Development of a 3D human osteoblast cell culture model for studying mechanobiology in orthodontics

Abstract

Summary Objectives Mechanobiology phenomena constitute a major element of the cellular and tissue response during orthodontic treatment and the implantation of a biomaterial. Better understanding these phenomena will improve the effectiveness of our treatments. The objective of this work is to validate a model of three-dimensional (3D) culture of osteoblasts to study mechanobiology. Materials and methods The hFOB 1.19 cell line was cultured either traditionally on a flat surface or in aggregates called spheroids. They were embedded in 0.8% low-melting agarose type VII and placed in a polyethylene terephthalate transwell insert. Compressive forces of 1 and 4 g/cm2 were applied with an adjustable weight. Proliferation was evaluated by measuring diameters, monitoring glucose levels, and conducting Hoechst/propidium iodide staining. Enzyme-linked immunosorbent assays focusing on the pro-inflammatory mediators interleukin (IL)-6 and IL-8 and bone remodelling factor osteoprotegerin were performed to evaluate soluble factor synthesis. quantitative reverse transcription-polymerase chain reaction was performed to evaluate bone marker transcription. Results The 3D model shows good cell viability and permits IL dosing. Additionally, three gene expression profiles are analysable. Limitations The model allows analysis of conventional markers; larger exploration is needed for better understanding osteoblast mechanobiology. However, it only allows an analysis over 3 days. Conclusion The results obtained by applying constant compressive forces to 3D osteoblastic cultures validate this model system for exploring biomolecule release and analysing gene transcription. In particular, it highlights a disturbance in the expression of markers of osteogenesis.

Effect of mechanical forces on the behavior of osteoblasts: a systematic review of in vitro studies

“…Human fetal (SV-HFO) [19][20][21][22][23][24] and adult osteoblast from the tibia, femur, 25,26 calvaria, 27 or from subchondral bone pieces 28 ; primary human osteoblasts from Cambrex Bio Science 4 ; osteoblast-like cells from tibia/femur/calvaria/ ilia 29 ; human bone marrow-derived osteoblasts (HBMDOs) from the femoral diaphysis 5 ; and Clonetics normal human osteoblasts (NHOst), 30 preosteoblasts, and osteoprogenitors 31 were used to evaluate the effect of tension. For compression assessment, the included studies used alveolar bone osteoblasts, 14,32 human fetal osteoblasts, 13 human tibia osteoblasts, 33 and commercial human osteoblast cell lines. 12…”

“…These cells are mechanical sensors that can transduce mechanical stimuli into biochemical signals (cell-to-cell communication or the production of paracrine factors). 13 Therefore, mechanical forces can affect the bone modeling/remodeling process. a wide range of in vitro experiments have investigated gene expression and proliferation changes of osteoblasts under different methods of loading application.…”

“…During typical orthodontic tooth movement, a compression side and a tension side are created by the applied force. 13 The effect of these two forces on osteoblasts has been investigated in several studies in vivo and in vitro. It has been shown that there are various factors affecting bone regeneration in the presence or absence of loading application, such as strain parameters (frequency, cycle number, and stimulation duration) 4 and scaffolds and medium growth factors.…”

Cyclic Stretching Triggers Cell Orientation and Extracellular Matrix Remodeling in a Periodontal Ligament 3D In Vitro Model

“…Nowadays, most PDL studies are still applying 2D culture systems that are extremely different from the topographically complex, 3D extracellular environment found in PDL. [17][18][19][20] The cells cultured in a 2D environment lack the effects of cell-to-matrix interactions that are known to define a gradient of soluble factors, discrete matrix fiber, adhesion point distribution, and steric cell spreading, all of which together greatly affect the cells in multiple aspects. [21,22] Underscoring the necessity of including a third dimension in PDL oriented assessments, a work of Tondon et al demonstrated that the cells spread in completely different directions under mechanical stretching performed on a 3D matrix compared to 2D substrates.…”

An Evaluation of Different 3D Cultivation Models on Expression Profiles of Human Periodontal Ligament Fibroblasts with Compressive Strain

“…It is interesting to note that the gene expression of cells behaves differently in three-dimensional cultivation than in two-dimensional cultivation [21,22]. In their studies, Brezulier et al (2020) were also able to demonstrate that PDLFs grown as spheroid culture in three dimensions show different reactions to mechanical strain than the same cell type after two-dimensional cultivation [23]. Thus, the question arises, to which extent the knowledge gained so far on 2D models regarding the response of PDLFs to mechanical forces in orthodontics corresponds to the physiology of the human organism and the actual response of PDLFs in the local tissue.…”