Osteocyte Characterization on Polydimethylsiloxane Substrates for Microsystems Applications

Abstract

In the body, osteocytes reside in lacunae, lenticular shaped cavities within mineralized bone. These cells are linked to each other and surface-residing osteoblasts via physical channels known as gap junctions. It has been suggested that osteocytes sense mechanical load applied to bone and relay that signal to osteoclasts and osteoblasts. Current in vitro and in vivo models of mechanotransduction face temporal and spatial barriers. Recent advances in polydimethylsiloxane (PDMS) based microfabrication techniques may be able to overcome some of these hurdles. However, before the bone research field can effectively utilize microsystems techniques, fundamental groundwork must be completed. This study characterized the behaviour of osteocytes on PDMS coated with collagen type I (CTI) and provides the framework for bone cell mechanotransduction studies using microsystems. The goal was to determine whether osteocytes were adversely affected by the substrate material by comparing their behaviour to a standard glass substrate. In addition, optimal culture conditions and time points for growing osteocytes on PDMS substrates were determined. Results of this study suggested that use of PDMS does not adversely affect osteocyte behaviour. Furthermore, the results demonstrated that osteocytes should be cultured for no less than 72 hours prior to experimentation to allow the establishment and maintenance of phenotypic characteristics. These results completed essential groundwork necessary for further studies regarding osteocytes in microsystems modelling utilizing PDMS.

Impact of Gap Junctional Intercellular Communication on MLO-Y4 Sclerostin and Soluble Factor Expression

“…This was significantly reduced in the presence of 18α-glycyrrhetinic acid treatment where only 13% of osteoctyes were in communication. 25 Figure 4 A and B shows osteocytes in communication intact, while Figure 4 C and D and display an inhibited environment.…”

“…All cells for loading were cultured 4 days prior to experimentation to allow the MLO-Y4 cells to exhibit their characteristic protein expression and morphology. 25 Cells were maintained in .5 ml of medium and fed every other day. Cleaning wipes were dipped in sterile water and wrapped around the edge of the petri dish to increase the humidity and lessen medium evaporation.…”

“…Previous studies indicated that this drug treatment did not alter the adhesion or viability of the MLO-Y4 cells. 25 …”

In vitro osteocytic microdamage and viability quantification using a microloading platform

“…These cells were maintained in .5 mL of medium, seeded 4 days prior to experimentation for reaching their characteristic protein expression and dendritic morphology, and fed every other day [22]. Two Kimwipes (Kimtech) were dipped in sterile water and wrapped around the perimeter of the plate to generate a humidified chamber.…”

“…The platform was fabricated, assembled, characterized, and a polydimethylsiloxane (PDMS) well substrate for in vitro cell culture was designed and fabricated. PDMS was chosen due to its optical clarity, ease of fabrication, and previous research indicating that the osteocytic cell line used in this work grew, proliferated, and exhibited the same characteristic protein expression on PDMS and polystyrene [22]. The fabricated platform was then used to study the lactate dehydrogenase (LDH) levels of cells at a range of strains due to mechanical displacement and substrate location.…”