流体静水压力对牙髓干细胞成牙/成骨分化的影响

Effect of hydrostatic pressure on odontogenic/osteogenic differentiation of dental pulp stem cells

背景:除了生物化学因素外,细胞力学被认为是影响干细胞行为和功能的关键因素。目的:探讨静水压力对人牙髓干细胞成牙/成骨分化潜能的影响。方法:通过酶消化法分离提取人牙髓干细胞,流式细胞仪检测细胞表面标志物的表达,成骨诱导培养14 d或成脂诱导培养21 d分别采用茜素红染色或油红O染色鉴定人牙髓干细胞的多向分化能力。利用自主研发的力学加载装置,对人牙髓干细胞加载不同的流体静水压(0,30,60,90,120,150 kPa),加压1 h,在第1,3,5,7天弃掉培养液,采用CCK-8法检测人牙髓干细胞的增殖活性;每天加压1 h并连续加压14 d,茜素红染色观察成骨矿化能力,Western blot法检测成牙/成骨分化相关基因的蛋白表达水平。结果与结论:牙髓干细胞高表达CD29、CD90、CD146、CD105,低表达CD45、CD34,具有成骨、成脂分化能力。流体静水压力在90,120 kPa时可以促进牙髓干细胞增殖,但150 kPa时明显抑制其增殖。流体静水压刺激14 d后,牙髓干细胞中牙本质涎磷蛋白、Ⅰ型胶原蛋白、骨钙素的蛋白表达量显著低于对照组(P<0.05),且随着静水压力值的增加,抑制其成牙/成骨分化的能力越明显。结果表明,流体静水压力参与牙髓干细胞成牙/成骨分化过程并具有负向调控作用。

BACKGROUND:Besides biochemical factors,cellular mechanics is increasingly recognized as the key factor affecting the behavior and function of stem cells.OBJECTIVE:To explore the effect of hydrostatic pressure on the odontogenic/osteogenic differentiation potential of human dental pulp stem cells.METHODS:Human dental pulp stem cells were isolated and extracted by enzyme digestion.The expression of cell surface markers was detected by flow cytometry.After osteogenic induction for 14 days or adipogenic induction for 21 days,alizarin red or oil red O staining was used to identify the mutidirectional differentiation of human dental pulp stem cells.The human dental pulp stem cells were loaded with hydrostatic pressure of different pressure values(0,30,60,90,120,and 150 kPa)by using the self-developed mechanical loading device,for 1 hour.At 1,3,5,and 7 days,the culture solution was discarded.CCK-8 assay was used to observe the proliferation of human dental pulp stem cells,1 hour per day,for 14 consecutive days.Alizarin red staining was applied to observe the osteogenic and mineralized abilities.Western blot assay was used to detect the protein expression levels of odontogenic/osteogenic differentiation related genes.RESULTS AND CONCLUSION:Dental pulp stem cells expressed high levels of CD29,CD90,CD146,CD105 and low levels of CD45 and CD34,and had the ability of osteogenic and adipogenic differentiation.Hydrostatic pressure could promote the proliferation of human dental pulp stem cells at 90 and 120 kPa,but the proliferation ability could be significantly inhibited when the pressure value was 150 kPa.After 14 days of hydrostatic pressure stimulation,the protein expression levels of dentin sialophosphoprotein,collagen type I,and osteocalcin in dental pulp stem cells were significantly lower than those in the control group(P<0.05),and their ability to inhibit differentiation became more obvious with the increase of hydrostatic pressure.The results show that hydrostatic pressure is involved in the process of dental pulp stem cell odontogenesis/osteogenesis and has a negative regulatory effect.