Osteocyte differentiation and the formation of an interconnected cellular network in vitro

Abstract

Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 10 6 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.

Primary Human Osteoblasts Cultured in a 3D Microenvironment Create a Unique Representative Model of Their Differentiation Into Osteocytes

“…Cells were used at passage 4-7 and mixed to the collagen gel solution. Final cell densities of 2.5 × 10 5 (low) and 1 × 10 6 cells/ml (high) were based on recent findings on osteocyte differentiation in traditional in vitro systems (McGarrigle et al, 2016).…”

“…The microarchitecture and the mechanical properties of the collagen fibrous hydrogels used in this study were characterized in previous studies (Moreno-Arotzena et al, 2015;Del Amo et al, 2018). In the present work, we selected the 6 mg/ml gel because its Young modulus (∼0.72 kPa, from Valero et al, 2018) was comparable to the one (∼0.58 kPa) that allowed a homogeneous cell distribution in a 3D matrix and enhanced osteocytic differentiation of MC3T3-E1 cells (McGarrigle et al, 2016).…”

“…Osteocytes exhibit exploratory dendrites that extend and retract from the surrounding extracellular matrix before forming a fully developed osteocyte network (McGarrigle et al, 2016). Cell primary protrusions longer than 10 µm were defined as dendrites, as previously described (Mullen et al, 2013).…”

“…However, osteoblasts can have three other possible fates but the mechanism regulating this transition is not clearly understood yet: they can become bone-lining cells (inactive osteoblasts), undergo apoptosis, or transdifferentiate into chondroid-depositing cells (Dallas and Bonewald, 2010). Gene expression profiles (Boukhechba et al, 2009;Sun et al, 2017) and immunohistochemistry stainings (Uchihashi et al, 2013;Sun et al, 2015;McGarrigle et al, 2016) in traditional 3D culture systems showed that the expression of osteoblast and osteocyte markers in vitro corresponded to the in vivo expression at the same differentiation stages (Franz-Odendaal et al, 2006).…”

“…Traditional in vitro cultures replicated essential features of the interaction between osteoblasts and osteocytes, such as osteoblast maturation and matrix mineralization. These systems consisted of 3D collagen hydrogels/sponges and human osteoblast-like cells (Atkins et al, 2009;Bernhardt et al, 2019;Skottke et al, 2019) or mouse clonal cell-lines (MC3T3-E1 and IDG-SW3) (Woo et al, 2011;McGarrigle et al, 2016), with the last ones usually preferred due to the excessive number of cells required. On a micro-scale, a 2D layer of MC3T3-E1 cells spontaneously formed a mineralized collagenous matrix in long-term cultures (up to 1 month) (Hao et al, 2018).…”