Ti nanorod arrays with a medium density significantly promote osteogenesis and osteointegration

Abstract

Ti implants are good candidates in bone repair. However, how to promote bone formation on their surface and their consequent perfect integration with the surrounding tissue is still a challenge. To overcome such challenge, we propose to form Ti nanorods on their surface to promote the new bone formation around the implants. Here Ti nanorod arrays (TNrs) with different densities were produced on pure Ti surfaces using an anodizing method. The influence of TNr density on the protein adsorption as well as on the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 pre-osteoblastic cells were assessed. The TNrs were also implanted into the bone defects in rabbits to test their application in promoting bone formation and osteointegration at the implant-bone interface. TNrs with the medium density were found to show the best capability in promoting the protein adsorption from surrounding medium, which in turn efficiently enhanced osteogenic differentiation in vitro and osteointegration in vivo. Our work suggests that growing TNrs with a medium density on the surface of traditional Ti implants is an efficient and facile method for promoting bone formation and osteointegration in bone repair.

Figure 1. An overview of Ti nanorod arrays (TNrs) in bone repair.

Left: a schematic diagram showing the fabrication of TNrs using an anodizing method. Top right: Ti and TNrs sheets are seeded with pre-osteoblasts (MC3T3-El) to study the cell response. Bottom right: Ti rods terminated with TNrs are implanted into bone defects in rabbit tibia to test their capability in promoting bone formation and osteointegration at the bone-implant interface.

Figure 2. Evaluation of the TNrs in terms of density, morphology and roughness.

SEM (a) and AFM (b) images of the TNrs with low density (LTNrs), medium density (MTNrs) and high density (HTNrs). (c) The density of TNrs. (d) The roughness of Ti and TNrs determined by AFM.

Figure 3. The protein adsorption and fluorescence images of MC3T3-E1 pre-osteoblasts seeded on Ti with different nanorod densities.

(a) The amount of protein adsorption on different substrates. The specimens are incubated in α-MEM (10% FBS) for 4 h to assay the early protein adsorption. n = 4. (b) The cell density on different substrates. The histogram for each specimen of counted cells after 30, 60 and 120 min of incubations. n = 5. (c) pre-osteoblast adhesion on the different substrates measured by counting cells stained with DAPI on a fluorescence microscope after 30, 60 and 120 min of incubations. All data are reported as the mean ± standard deviation, *p < 0.05 and **p < 0.01 compared with the pure Ti, LTNr and HTNr surfaces.

Figure 4. Analysis of the cell proliferation and differentiation on the surface of different substrates (Ti, LTNrs, MTNrs and HTNrs).

(a) Proliferation (MTT assay) of pre-osteoblasts on different substrates after incubation for 1, 3 and 5 days (n = 4). (b) Differentiation (ALP activity) of MC3T3-E1 cells on different substrates after 14 days of culture (n = 4). (c) The Runx2 gene expression on TNrs by primary pre-osteoblasts after incubation of 7 and 14 days. *p < 0.05 compared to pure Ti. **p < 0.01 compared to pure Ti.

Figure 5. Analysis of newly formed bone on the surface of cylindrical rods modified with nanorods with a medium density.

(a) Push out forces of Ti and MTNrs after implanted into rabbit 12 weeks. This data is reported as the mean ± standard deviation. **p < 0.01 compared with the pure Ti surface. (b,c) Masson staining of the sections of implants showing MTNrs (c) can efficiently induce the formation of new bone tissue in comparison with pure Ti (b).