大鼠牙髓干细胞在微弧氧化钛表面诱导分化成骨的研究
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摘要
目的：
探讨大鼠牙髓干细胞(DPSCs)在微弧氧化(MAO)钛表面诱导分化成骨的能力,并通过与大鼠骨髓间充质细胞(BMSCs)成骨能力的比较,为DPSCs成为种植与组织工程联合应用的新型种子细胞提供理论依据。
方法：
1.大鼠DPSCs的分离,培养,鉴定及诱导分化：采用组织块酶消化法和有限稀释克隆化培养分离大鼠DPSCs,免疫荧光方法鉴定细胞表型。体外诱导DPSCs分别向成骨细胞和脂肪细胞分化。
2. DPSCs在MAO钛表面的生物活性研究：分为DPSCs-MAO组,DPSCs成骨诱导组及DPSCs无诱导组。扫描电镜(SEM)观察细胞生长情况,生物化学检测和实时定量PCR检测DPSCs的分化能力。
3.大鼠DPSCs与大鼠BMSCs在MAO钛表面的成骨能力比较：分为DPSCs-MAO组,BMSCs-MAO组及DPSCs-smooth组。SEM观察细胞生长形态,能谱分析仪(EDS)分析MAO钛表面元素变化,生物化学检测和实时定量PCR检测比较DPSCs与BMSCs的成骨分化能力差别。
结果：
1.大鼠DPSCs克隆化分离纯化培养后,细胞增殖较快,呈集落样生长,细胞形态为长梭型；免疫荧光结果显示大鼠DPSCs阳性表达CD44,STRO-1和Ⅰ型胶原,阴性表达CD34。大鼠DPSCs具有向成骨细胞和成脂肪细胞分化的能力。
2.SEM结果显示MAO钛片表面粗糙多孔,均匀分布着5-12μm的微孔,大鼠DPSCs在MAO钛片表面生长良好,可见细胞突起向MAO钛片表面的微孔中生长。DPSCs-MAO组ALP活力各时间点都比DPSCs无诱导组明显增强(P<0.05),DPSCs-MAO组与DPSCs成骨诱导组各时间点ALP活力相接近(P>0.05)。细胞培养第21d, DPSCs-MAO组与DPSCs无诱导组,DPSCs成骨诱导组与DPSCs无诱导组相比,Runx2、Osterix、DSPP和DMP-1表达增强,差别具有统计学意义(P<0.05); DPSCs-MAO组与DPSCs成骨诱导组基因表达相接近,差别无显著意义(P>0.05)。DPSCs-MAO组与DPSCs成骨诱导组钙结节茜素红染色阳性,钙结节面积差别无统计学意义(P>0.05), DPSCs无诱导组茜素红染色阴性表达。
3.MTT结果显示DPSCs-MAO组细胞活力比DPSCs-smooth组明显增强(P<0.05)。DPSCs-MAO组与BMSCs-MAO组细胞活力统计学分析差别无显著性意义(P>0.05)。细胞培养第7d, SEM显示,DPSCs-MAO与BMSCs-MAO细胞形态呈分化趋势,EDS结果显示,MAO钛表面钙离子含量升高,磷离子含量降低。DPSCs-MAO组各时间点ALP活力比DPSCs-smooth组明显增强,差别具有统计学意义(P<0.05), DPSCs-MAO组ALP活力与BMSCs-MAO组比较,除第5d及第13d有统计学意义外,其他时间点无显著性差异(P>0.05)。DPSCs-MAO组与BMSCs-MAO组钙结节茜素红染色阳性,钙结节面积差别无统计学意义(P>0.05)。DPSCs-MAO组与BMSCs-MAO组实时定量PCR检测Runx2, Osterix, DSPP和DMP-1基因表达趋势相接近,两组间差别不具有统计学意义(P>0.05)。
结论：
大鼠DPSCs在粗糙多孔,富含钙、磷元素的MAO钛表层有良好的生物相容性和生物活性,大鼠DPSCs的分化能力可能受到MAO钛的调节,大鼠DPSCs在MAO钛表面具有与大鼠BMSCs相似的成骨分化潜能。
Objectives:
To explore the osteogenic differentiation potential of rat dental pulp stem cells (DPSCs) on micro-arc oxidation (MAO) titanium surface, and compare osteogenesis with rat bone marrow stromal cells (BMSCs), to provide the theory basis for DPSCs being the new seed cells in implant and tissue engineering combined application.
Methods:
1. The separation, culturing, identification and inducing differentiation of rat DPSCs. DPSCs were cultured by explants combined with enzyme digestion technique. Single cell line of DPSCs was isolated by limited dilution of cultured cells and identified by immunofluorescence. Inducing differentiation of DPSCs to osteoblast and adipocyte.
2. The study of biological activity of DPSCs on MAO titanium surface. Groups are: group DPSCs- MAO, group DPSCs- bone condition induction and group DPSCs- no induction. At different time points during cells cultured, scanning electronic microscopy (SEM), biochemical tests and real-time PCR to observe differentiation potential of DPSCs.
3. The osteogenic differentiation potential comparison between DPSCs and BMSCs on MAO titanium surface. Groups are:group DPSCs- MAO, group BMSCs- MAO and DPSCs- smooth. At different time points during cells cultured, SEM, energy dispersive spectroscope (EDS), biochemical tests and real-time PCR to observe the difference osteogenic differentiation potential between DPSCs and BMSCs.
Results:
1. DPSCs showed a high proliferation rate and arranged closely to form colony, besides the cells expanded as spindle-shape. Immunofluorescence analysis showed that DPSCs expressed CD44, STRO-1 and collagen I while CD34 was absent. DPSCs had the ability to differentiate into osteoblasts and adipocytes.
2. SEM observation showed that the surface of MAO film was rough and porous, uniform distribution of 5-12μm microporous. And DPSCs were prone to grow toward micropore. ALP activity of DPSCs- MAO and DPSCs- bone condition induction were no significant difference (P>0.05), but there were significant difference with DPSCs-no induction (P<0.05). Cells cultured 21 days, the expression of Runx2, Osterix, DSPP and DMP-1 were increased between DPSCs- MAO and DPSCs- no induction, as the same between DPSCs- bone condition induction and DPSCs- no induction (P< 0.05); the expression of genes in DPSCs- MAO and DPSCs- bone condition induction were similar (P>0.05). Mineralization ability of DPSCs- MAO and DPSCs- bone condition induction, the calcium nodule area was no significant difference (P>0.05) and DPSCs- no induction was negative.
3. MTT results showed that the cell viability of DPSCs- MAO was significantly enhanced compared to the DPSCs- smooth (P<0.05). The cell viability of DPSCs- MAO and BMSCs- MAO was no significance difference (P>0.05). Cells cultured 7 days, SEM showed both cells expressed the trend to differentiation on DPSCs- MAO and BMSCs-MAO. EDS showed that Ca was raised and P was declined because of cells cultured. The results showed higher ALP activity in DPSCs- MAO than in DPSCs- smooth (P< 0.05). Compared DPSCs- MAO and BMSCs- MAO, there was no significance difference except at the 5th and 13th day (P>0.05). The alizarin red positive mineralized area in DPSCs- MAO and BMSCs- MAO were no significant difference (P>0.05). The RT- PCR results showed that the expression levels of Runx-2, Osterix, DSPP and DMP-1 were similar. The expression levels of genes showed no significant difference between DPSCs- MAO and BMSCs- MAO (P>0.05).
Conclusion:
Rat DPSCs have good biocompatibility and biological activity on MAO titanium which was rough, porous and rich in calcium and phosphorus. The DPSCs differentiation may be regulated by MAO titanium surface. The osteogenesis potential of DPSCs on the MAO titanium like the BMSCs did.
探讨大鼠牙髓干细胞(DPSCs)在微弧氧化(MAO)钛表面诱导分化成骨的能力,并通过与大鼠骨髓间充质细胞(BMSCs)成骨能力的比较,为DPSCs成为种植与组织工程联合应用的新型种子细胞提供理论依据。
方法：
1.大鼠DPSCs的分离,培养,鉴定及诱导分化：采用组织块酶消化法和有限稀释克隆化培养分离大鼠DPSCs,免疫荧光方法鉴定细胞表型。体外诱导DPSCs分别向成骨细胞和脂肪细胞分化。
2. DPSCs在MAO钛表面的生物活性研究：分为DPSCs-MAO组,DPSCs成骨诱导组及DPSCs无诱导组。扫描电镜(SEM)观察细胞生长情况,生物化学检测和实时定量PCR检测DPSCs的分化能力。
3.大鼠DPSCs与大鼠BMSCs在MAO钛表面的成骨能力比较：分为DPSCs-MAO组,BMSCs-MAO组及DPSCs-smooth组。SEM观察细胞生长形态,能谱分析仪(EDS)分析MAO钛表面元素变化,生物化学检测和实时定量PCR检测比较DPSCs与BMSCs的成骨分化能力差别。
结果：
1.大鼠DPSCs克隆化分离纯化培养后,细胞增殖较快,呈集落样生长,细胞形态为长梭型；免疫荧光结果显示大鼠DPSCs阳性表达CD44,STRO-1和Ⅰ型胶原,阴性表达CD34。大鼠DPSCs具有向成骨细胞和成脂肪细胞分化的能力。
2.SEM结果显示MAO钛片表面粗糙多孔,均匀分布着5-12μm的微孔,大鼠DPSCs在MAO钛片表面生长良好,可见细胞突起向MAO钛片表面的微孔中生长。DPSCs-MAO组ALP活力各时间点都比DPSCs无诱导组明显增强(P<0.05),DPSCs-MAO组与DPSCs成骨诱导组各时间点ALP活力相接近(P>0.05)。细胞培养第21d, DPSCs-MAO组与DPSCs无诱导组,DPSCs成骨诱导组与DPSCs无诱导组相比,Runx2、Osterix、DSPP和DMP-1表达增强,差别具有统计学意义(P<0.05); DPSCs-MAO组与DPSCs成骨诱导组基因表达相接近,差别无显著意义(P>0.05)。DPSCs-MAO组与DPSCs成骨诱导组钙结节茜素红染色阳性,钙结节面积差别无统计学意义(P>0.05), DPSCs无诱导组茜素红染色阴性表达。
3.MTT结果显示DPSCs-MAO组细胞活力比DPSCs-smooth组明显增强(P<0.05)。DPSCs-MAO组与BMSCs-MAO组细胞活力统计学分析差别无显著性意义(P>0.05)。细胞培养第7d, SEM显示,DPSCs-MAO与BMSCs-MAO细胞形态呈分化趋势,EDS结果显示,MAO钛表面钙离子含量升高,磷离子含量降低。DPSCs-MAO组各时间点ALP活力比DPSCs-smooth组明显增强,差别具有统计学意义(P<0.05), DPSCs-MAO组ALP活力与BMSCs-MAO组比较,除第5d及第13d有统计学意义外,其他时间点无显著性差异(P>0.05)。DPSCs-MAO组与BMSCs-MAO组钙结节茜素红染色阳性,钙结节面积差别无统计学意义(P>0.05)。DPSCs-MAO组与BMSCs-MAO组实时定量PCR检测Runx2, Osterix, DSPP和DMP-1基因表达趋势相接近,两组间差别不具有统计学意义(P>0.05)。
结论：
大鼠DPSCs在粗糙多孔,富含钙、磷元素的MAO钛表层有良好的生物相容性和生物活性,大鼠DPSCs的分化能力可能受到MAO钛的调节,大鼠DPSCs在MAO钛表面具有与大鼠BMSCs相似的成骨分化潜能。
Objectives:
To explore the osteogenic differentiation potential of rat dental pulp stem cells (DPSCs) on micro-arc oxidation (MAO) titanium surface, and compare osteogenesis with rat bone marrow stromal cells (BMSCs), to provide the theory basis for DPSCs being the new seed cells in implant and tissue engineering combined application.
Methods:
1. The separation, culturing, identification and inducing differentiation of rat DPSCs. DPSCs were cultured by explants combined with enzyme digestion technique. Single cell line of DPSCs was isolated by limited dilution of cultured cells and identified by immunofluorescence. Inducing differentiation of DPSCs to osteoblast and adipocyte.
2. The study of biological activity of DPSCs on MAO titanium surface. Groups are: group DPSCs- MAO, group DPSCs- bone condition induction and group DPSCs- no induction. At different time points during cells cultured, scanning electronic microscopy (SEM), biochemical tests and real-time PCR to observe differentiation potential of DPSCs.
3. The osteogenic differentiation potential comparison between DPSCs and BMSCs on MAO titanium surface. Groups are:group DPSCs- MAO, group BMSCs- MAO and DPSCs- smooth. At different time points during cells cultured, SEM, energy dispersive spectroscope (EDS), biochemical tests and real-time PCR to observe the difference osteogenic differentiation potential between DPSCs and BMSCs.
Results:
1. DPSCs showed a high proliferation rate and arranged closely to form colony, besides the cells expanded as spindle-shape. Immunofluorescence analysis showed that DPSCs expressed CD44, STRO-1 and collagen I while CD34 was absent. DPSCs had the ability to differentiate into osteoblasts and adipocytes.
2. SEM observation showed that the surface of MAO film was rough and porous, uniform distribution of 5-12μm microporous. And DPSCs were prone to grow toward micropore. ALP activity of DPSCs- MAO and DPSCs- bone condition induction were no significant difference (P>0.05), but there were significant difference with DPSCs-no induction (P<0.05). Cells cultured 21 days, the expression of Runx2, Osterix, DSPP and DMP-1 were increased between DPSCs- MAO and DPSCs- no induction, as the same between DPSCs- bone condition induction and DPSCs- no induction (P< 0.05); the expression of genes in DPSCs- MAO and DPSCs- bone condition induction were similar (P>0.05). Mineralization ability of DPSCs- MAO and DPSCs- bone condition induction, the calcium nodule area was no significant difference (P>0.05) and DPSCs- no induction was negative.
3. MTT results showed that the cell viability of DPSCs- MAO was significantly enhanced compared to the DPSCs- smooth (P<0.05). The cell viability of DPSCs- MAO and BMSCs- MAO was no significance difference (P>0.05). Cells cultured 7 days, SEM showed both cells expressed the trend to differentiation on DPSCs- MAO and BMSCs-MAO. EDS showed that Ca was raised and P was declined because of cells cultured. The results showed higher ALP activity in DPSCs- MAO than in DPSCs- smooth (P< 0.05). Compared DPSCs- MAO and BMSCs- MAO, there was no significance difference except at the 5th and 13th day (P>0.05). The alizarin red positive mineralized area in DPSCs- MAO and BMSCs- MAO were no significant difference (P>0.05). The RT- PCR results showed that the expression levels of Runx-2, Osterix, DSPP and DMP-1 were similar. The expression levels of genes showed no significant difference between DPSCs- MAO and BMSCs- MAO (P>0.05).
Conclusion:
Rat DPSCs have good biocompatibility and biological activity on MAO titanium which was rough, porous and rich in calcium and phosphorus. The DPSCs differentiation may be regulated by MAO titanium surface. The osteogenesis potential of DPSCs on the MAO titanium like the BMSCs did.
引文
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