The roles of titanium surface micro/nanotopography and wettability on the differential response of human osteoblast lineage cells

Abstract

Surface micro- and nanostructural modifications of dental and orthopedic implants have shown promising in vitro, in vivo and clinical results. Surface wettability has also been suggested to play an important role in osteoblast differentiation and osseointegration. However, the available techniques to measure surface wettability are not reliable on clinically relevant, rough surfaces. Furthermore, how the differentiation state of osteoblast lineage cells impacts their response to micro/nanostructured surfaces, and the role of wettability on this response, remain unclear. In the current study, surface wettability analyses (optical sessile drop analysis, environmental scanning electron microscopic analysis and the Wilhelmy technique) indicated hydrophobic static responses for deposited water droplets on microrough and micro/nanostructured specimens, while hydrophilic responses were observed with dynamic analyses of micro/nanostructured specimens. The maturation and local factor production of human immature osteoblast-like MG63 cells was synergistically influenced by nanostructures superimposed onto microrough titanium (Ti) surfaces. In contrast, human mesenchymal stem cells cultured on micro/nanostructured surfaces in the absence of exogenous soluble factors exhibited less robust osteoblastic differentiation and local factor production compared to cultures on unmodified microroughened Ti. Our results support previous observations using Ti6Al4V surfaces showing that recognition of surface nanostructures and subsequent cell response is dependent on the differentiation state of osteoblast lineage cells. The results also indicate that this effect may be partly modulated by surface wettability. These findings support the conclusion that the successful osseointegration of an implant depends on contributions from osteoblast lineage cells at different stages of osteoblast commitment.

Figure 1

Static and dynamic evaluations of surface wettability. (A) Micrograph of a sessile water drop used to measure contact angles. (B) ESEM image of the condensation of water on the surface of Ti specimens for contact angle assessment. (C) Photograph of the Wilhelmy plate setup revealing the use of a tensiometer to suspend a rectangular Ti specimen that is partially immersed in a water reservoir. (D) Example of a typical DCA 10-loop hysteresis cycle, showing the advancing and receding curves.

Figure 2

SE images of (A) microrough Ti specimens (SLA) and (B) microrough specimens that were subsequently heat-treated to superimpose oxidation-induced nanostructures on the surface (NMSLA). SLA surfaces possessed peaks and valleys in the order of tens of micrometers as a result of the sandblasting process, with some sharp sub-microscale features left from the acid-etch treatment. After the nanomodification oxidation treatment for 90 minutes at 740 °C in flowing synthetic air, the NMSLA surfaces possessed high and homogeneous concentrations of nanostructures.

Figure 3

Average surface roughness (S_a) values of original and nanomodified surfaces measured by laser confocal microscopy (LCM, black bars) and atomic force microscopy (AFM, grey bars). AFM scans were not possible on microrough SLA and NMSLA specimens, due to z-height tool limitations, thus requiring the evaluation of microsmooth PT and NMPT specimens. * refers to a statistically-significant p value below 0.05 vs. PT; # refers to a statistically-significant p value below 0.05 vs. NMPT.

Figure 4

Surface elemental compositions of the SLA and NMSLA specimens measured by XPS. All surfaces were mainly composed of Ti, O and C. N was also present at low levels on the SLA surfaces, while NMSLA surfaces only had traces (T) of N. Traces of other contaminants such as Ca and Cl were found on the surface of SLA specimens, but these were not detectable (ND) on the NMSLA surfaces. * refers to a statistically-significant p value below 0.05 vs. SLA.

Figure 5

Static and dynamic contact angle analyses on SLA and NMSLA specimens. (A) Optical sessile-drop water contact observations on the surfaces of SLA and NMSLA specimens showed hydrophobic static responses. (B) ESEM image showing condensed water droplets on the surfaces of SLA and NMSLA specimens for contact angle evaluations. Some of the smaller droplets exhibited complete wetting of the surface. (C–D) Force graphs of 10-loop Wilhelmy experiments showed extremely negative F/L values for the initial advancing loop of (C) SLA and (D) NMSLA specimens. Subsequent immersion-emersion loops on the SLA specimens continued to show negative values without reaching equilibrium. In contrast, NMSLA specimens presented positive F/L values for all following loops without evidence of appreciable hysteresis. (E) Sterilized SLA specimens were ultrasonically cleaned after dynamic contact angle analysis and reanalyzed, showing a slightly negative F/L value for the initial loop and positive values for the subsequent loops without appreciable hysteresis.

Figure 6

Contact angles measured by sessile-drop technique (CA), ESEM, or calculated from the measured F/L values from different advancing and receding loops (L) from dynamic analyses of the autoclaved SLA and NMSLA specimens.

Figure 7

Effects of micro and nanoscale surface modifications on immature MG63 osteoblast-like cells and human MSCs relative to microsmooth PT controls (dashed line). Osteoblasts and MSCs were plated on PT controls, SLA, and NMSLA surfaces and grown to confluence. The nanomodification involves surface oxidation in flowing synthetic air for 90 minutes at 740 °C. At confluence, (A) cell number, (B) osteocalcin, (C) osteoprotegerin, and (D) VEGF levels were measured. Data represented are the mean ± SE of six independent samples. * refers to a statistically-significant p value below 0.05 vs. PT; # refers to a statistically-significant p value below 0.05 vs. SLA-MG63; $ refers to a statistically-significant p value below 0.05 vs. NMSLA-MG63; ^ refers to a statistically-significant p value below 0.05 vs. SLA-MSC.