于C组(P < 0.05 ~ P < 0.01)。采用Von Kossa及茜素红S对牙周膜干细胞进行处理后,可见E组细胞已大量合成并释放磷酸盐和钙盐,且着色严重程度明显高于其余各组;A、C组细胞的Von Kossa及茜素红S染色深度高于B、D组,且A组染色深度高于C组。
      结论: 微地貌FA-PCL纳米纤维三维支架可有效促进牙周膜干细胞增殖,改善牙周膜干细胞的矿化及分化。

       

      Abstract:
      Objective To analyze the effects of micro-geomorphic fluorapatite (FA)-polycaprolactone (PCL) nanofiber three-dimensional scaffolds on the proliferation, mineralization and differentiation of periodontal ligament stem cells.
      Methods The rat models were constructed and divided into three groups. The negative control group were implanted with collagen gel, the observation group were implanted with micro topography FA-PCL nanofiber three-dimensional scaffold, and the blank control group did not do any treatment. The periodontal ligament remodeling related factors were compared among three groups. Periodontal ligament stem cells were isolated from periodontal tissue of young permanent teeth extracted by orthodontic treatment. The cells were randomly divided the A-F groups. The A group were treated with 10 ng/ml final concentration of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) + FA-PCL. The B group were treated with 10 ng/ml final concentration of PG-LPS + PCL. The C group were treated with 10 μg/ml PG-LPS + FA-PCL. The D group were treated with 10 μg/ml PG-LPS + PCL. The E group were treated with FA-PCL. The F group were traeted with PCL. The changes of cell proliferation, differentiation and mineralization activity in A-F groups were observed.
      Results There were statistically significant in the number of trabeculae (TB.N), tissue mineral density (TMD), bone volume fraction (BV/TV), bone mineral density (BMD), trabecular thickness (TB.Th) and trabecular separation (TB.SP) among the blank control group, negative control group and observation group, while the levels of Tb.N TMD, BV/TV, BMD andTb.Th were the highest, and the Tb.SP was the lowest in the observation group (P < 0.05 ~ P < 0.01). There was no statistical significance in the HE staining pathological score, IL-6 and IL-1B levels among the blank control group, negative control group and observation group (P > 0.05), while the level of MCP-1 in the observation group was the lowes (P < 0.05). The cell proliferation ability of the A and B groups was higher than that of the other groups at 24h and 48h (P < 0.05), but there was no statistical significance in the cells proliferation between A group and B group (P > 0.05). The expression levels of Runx2 and SPP1 ALP in E group were the highest (P < 0.05), which in the A and C groups were higher that in B, D and F groups (P < 0.05), and the levels of various indicators in the A group were higher than those in C group (P < 0.05). After the periodontal ligament stem cells were treated with von Kossa and alizarin red S, it can be seen that the cells in E group had synthesized and released a large mount of phosphate and calcium salts, and the severity of staining in the E group was significantly higher than that in the other groups. The staining depth of von Kossa and alizarin red S in the A and C groups were higher than those in B and D groups, and which in A group was higher than that in C group.
      Conclusions The micro topography fluoroapatite polycaprolactone nanofiber three-dimensional scaffold can effectively promote the proliferation of periodontal ligament stem cells, and improve the mineralization and differentiation of periodontal ligament stem cells.

       

    /

    返回文章
    返回