Alleviation of commercial collagen sponge- and membrane-induced apoptosis and dysfunction in cultured osteoblasts by an amino acid derivative.
Alleviation of commercial collagen sponge- and membrane-induced apoptosis and dysfunction in cultured osteoblasts by an amino acid derivative.
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2010-09
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本体外研究的目的是确定骨增量手术中使用的商业胶原材料是否诱导氧化应激介导的对成骨细胞活力和功能的不利影响，并确定N-乙酰半胱氨酸（NAC），一种抗氧化剂氨基酸衍生物，是否可以减轻这些影响。材料和方法用NAC处理商业胶原海绵（Collaplug）和膜（BioGide）。将大鼠颅骨来源的成骨细胞直接接种在这些材料上，有或没有NAC预处理。通过流式细胞术细胞活力测定、粘附细胞形态和活性氧（ROS）定位的激光共聚焦显微镜分析以及碱性磷酸酶染色进行细胞毒性评价。结果细胞接种后24小时在胶原海绵和膜上的存活率均低于40%，NAC预处理可使细胞存活率增加至50%。细胞死亡以凋亡为特征。在第1天，贴壁细胞在未处理的海绵和膜上的定殖是稀疏的，并且细胞是圆形的，小的，并且充满强烈且紧密堆积的细胞内ROS。相比之下，NAC预处理的材料具有致密的细胞集落，包括良好分散的成骨细胞和充分发育的细胞骨架和细胞过程，几乎没有ROS生成。在培养的第7天，NAC预处理的胶原海绵和膜产生了扩大的碱性磷酸酶阳性区域，分别占表面积的60%和80%，而未处理的胶原材料具有有限的碱性磷酸酶活性（7%或更低）。结论：商业胶原海绵和膜诱导相当多的细胞死亡，受损的初始功能，并在附着的成骨细胞中产生非凡的细胞内ROS，而NAC预处理大大改善了这些影响。NAC的解毒能力在动物模型中使用胶原基质材料对骨再生的潜在益处应通过进一步研究来证实。
PURPOSE The objectives of this in vitro study were to determine whether the commercial collagen material used in bone augmentation procedures induces oxidative stress-mediated adverse effects on the viability and function of osteoblasts and to determine whether N-acetyl cysteine (NAC), an antioxidant amino acid derivative, can alleviate these effects. MATERIALS AND METHODS Commercial collagen sponge (Collaplug) and membrane (BioGide) were treated with NAC. Rat calvaria-derived osteoblasts were directly seeded on these materials with or without NAC pretreatment. Cytotoxic evaluation was performed by flowcytometric cell viability assay, confocal laser microscopic analysis of attached cell morphology and reactive oxygen species (ROS) localization, and alkaline phosphatase staining. RESULTS Cell viability was less than 40% on both collagen sponge and membrane 24 hours after seeding and increased to 50% with NAC pretreatment. Cell death was characterized by apoptosis. Colonization of attached cells was sparse on the untreated sponge and membrane on day 1, and the cells were round, small, and filled with intense and closely packed intracellular ROS. In contrast, NAC-pretreated material had dense cell colonies consisting of well-spread osteoblasts and fully developing cytoskeleton and cellular processes with little ROS generation. On day 7 of culture, NAC-pretreated collagen sponge and membrane yielded an expanded alkaline phosphatase-positive area occupying 60% and 80% of the surface area, respectively, whereas the untreated collagen materials had limited alkaline phosphatase activity (7% or less). CONCLUSIONS Commercial collagen sponge and membrane induced considerable cell death, impaired initial function, and generated extraordinary intracellular ROS in attached osteoblasts, whereas NAC pretreatment substantially ameliorated these effects. The potential benefits of NAC's detoxifying capacity on bone regeneration using collagen matrix materials in an animal model should be confirmed with further study.