Dhaliwal Jagjit Singh, Marulanda Juliana, Li Jingjing, Alebrahim Sharifa, Feine Jocelyne Sheila, Murshed Monzur
Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
PAPRSB, Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE1410, Brunei Darussalam.
Int J Implant Dent. 2017 Dec;3(1):27. doi: 10.1186/s40729-017-0083-5. Epub 2017 Jun 27.
An ideal implant should have a surface that is conducive to osseointegration. In vitro cell culture studies using disks made of same materials and surface as of implants may provide useful information on the events occurring at the implant-tissue interface. In the current study, we tested the hypothesis that there is no difference in the proliferation and differentiation capacities of osteoblastic cells when cultured on titanium disks mimicking the surface of 3M™ESPE™ MDIs or standard (Ankylos®) implants.
Cells were grown on disks made of the same materials and with same surface texture as those of the original implants. Disks were sterilized and coated with 2% gelatin solution prior to the cell culture experiments. C2C12 pluripotent cells treated with 300 ng/ml bone morphogenetic protein 2 BMP-2 and a stably transfected C2C12 cell line expressing BMP2 were used as models for osteogenic cells. The Hoechst 33258-stained nuclei were counted to assay cell proliferation, while alkaline phosphatase (ALPL) immunostaining was performed to investigate osteogenic differentiation. MC3T3-E1 cells were cultured as model osteoblasts. The cells were differentiated and assayed for proliferation and metabolic activities by Hoechst 33258 staining and Alamar blue reduction assays, respectively. Additionally, cultures were stained by calcein to investigate their mineral deposition properties.
Electron microscopy showed greater degree of roughness on the MDI surfaces. Nuclear counting showed significantly higher number of C2C12 cells on the MDI surface. Although immunostaining detected higher number of ALPL-positive cells, it was not significant when normalized by cell numbers. The number of MC3T3-E1 cells was also higher on the MDI surface, and accordingly, these cultures showed higher Alamar blue reduction. Finally, calcein staining revealed that the MC3T3-E1 cells grown on MDI surfaces deposited more minerals.
Although both implant surfaces are conducive for osteoblastic cell attachment, proliferation, and extracellular matrix mineralization, cell proliferation is higher on MDI surfaces, which may in turn facilitate osseointegration via increased ECM mineralization.
理想的植入物应具有有利于骨整合的表面。使用与植入物相同材料和表面制成的圆盘进行体外细胞培养研究，可能会为植入物-组织界面发生的事件提供有用信息。在本研究中，我们检验了这样一个假设：当在模拟3M™ ESPE™ MDI或标准（Ankylos®）植入物表面的钛圆盘上培养时，成骨细胞的增殖和分化能力没有差异。
细胞在与原始植入物相同材料和表面纹理的圆盘上生长。在细胞培养实验之前，将圆盘灭菌并用2%明胶溶液包被。用300 ng/ml骨形态发生蛋白2（BMP-2）处理的C2C12多能细胞和表达BMP2的稳定转染C2C12细胞系用作成骨细胞模型。通过对Hoechst 33258染色的细胞核进行计数来测定细胞增殖，同时进行碱性磷酸酶（ALPL）免疫染色以研究成骨分化。将MC3T3-E1细胞作为成骨细胞模型进行培养。分别通过Hoechst 33258染色和Alamar蓝还原试验对细胞进行分化并测定其增殖和代谢活性。此外，用钙黄绿素对培养物进行染色以研究其矿物质沉积特性。
电子显微镜显示MDI表面的粗糙度更高。细胞核计数显示MDI表面的C2C12细胞数量显著更多。尽管免疫染色检测到ALPL阳性细胞数量更多，但按细胞数量进行标准化后并不显著。MDI表面的MC3T3-E1细胞数量也更多，因此，这些培养物显示出更高的Alamar蓝还原率。最后，钙黄绿素染色显示在MDI表面生长的MC3T3-E1细胞沉积了更多矿物质。
虽然两种植入物表面都有利于成骨细胞附着、增殖和细胞外基质矿化，但MDI表面的细胞增殖更高，这可能进而通过增加细胞外基质矿化促进骨整合。
