Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells

doi:10.1016/j.jmst.2016.08.009

材料科学与技术 ›› 2016, Vol. 32 ›› Issue (9): 944-949.DOI: 10.1016/j.jmst.2016.08.009

收稿日期:2015-11-30 接受日期:2016-04-08 出版日期:2016-09-20 发布日期:2016-11-02

Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells

Xu Shao¹,Zhou Zhiyu²,Gao Manman²,Zou Changye³,Che Yinglin¹,Cody Bünger⁴,Zou Xuenong^2,^*(),Zhou Lei^5,^**()

¹ Department of Stomatology, The Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China
² Guangdong Provincial Key Laboratory of Orthopedics and Traumatology/Orthopedic Research Institute, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510075, China
³ Department of Musculoskeletal Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
⁴ Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Aarhus University Hospital, Aarhus 8000, Denmark
⁵ Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou 510280, China

Received:2015-11-30 Accepted:2016-04-08 Online:2016-09-20 Published:2016-11-02
Contact: Zou Xuenong,Zhou Lei

摘要/Abstract

Abstract:

Titanium (Ti) nanorods fabricated using selective corrosion of Ti substrate by anodic technology show better biocompatibility with pre-osteoblast cells. The current study investigated the response of the murine pre-osteoblast cell MC3T3-E1 on Ti nanorod topography and untreated Ti surfaces by means of examination of the morphology and osteogenic differentiation responsible for the pre-osteoblast reaction. The morphology of MC3T3-E1 cells was observed using scanning electron microscopy, and alkaline phosphatase (ALP) activity was measured using a colorimetric assay after incubation for 7, 14, and 21 days. The expression of three osteogenic differentiation markers including ALP, osteocalcin (OCN), and collagen type 1A1 (COL1A1) and two transcription factors including runt related transcription factor 2 (Runx2) and osterix (Osx) at different time points was detected using real-time polymerase chain reaction analysis in both groups. Osx was used to confirm the protein level. The results showed that Ti nanorod surfaces provided prolonged higher levels of ALP activity compared with unmodified Ti surface on the 14th and 21st days. Gene expression analysis of ALP, OCN, and COL1A1 showed significant upregulation with modified nanorod topography after incubation for 14 and 21 days. Osteogenic transcription factors of Runx2 and Osx exhibited changes consistent with the osteogenic differentiation markers, and this may contribute to the persistently active differentiation of MC3T3-E1 cells in the Ti nanorod group. These results demonstrated that the current nanostructured surface may be considered bioadaptive topography to control cellular behaviors and osteoblast differentiation. The in vivo performance and applicability are further required to investigate osseointegration between implant and host bone in the early stages for prevention of aseptic implant loosening.

Key words: Titanium, Nanorods, Osteointegration, Osteoblast differentiation

. [J]. 材料科学与技术, 2016, 32(9): 944-949.

Xu Shao,Zhou Zhiyu,Gao Manman,Zou Changye,Che Yinglin,Cody Bünger,Zou Xuenong,Zhou Lei. Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells[J]. J. Mater. Sci. Technol., 2016, 32(9): 944-949.

图/表 6

Table 1 Primer sequences of genes

Gene symbol	Forward primer	Reversed primer
GAPDH	CTTGGGCTACACTGAGGACC	CATACCAGGAAATGAGCTTGAC
ALP	TGGAAGGAGGCAGGATTGA	ATCAGCAGTAACCACAGTCA
OCN	CAGACAAGTCCCACACAG	GTGAGGTCAGAGAGACAG
RUNX2	AATGCCTCCGCTGTTATGA	TTGTGAAGACTGTTATGGTCAAG
COL1A1	CCTGGCAAAGACGGACTCA	GGGCTGCGGATGTTCTCAAT
Osterix	TCTCAAGCACCAATGGACT	AGGAAATGAGTGAGGGAAGG

Fig. 1. Microscopic images of untreated and titanium nanorods surface with and without cellular adhesion of preosteoblast MC3T3-E1. SEM images of untreated titanium surface (A), nanorods titanium surface (B), SEM images of MC3T3-E1 cultured on untreated titanium surface (C) and on the titanium nanorods surface (D) after 24?h of incubation. Fluorescence microscopy images of cytoskeletal actin (green) and nucleus-staining DAPI (blue) for MC3T3-E1 cells cultured on untreated titanium surface (E) and on the titanium nanorods surface (F) after 24?h of culture incubation.

Fig. 2. ALP activity of preosteoblast MC3T3-E1 detected after 7, 14 and 21 days of incubation on untreated titanium and titanium nanorods surface. Con: untreated titanium, Ti: titanium nanorods. *p?<?0.05, **p?<?0.01.

Fig. 3. Osteogenic differentiation markers expression analysis of preosteoblast MC3T3-E1 cultured on untreated titanium and titanium nanorods surface after 7, 14 and 21 days of incubation using real-time PCR: (A) ALP, (B) OCN, (C) COL1A1. Con: untreated titanium, Ti: titanium nanorods. *p?<?0.05, **p?<?0.01.

Fig. 4. Runx2 expression analysis of preosteoblast MC3T3-E1 cultured on untreated titanium and titanium nanorods surface after 7, 14 and 21 days of incubation using real-time PCR. *p?<?0.05.

Fig. 5. Osterix expression analysis of preosteoblast MC3T3-E1 cultured on untreated titanium and titanium nanorods surface after 7, 14 and 21 days of incubation with real-time PCR (A) and with western-blot (B). *p?<?0.05.

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Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells

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