Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D

Abstract

Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.

In vitro effects of particulate bone substitute materials on the resorption activity of human osteoclasts

“…Due to the lack of any other cell type capable of bone resorption, we were convinced that we were able to culture functional (bone resorbing) human osteoclasts. The observation of osteoclast-derived bone resorption by SEM corresponded to published information (Kleinhans et al, 2015;Sabokbar et al, 2003;Sabokbar et al, 1998).…”

“…Similarly, Perotti et al (2009) reports slower bone resorption of osteoclasts on Bio-Oss plates than on bovine bone. As these results are based on different study setups (processing of biomaterial, cultivation length and cell line used), in future, a standard assay for the evaluation of cell based resorption effects should be established to facilitate the comparison of these various reports (Kleinhans et al, 2015). For example, Schilling et al (2004) recommend the simultaneous use of dentin as a control, so as to allow comparison of biomaterials.…”

From the Clinical Problem to the Basic Research—Co-Culture Models of Osteoblasts and Osteoclasts

“…Later on, more complex 3D systems integrating different cell types, fluidics, and mechanics should be established to gain deeper insight into the bone remodeling process or pathological state [89,90]. …”

Osteoclast Formation within a Human Co-Culture System on Bone Material as an In Vitro Model for Bone Remodeling Processes

“…hMCs were isolated as described previously in Kleinhans et al 2015 [10]. Briefly, cells were obtained by Ficoll-Paque ™ Plus (GE Healthcare; Chalfont St Giles, UK) density gradient centrifugation at 485× g for 30 min at 4 • C (without brake).…”

“…Functional osteoclasts are bone-resorbing cells. Even in vitro osteoclasts are able to develop this resorption activity when they are cultured on bone or bone-like materials [10]. To simulate the in vivo situation of osteoclastogenesis, a co-culture system with human monocytes or osteoclasts and supporting cells such as mesenchymal stem cells (MSCs) or osteoblasts in appropriate culture conditions, especially media supplementation and bone or bone-like materials, have to be combined.…”