Thangakumaran S, Sudarsan Sabitha, Arun K V, Talwar Avaneendra, James Johnson R
Department of Periodontics and Implantology, Ragas Dental College and Hospital, Chennai, India.
Indian J Dent Res. 2009 Jan-Mar;20(1):7-12. doi: 10.4103/0970-9290.49048.
The enamel matrix derivative (EMD) has been used in combination with barrier membranes to optimize regeneration in vertical osseous defects. However, the osteoblast response when exposed to the EMD/barrier membrane combination has not yet been evaluated. The osteoblast behavior when exposed to a combination of regenerative materials must be evaluated to fully understand their effect on bone regeneration. Therefore, the present study was undertaken to estimate the initial adhesion and alkaline phosphatase (ALP) activity of an osteoblast cell line (SaOS-2) when exposed to four commercially available resorbable membranes and determine if the addition of EMD had any modulatory effect on osteoblast behavior.
5 x 104 SaOS-2 cells between passages 7-10 were cultured in two 24-well culture plates. Plate A was used for the adhesion assay and Plate B was used for the ALP assay. A MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay was done after 24 hours to determine the adhesion of the osteoblastic cells to four barrier membranes: 1) a non cross-linked porcine Type I and III collagen membrane (BG), 2) a weakly cross-linked Type I collagen membrane (HG), 3) a glutaraldehyde cross-linked bovine Type I collagen (BM), and 4) a resorbable polymer membrane (CP). Osteoblast differentiation was studied using an ALP assay with p-nitro phenyl phosphate as the substrate at 24 hours, 72 hours, and 1 week. A total of 50 microg/ml of EMD dissolved in 10 mM acetic acid was added into each well and the entire experimental protocol outlined above was repeated.
The osteoblast adhesion to collagen barriers showed a statistically insignificant reduction following the addition of EMD. Adhesion to the polymer barrier, although significantly lower when compared with collagen barriers, was unaffected by the addition of EMD. ALP activity after 1 week among the various groups was as follows: EMD alone (75.59+/-2.5)>EMD/BG(64.78+/-3.04)>EMD/HG(55.40+/-3.89) approximately EMD/BM(54.75+/-4.17)>BG (51.32+/-2.76)>HG(49.92+/-2.4)>BM(48.14+/-1.4)>Control(46.29+/-1.39)>EMD/CP (37.46+/-3.54)>CP(32.12+/-1.49)
There was no additive effect on osteoblast adhesion/ALP activity following exposure to an EMD/polymer combination. EMD/collagen positively influences osteoblast differentiation in a time dependent manner.
釉基质衍生物（EMD）已与屏障膜联合使用，以优化垂直骨缺损的再生。然而，暴露于EMD/屏障膜组合时成骨细胞的反应尚未得到评估。必须评估成骨细胞暴露于再生材料组合时的行为，以充分了解它们对骨再生的影响。因此，本研究旨在评估成骨细胞系（SaOS-2）暴露于四种市售可吸收膜时的初始黏附力和碱性磷酸酶（ALP）活性，并确定添加EMD是否对成骨细胞行为有任何调节作用。
将5×10⁴处于第7 - 10代的SaOS-2细胞接种于两个24孔培养板中培养。A板用于黏附试验，B板用于ALP试验。24小时后进行MTT（3-[4,5-二甲基噻唑-2]-2,5-二苯基四氮唑溴盐）试验，以确定成骨细胞与四种屏障膜的黏附情况：1）非交联猪I型和III型胶原膜（BG），2）弱交联I型胶原膜（HG），3）戊二醛交联牛I型胶原膜（BM），4）可吸收聚合物膜（CP）。在24小时、72小时和1周时，以对硝基苯磷酸为底物进行ALP试验，研究成骨细胞分化情况。将溶于10 mM乙酸的50 μg/ml EMD加入每个孔中，并重复上述整个实验方案。
添加EMD后，成骨细胞对胶原屏障的黏附力在统计学上有不显著的降低。与胶原屏障相比，对聚合物屏障的黏附力虽显著较低，但不受添加EMD的影响。1周后各实验组的ALP活性如下：单独EMD（75.59±2.5）>EMD/BG（64.78±3.04）>EMD/HG（55.40±3.89）≈EMD/BM（54.75±4.17）>BG（51.32±2.76）>HG（49.92±2.4）>BM（48.14±1.4）>对照组（46.29±1.39）>EMD/CP（37.46±3.54）>CP（32.12±1.49）
暴露于EMD/聚合物组合后，对成骨细胞黏附力/ALP活性没有相加作用。EMD/胶原以时间依赖性方式对成骨细胞分化有积极影响。
