Low oxygen tension is a more potent promoter of chondrogenic differentiation than dynamic compression

Abstract

During fracture healing and microfracture treatment of cartilage defects mesenchymal stem cells (MSCs) infiltrate the wound site, proliferate extensively and differentiate along a cartilaginous or an osteogenic lineage in response to local environmental cues. MSCs may be able to directly sense their mechanical environment or alternatively, the mechanical environment could act indirectly to regulate MSC differentiation by inhibiting angiogenesis and diminishing the supply of oxygen and other regulatory factors. Dynamic compression has been shown to regulate chondrogenesis of MSCs. In addition, previous studies have shown that a low oxygen environment promotes in vitro chondrogenesis of MSCs. The hypothesis of this study is that a low oxygen environment is a more potent promoter of chondrogenic differentiation of MSCs embedded in agarose hydrogels compared to dynamic compression. In MSC-seeded constructs supplemented with TGF-β3, GAG and collagen accumulation was higher in low oxygen conditions compared to normoxia. For normoxic and low oxygen culture GAG accumulation within the agarose hydrogel was inhomogeneous, with low levels of GAG measured in the annulus of constructs maintained in normoxic conditions. Dynamic compression did not significantly increase GAG or collagen accumulation in normoxia. However under low oxygen conditions, dynamic compression reduced GAG accumulation compared to free-swelling controls, but remained higher than comparable constructs maintained in normoxic conditions. This study demonstrates that continuous exposure to low oxygen tension is a more potent pro-chondrogenic stimulus than 1 h/day of dynamic compression for porcine MSCs embedded in agarose hydrogels.

Introduction

Mesenchymal stem cells (MSCs) are multipotent progenitor cells found in various tissues of adults that have the ability to proliferate extensively while maintaining their multipotent differentiation capabilities (Kadiyala et al., 1997, Bruder et al., 1997, Pittenger et al., 1999). During fracture healing and microfracture treatment of cartilage defects, MSCs from the bone marrow and other surrounding soft tissues infiltrate the wound site, proliferate extensively and differentiate along a cartilaginous or an osteogenic lineage in response to local environmental cues such as growth factors and cytokines. The mechanical environment is also known to regulate the mechanisms of repair following bone fracture. When there is excess motion at the site of injury the predominant mechanism of bone regeneration is through endochondral ossification. Conversely, when motion is minimized, healing primarily occurs through intramembranous ossification. A number of different hypotheses have been proposed to explain how the mechanical environment regulates endochondral ossification during fracture healing. MSCs may be able to directly sense their mechanical environment within a regenerating tissue and differentiate based on the local magnitude of shear strain, hydrostatic pressure, tensile strain, compressive strain and/or fluid flow they experience (Pauwels, 1980, Prendergast et al., 1997, Carter et al., 1998, Claes and Heigele, 1999, Garcia et al., 2002, Lacroix and Prendergast, 2002, Kelly and Prendergast, 2005, Geris et al., 2003). In conjunction or perhaps alternatively, the mechanical environment could also act indirectly to regulate MSC differentiation by inhibiting angiogenesis and hence the supply of oxygen and other factors to the wound site (Ferguson et al., 1999).

Uncoupling the different roles the mechanical environment can have on endochondral ossification during fracture healing has proved challenging using in vivo and in silico models. Such studies have demonstrated that fracture healing is strongly influenced by mechanical factors, such as loading, fixation stiffness and gap size (Choe et al., 1998, Goodship and Cunningham, 2001, Lacroix and Prendergast, 2002). Pauwels (1980) proposed that stress and strain invariants guide the differentiation pathway, whereby hydrostatic pressure results in cartilage formation; while distortional strain, or elongation, favored fibrous tissue formation. A similar concept has been adopted by Carter et al. (1998) to explain a number of mechanobiological processes (e.g. Carter and Wong, 2003, Carter et al., 2004). Further developing this concept, fluid flow and shear strain have also been hypothesized as the stimuli that direct tissue differentiation over time (Prendergast et al., 1997). These biophysical stimuli may directly act to regulate differentiation, or they may interact with biological factors such as growth factors or cytokines. A number of studies have suggested that the mechanical environment during fracture healing can also regulate the neo-vascularization necessary for new bone formation (Geris et al., 2008, Shefelbine et al., 2005, Chen et al., 2009). The hypoxic environment generated by poor vascularity has been shown to promote a more chondrogenic rather than an osteogenic phenotype (Hirao et al., 2006). More recently, computational models of tissue differentiation have been extended to include factors such as angiogenesis (Checa and Prendergast, 2009), providing further support for the hypothesis that capillary formation is indeed mechano-regulated. However, the relative roles played by the local oxygen tension and mechanical signals in regulating endochondral ossification have yet to be elucidated.

A number of in vitro models have been developed, primarily in the field of tissue engineering, to investigate the roles of environmental factors such as mechanical signals and oxygen tension on the differentiation pathway of MSCs. For example, dynamic compression has been shown to regulate chondrogenesis of bone marrow derived MSCs encapsulated in agarose gel (Angele et al., 2004, Huang et al., 2004, Campbell et al., 2006, Mauck et al., 2007, Terraciano et al., 2007, Thorpe et al., 2008, Kisiday et al., 2009, Huang et al., 2010, Thorpe et al., 2010). Similar studies have shown that a low oxygen environment promotes chondrogenesis of MSCs (Lennon et al., 2001, Scherer et al., 2004, Robins et al., 2005, Wang et al., 2005, Krinner et al., 2009, Kanichai et al., 2008). As already described, the mechanical environment within a fracture callus can promote endochondral ossification in one of two ways. Firstly, the biophysical stimuli that act on the MSCs within the defect could directly promote differentiation along the chondrogenic pathway. Secondly, by preventing neo-vascularization and promoting a local hypoxic environment, mechanical loading could indirectly promote chondrogenesis of MSCs. The objective of this study is to determine the relative roles of mechanical signals and oxygen tension on chondrogenesis of bone marrow (BM) derived MSCs using a well developed tissue engineering model. We hypothesized that a low oxygen environment is a more potent promoter of chondrogenic differentiation of MSCs compared to dynamic compression.