Int J Oral Maxillofac Implants. 2020 Mar/Apr;35(2):320-329. doi: 10.11607/jomi.7606.
The aim of this study was to compare how two innovative laser titanium surfaces and sandblasted and acid-etched surfaces influence human osteoblast behavior during osteogenesis and the initial phases of bone deposition.
Human osteoblasts from human adipose stem cells were sorted by flow cytometric analysis and induced to differentiate. After 40 days, the osteogenic differentiation was detected by alizarin red staining, and the alkaline phosphatase (ALP) was evaluated with western blot (WB) and real-time reverse transcriptase-polymerase chain reaction (RT-PcR) analysis. After confluence, human osteoblasts were cultured onto two different innovative laser-obtained titanium surfaces (L1 and L2) and compared with one sandblasted and acid-etched (SBAE) surface as the control. At different times, human osteoblast behavior was evaluated with cell proliferation viability assay (MTT), scanning electron microscopy (SEM), energy-dispersive x-rays (EDAX), osteogenic markers with RT-PcR, and WB analysis of matrix extracellular phosphoglycoprotein (MEPE), ALP, and osteocalcin (OCN).
After cell sorting, the human osteoblasts from human adipose stem cells showed increasing values of ALP mRNA and protein expression. The osteogenic differentiation was confirmed by quantitative alizarin red staining assay. Profilometric and SEM analysis showed relevant differences between SBAE, L1, and L2 specimens. After 20 days of culture onto titanium samples, SEM evaluation showed a small number of human osteoblasts and isolated sites of bone matrix deposition in SBAE specimens. At the same time, on L1 surfaces, only an osteoblast mono-layer with initial bone deposition was found, while on L2 specimens, there was a thick network with flattened large stellate cells, many cellular interconnections with strong titanium adhesion, and a large complex mineralized structure of crystal bone. After 20 days, for all titanium samples, human osteoblasts culturing EDAX analysis showed the absence of impurities and a higher bone matrix deposition in L2 specimens compared with L1 and SBAE samples.
The innovative microtopography and nanotopography laser-induced surface showed high biocompatibility with primary human osteoblast cultures and the absence of impurities. The innovative laser texture was capable of influencing the osteogenic process, confirming the critical role of titanium surface characteristics in the cell adhesion and bone deposition during the early phases of osseointegration. The association of human adipose stem cells and titanium surfaces laser-induced with an innovative procedure could generate promising improvements and developments in orthopedics, maxillofacial, and dental implant surgery.
本研究旨在比较两种创新的激光钛表面与喷砂酸蚀表面在成骨和初始骨沉积阶段对人成骨细胞行为的影响。
通过流式细胞术分析对人脂肪干细胞来源的人成骨细胞进行分选，并诱导其分化。40 天后，通过茜素红染色检测成骨分化，通过 Western blot（WB）和实时逆转录聚合酶链反应（RT-PCR）分析评估碱性磷酸酶（ALP）。当细胞达到汇合状态后，将人成骨细胞培养在两种不同的创新激光获得的钛表面（L1 和 L2）上，并与喷砂酸蚀（SBAE）表面作为对照进行比较。在不同时间点，通过细胞增殖活力测定（MTT）、扫描电子显微镜（SEM）、能谱分析（EDAX）、RT-PCR 分析成骨标志物以及 WB 分析基质细胞外磷糖蛋白（MEPE）、ALP 和骨钙素（OCN）来评估人成骨细胞的行为。
细胞分选后，人脂肪干细胞来源的人成骨细胞的 ALP mRNA 和蛋白表达水平逐渐增加。通过定量茜素红染色测定证实了成骨分化。轮廓测定和 SEM 分析显示 SBAE、L1 和 L2 标本之间存在显著差异。培养 20 天后，SEM 评价显示 SBAE 标本中仅有少量人成骨细胞和孤立的骨基质沉积部位。与此同时，在 L1 表面仅发现单层成骨细胞和初始骨沉积，而在 L2 标本上则发现了厚的网络，其中有扁平的大型星状细胞、许多细胞连接和强烈的钛附着，以及复杂的结晶骨矿化结构。培养 20 天后，对于所有钛样本，人成骨细胞的 EDAX 分析显示 L2 样本中没有杂质，并且骨基质沉积更高，而 L1 和 SBAE 样本则没有。
创新的微形貌和纳米形貌激光诱导表面与人原代成骨细胞培养具有高度的生物相容性，且无杂质。创新的激光纹理能够影响成骨过程，证实了钛表面特性在早期骨整合阶段细胞黏附和骨沉积中的关键作用。人脂肪干细胞与钛表面激光诱导的联合应用可能会在矫形、颌面和牙科植入手术方面带来有前景的改进和发展。
