The Mechanisms Of Implant Micro/nano-textured Topography Effects On Osteoblasts Behaviors

Micro/nano-textured topographies (MNTs) combined with nanotubes and micropittedtopography can provide more abundant topographical cues on both micro and nanoscalesimilar to natural bone extracellular matrix and may exhibit more pronounced effects onosteoblasts biofunctions. However, the mechanisms mediating the response of cells totopographical cues are largely unknown. In our study, we fabricated the hierarchicalmicro/nano-textured topography with etching and subsequent anodization methods. TheMG63cells were seeded on the samples and osteoblasts multiple biofunctions wereassessed. Furthermore, the intracellular signaling pathways were studied using molecularbiology methods. Our study serves to provide precise understanding of biological effect oftopographical cues and better optimize implants surface modification systematically.Part IFabrication micro/nano-textured topography on titanium surface [Objective] To establish the hierarchical micro/nano-textured topography (MNTs)according to the former established protocols of our lab, and provide sample model forfollowing experiments.[Methods] Pure titanium was used as the substrate. After polishing, the samples wereetched with hydrofluoric acid for30min and anodized with a DC power supply at5and20V for1h to fabricate the MNTs. The morphology of the samples was inspected byfield-emission scanning electron microscopy.[Results] The titanium surface after etching and anodization exhibited a micro/nano-textured topography combined micropitted with nanotubes. The average diameter of thenanotubes was about30and100nm respectively.[Conclusion] The hierarchical micro/nano-textured topography can be formed withetching and anodization methods. Part IIThe effects of micro/nano-textured topography on osteoblasts behaviors[Objectives] To examine the influence of the micro/nano-textured topography (MNTs) onosteoblasts behaviors.[Methods] MG63cells were seeded on the samples, and the cell morphology wasobserved with scanning electron microscopy. Cell proliferation was assessed with MTTassay. Osteogenesis-related genes were examined by florescent real-time quantitativeRT-PCR. Collagen production was assayed by sirius red staining and extracellular matrix(ECM) mineralization was assessed with alizarin red staining.[Results] MG63cells spread well on the samples and exhibit longer cell morphology onlarger nanotubular diameter. The proliferation rate was not obviously affected by MNTs. The MNTs significantly enhanced the osteogenesis-related genes expression, collagensecretion ability and ECM mineralization. The promoting effects of osteoblastsdifferentiation were more evident on the MNTs with100nm diameter nanotubes.[Conclusion] The MNTs promoted the osteoblasts differentiation and the promotingeffects were more obvious on the100nm diameter nanotubes. Part IIIThe role of the Wnt/-catenin pathway in the effect ofmicro/nano-textured topography on MG63behaviors[Objective] To assess the role of the Wnt/-catenin signaling cascade in the response ofosteoblasts to the implant micro/nano-textured topography.[Methods] MG63cells were cultured on the MNTs and the transcriptional expressions ofWnt/-catenin pathway receptors, activators, and inhibitors are measured by florescentreal-time quantitative RT-PCR.-catenin signaling activity, cell proliferation, apoptosisrate, cell differentiation including osteogenesis-related gene expression, alkalinephosphatase expression and collagen secretion ability are studied in the absence andpresence of exogenous Dickkopf1(Dkk1) on the MNTs and exogenous Wnt3a on thesmooth surface.[Results] The expressions of the Wnt/-catenin pathway receptor low density lipoproteinreceptor-related protein6and pathway ligand Wnt3a are up-regulated by the MNTswhereas those of the pathway inhibitors including Dkk1/2and secreted frizzled-relatedprotein1/2are down-regulated by the MNTs, indicating regulation of the Wnt/-cateninpathway modulators to activate the pathway. Consequently, the-catenin signalingactivity is enhanced by the MNTs as well as cell differentiation in terms of osteogenesis-related gene expressions and alkaline phosphatase and collagen products. Onthe smooth surface, exogenous Wnt3a stimulates-catenin signaling and celldifferentiation while exogenous Dkk1attenuates the enhancement by the MNTs.[Conclusion] The results demonstrate that the implant topography regulates the product ofWnt/-catenin pathway modulators form the cells and in turn activated the cellWnt/-catenin pathway promoting osteoblast differentiation. PartIVThe role of integrin-linked kinase/-catenin pathway in theenhanced MG63differentiation by micro/nano-textured topography[Objective] To confirm the role of integrin linked kinase (ILK)/-catenin pathway inmediating the signals of topographical cues to osteoblasts and effects of the pathway onosteoblasts differentiation.[Methods] Human MG63osteoblasts are cultured on the MNTs to assess the celldifferentiation in terms of collagen secretion, extracellular matrix mineralization, andosteogenesis-related gene expression. The expression of-catenin, ILK and integrin1and3is assayed by real-time PCR and the protein levels of-catenin, phosphorylatedglycogen synthase kinase3p-GSK and ILK are determined by western blot. TheILK silenced MG63induced by small interfering RNA is cultured on the samples and thecell functions and the levels of-catenin, GSK and p-GSK are determined.[Results] The MNTs enhance MG63differentiation and it is related to the higherexpression of integrin and ILK, which activate the-catenin signaling by initiating-catenin expression and inhibiting its degradation by phosphorylating GSK ILKsilencing attenuates the-catenin signaling activation and the enhanced MG63differentiation by the MNTs. [Conclusion] The results demonstrate the role of the ILK/-catenin pathway in mediatingthe signals from topographical cues to osteoblasts to tailor differentiation and provide newtarget points for biomaterials modification and biofunctionalization to attain better clinicalperformance. PartVThe Role of Integrin-Linked Kinase/ERK1/2Pathway in the OsteoblastFunctions on Micro/Nano-Textured Titanium Surface[Objective] To more comprehensively depict the ILK related signal network and test thewheter the extracellular signal-regulated kinases (ERK1/2) are another molecular eventdownstream of ILK.[Methods] The levels of ILK and ERK1/2signaling activity in MG63cells on the MNTsare examined by western blot. The ILK expression is down-regulated using ILK specificsiRNA (ILKsi) and the ERK1/2activity is determined again. MG63cell viability anddifferentiation are studied with or without ERK1/2inhibitor U0126to confirm the role ofthe ERK1/2signlaing in the cell functions.[Results] Both ILK and ERK1/2activities are up-regulated by the MNTs. ILKsisignificantly attenuates the phosphorylated ERK1/2activity on the MNTs. Blocking theERK1/2signliang by U0126dignificantly down-regulates the cell viability,osteogenesis-related gene expression, ALP production, collagen secretion and matrixmineralization.[Conclusion] The data demonstrated that ILK/ERK1/2pathway plays an important rolein mediating osteoblast functions in response to biomaterial surface topography.