Effects of interfacial micromotions on vitality and differentiation of human osteoblasts

Abstract

Objectives: Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed.

Methods: Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated.

Results: Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio.

Conclusion: Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be considered.Cite this article: J. Ziebart, S. Fan, C. Schulze, P. W. Kämmerer, R. Bader, A. Jonitz-Heincke. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res 2018;7:187-195. DOI: 10.1302/2046-3758.72.BJR-2017-0228.R1.

Keywords: Endoprosthesis; Micromotion; Osseointegration; Osteoblast; Osteoblast differentiation.

Fig. 1

System for application of micromotions. The stage supporting the six-well plate is fixed on top of the linear piezo positioning system. The lid of the system holds the titanium alloy (Ti6Al4V) cylinders mounted on slight bearings and resting on the samples. There are three experimental groups: osteoblasts seeded on collagen scaffolds (unloaded control); Ti6Al4V cylinders resting on collagen scaffolds with osteoblasts under static loading conditions (loaded control); and the six-well plate containing collagen scaffolds with osteoblasts, which is moved by the linear positioning system relative to static Ti6Al4V cylinders (micromotions, 25 µm to 100 µm).

Fig. 2

Characteristics of applied micromotions. Depiction of a 50 µm sine (light orange function) and 50 µm triangle (dark orange function) micromotion applied with 1 Hz frequency. The table shows the maximum velocity and maximum acceleration of the applied micromotions resulting from the magnitude and waveform of the micromotions applied with 1 Hz frequency.

Cell viability of human osteoblasts: a) Live/Dead staining of osteoblasts growing on collagen material. Live cells appear green; nuclei of dead cells fluoresce red; white bar represents 100 µm. b) Graph showing the relative cell count of live and dead cells in unloaded samples and samples treated with micromotions compared with loaded controls. c) Graph showing the metabolic activity of osteoblasts in unloaded samples and samples treated with micromotions compared with loaded controls. *p < 0.05, †p < 0.01 compared with loaded control (Wilcoxon’s signed-rank test); ‡p < 0.05, §p < 0.01, ¶p < 0.001 comparison between different groups (Kruskal–Wallis test and Dunn’s multiple comparison test).

Expression of osteogenic markers in human osteoblasts: a) fold change of osteogenic genes collagen type I (COL1A1), alkaline phosphatase (ALP) and osteocalcin (OC); b) pro-collagen type I synthesis; and c) ALP activity of unloaded osteoblasts and osteoblasts treated with micromotions normalized to loaded samples (*p < 0.05 compared with loaded control (Wilcoxon’s signed-rank test); §p < 0.01, ¶p < 0.001 comparison between different groups (Kruskal–Wallis test and Dunn’s multiple comparison test).

Expression of genes responsible for bone remodelling in human osteoblasts: a) fold change of tissue inhibitor of metalloproteinase-1 (TIMP-1) versus matrix metalloproteinase-1 (MMP-1); and b) osteoprotegerin (OPG) versus receptor activator of NF-κB ligand (RANKL) in unloaded and loaded osteoblasts and osteoblasts treated with micromotions (*p < 0.05 comparison between different groups (Kruskal–Wallis test and Dunn’s multiple comparison test). The colours correspond with the extent of strain exerted on the cells: unloaded control (white), no strain exerted on the cells; loaded control (light orange), affected by the static loading; micromotion (dark orange), strain on the cells (loading and micromotions).