Establishment and Evaluation of an In Vitro System for Biophysical Stimulation of Human Osteoblasts

Abstract

While several studies investigated the effects of mechanical or electrical stimulation on osseointegration and bone fracture healing, little is known about the molecular and cellular impact of combined biophysical stimulation on peri-implant osseointegration. Therefore, we established an in vitro system, capable of applying shear stress and electric fields simultaneously. Capacitively coupled electric fields were used for electrical stimulation, while roughened Ti6Al4V bodies conducted harmonically oscillating micromotions on collagen scaffolds seeded with human osteoblasts. Different variations of single and combined stimulation were applied for three days, while samples loaded with Ti6Al4V bodies and untreated samples served as control. Metabolic activity, expression of osteogenic markers and bone remodeling markers were investigated. While combined stimulation showed no substantial benefit compared to sole mechanical stimulation, we observed that 25 µm micromotions applied by roughened Ti6Al4V bodies led to a significant increase in gene expression of osteocalcin and tissue inhibitor of metalloprotease 1. Additionally, we found an increase in metabolic activity and expression of bone remodeling markers with reduced procollagen type 1 synthesis after 100 mVRMS electrical stimulation. We were able to trigger specific cellular behaviors using different biophysical stimuli. In future studies, different variations of electrical stimulation will be combined with interfacial micromotions.

Electronic Bone Growth Stimulators for Augmentation of Osteogenesis in In Vitro and In Vivo Models: A Narrative Review of Electrical Stimulation Mechanisms and Device Specifications

“…Procollagen protein synthesis has been measured using the biomarker type I C-terminal collagen propeptide (CICP) via ELISA (Stephan et al, 2020). The ALP gene has been quantified using p-nitrophenyl phosphate (pNpp) with microplate analysis at a wavelength of 405 nm (Stephan et al, 2020).…”

“…This has been measured by [ 35 H]thymidine and [ 3 S]sulfate uptake in chondrocytes and safranin-O staining ( Brighton et al, 1984 ). Additionally, procollagen protein synthesis is a marker of collagen 1 (COL-1), which is a key component to bone formation, and alkaline phosphatase (ALP) is a biomarker for osteoblasts ( Stephan et al, 2020 ). Procollagen protein synthesis has been measured using the biomarker type I C-terminal collagen propeptide (CICP) via ELISA ( Stephan et al, 2020 ).…”

“…Additionally, procollagen protein synthesis is a marker of collagen 1 (COL-1), which is a key component to bone formation, and alkaline phosphatase (ALP) is a biomarker for osteoblasts ( Stephan et al, 2020 ). Procollagen protein synthesis has been measured using the biomarker type I C-terminal collagen propeptide (CICP) via ELISA ( Stephan et al, 2020 ). The ALP gene has been quantified using p-nitrophenyl phosphate (pNpp) with microplate analysis at a wavelength of 405 nm ( Stephan et al, 2020 ).…”

“…Procollagen protein synthesis has been measured using the biomarker type I C-terminal collagen propeptide (CICP) via ELISA ( Stephan et al, 2020 ). The ALP gene has been quantified using p-nitrophenyl phosphate (pNpp) with microplate analysis at a wavelength of 405 nm ( Stephan et al, 2020 ). Another marker for osteogenesis is intracellular calcium levels, which contribute to cytoskeletal organization, cell motility, and cell growth and has been measured using flow cytometry ( Li et al, 2019 ).…”

Anodic TiO2 Nanotubes: Tailoring Osteoinduction via Drug Delivery

“…Further, despite a long history of clinical trials, the mechanism linking electric stimuli and its sensing on the cell surface is still elusive. Therefore, during recent decades, the mechanism of EF (e.g., how cells sense and react to EFs) affecting cell adhesion, migration, proliferation, and differentiation has been extensively investigated in various primary cell/cell lines [ 206 , 207 , 208 , 209 , 210 , 211 ] including epithelial, mesenchymal, and neural cells [ 212 , 213 , 214 , 215 ]. Exposure of the cells to an electric current, if a certain current value threshold is exceeded, leads to cell death, while currents lower than 11 As/m 2 have shown no decrease in cell viability [ 216 ].…”