Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression

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Abstract

Elucidating the intracellular signaling cascades which lead to differentiation programs can be a daunting but necessary task. Even more so when the nature of the differentiating stimuli can elicit different biochemical responses yet achieve the same functional outcome. In the field of cartilage and bone regeneration the importance of the extracellular signal-regulated kinase (ERK) pathway has been a controversial issue as of late. Whether differentiation results from a soluble chemical induction or a microenvironmental cue on the cells seems to have a determining effect on the role that this pathway plays in ultimate cell fate. Here we explore the role of the ERK1/2 pathway on the mechanical induction of chondrogenesis of bone marrow mesenchymal stem cells (MSC). The cells were encapsulated in fibrin gel scaffolds and subjected to a dynamic mechanical compression stimulus previously demonstrated to induce chondrogenic differentiation of the cells with and without the addition of PD98059, a selective inhibitor for the ERK1/2 pathway. Samples were then analyzed by RT-PCR and histochemical staining for markers of both chondrogenic and osteogenic differentiation. Our results show that dynamic compression induces the chondrogenic differentiation of the cells and that inhibition of the ERK1/2 pathway completely abolishes this chondrogenic response. On the other hand, inhibition of ERK1/2 under dynamic compression augments the osteogenic response of the cells and significantly increases their expression of alkaline phosphatase (ALP), collagen type I (COLI) and osteocalcin (OCN) (P < 0.05). These results were confirmed by the histochemical staining where dynamically compressed samples show staining for sulfated glycosaminoglycans (sGAG) while the inhibited and compressed samples show no sGAG but present positive staining for microcalcifications. These results would suggest that the activation of ERK1/2 can determine the ultimate cell fate between the chondrogenic and osteogenic programs in cells stimulated under dynamic unconfined mechanical compression.

Highlights

► Dynamic cyclic compression upregulates the chondrogenic response of MSC. ► Compression-induced chondrogenesis of MSC is ERK1/2 activation-dependent. ► ERK1/2 activation is essential for the compression-induced chondrogenesis of MSC. ► Inhibition of ERK phosphorylation abolishes compression-induced chondrogenesis. ► ERK inhibition augments the osteogenic response of mechanically stimulated cells.

Introduction

In the field of regenerative medicine and bioengineering, the term functional tissue engineering (FTE) has become the concept by which ultimate success of any engineered tissue is evaluated [1]. Successful FTE involves many key concepts including adequate cell source selection, biomaterial scaffolding and differentiation/phenotypic maintenance parameters, as well as effective delivery method for the engineered tissue. And in no other tissue is this as crucial as in the mechanical-bearing tissues of the body where the ultimate structure and mechanical properties of the engineered tissue must be able to withstand the long-term onslaught of constant mechanical loading. For these types of tissues, the determination of in vivo force pattern exposure of repair tissues [2] must be taken into consideration as well. The type and magnitude of these forces has been extensively modeled for some systems in the human body. For functional tissue engineering purposes, these models are used in the design of bioreactors, which recreate the physiological environment of a specific tissues with the hopes of imparting that ultimate mechanical functionality to the tissue [3]. Our lab has previously shown that unconfined dynamic compression by itself is capable of eliciting a chondrogenic response from mesenchymal stem cells (MSC) seeded in different biomaterial scaffolds [4], [5]. Similarly, other researchers have demonstrated the ability of biomaterial scaffold chemistry and mechanical properties to induce specific differentiation pathways and guide the ultimate fate of the cells in them [6], [7], [8].

However, little is known about the underlying biochemical cascades elicited by the use of mechanical stimuli as part of any differentiation protocol and how they may overlap, substitute or bypass common pathways activated by soluble factor induction. The field of mechanobiology, and mechanotransduction within it, focuses primarily on the role of different mechanical forces in the activation of these intracellular cascades and ultimate cell fate [9]. One of the most investigated signal transduction pathways is the Mitogen Activated Protein Kinases (MAPK), which convert environmental cues into biochemical responses and are known to be essential in cellular growth, differentiation, and response to stresses [10]. Like soluble factor induction, mechanical impulses applied to the cells might have an impact on one or more of the three major MAPK pathways: p38, p42/44 and c-Jun N-terminal kinase (JNK). We recently reported the essential role that the activation of the p42/44 pathway plays on both the chondrogenesis and osteogenesis of mesenchymal stem cells (MSC) stimulated with TGF-β3 [11]. Lund et al., [12] showed that when subjected to microenvironmental cues within a type-1 collagen scaffold, the inhibition of the ERK1/2 pathway actually resulted in an augmented osteogenic response from the cells and an inhibited chondrogenic differentiation concurrently. The authors suggested that it was the three-dimensional configuration that led to the up-regulation of the chondrogenic and osteogenic expression of the cells rather than any biochemical stimulation (as no differentiation moieties were employed) or the collagen I substrate used in the scaffold [12]. Similarly, the activation of ERK1/2 has been reported by other researchers to be at least partially non-critical for the hydrostatic pressure-induced osteogenesis of bone marrow mesenchymal stem cells [13]. However, other reports combining both biochemical and mechanical stimuli show opposing results to the ones presented with solely the use of mechanical/physical cues. Kim et al., [14] showed that the expression of osteogenic markers osteocalcin and osteopontin were up-regulated in MSCs encapsulated within poly(lactic-glycolic acid) scaffolds and subjected to cyclic hydrostatic pressure in the presence of osteogenic media over those of the unloaded constructs in the same media. This study showed that osteogenesis was suppressed in the presence of U0126, a potent selective inhibitor of p42/44 MAPK, in both compressed and non-compressed cellular constructs.

The intricacies of this phenomenon were further highlighted by studies demonstrating no additive effect when both mechanical stimulation and exogenous cytokine supplementation are combined in the osteogenesis and chondrogenesis of stem cells [15], [16]. Based off of mechanical stimulation alone, we experiments performed in our laboratory showed the potential for dynamic compressive loading to induce chondrogenic differentiation of various stem cell populations [17], [4]. Dynamic compression led to significant increase in the expression of both TGF-β 1 and II receptors and potential involvement of p42/44 MAPK due to the rapid upregulation in the expression of c-Fos [5], which is a known downstream target of p42/44 MAPK [18]. Moreover, Fritz showed that cyclic unconfined mechanical compression activated the p42/44 pathway but not the p38 or JNK pathways in the stem cells [17] and the inhibition of the p42/44 pathway did not result in the activation of any of the other two MAPK cascades and completely abolished the chondrogenic response seen in the cells [17]. However, several questions still remain as to the underlying mechanisms that these differentiation stimuli activate and to what extent they may augment, suppress or overlap each other.

Given the apparent dichotomy in the role the ERK1/2 pathways plays in the mechanical and biochemical stimulation of chondrogenesis and osteogenesis in stem cells, it is necessary to further investigate and isolate the response of the cells to each stimulus independently in order to shed light on the complex phenomena behind these processes. With this purpose in mind, the current project sought to investigate the role of the ERK1/2 pathway in the chondrogenesis and osteogenesis of MSCs subjected to cyclic unconfined mechanical compression within fibrin gel scaffolds.

Section snippets

Cell culture

All cell culture reagents were purchased from Invitrogen (Carlsbad, CA) unless otherwise specified. Inhibitor PD98059 and fibrinolytic inhibitor aprotinin were purchased from Sigma–Aldrich (St. Louis, MO). Bone marrow-derived mesenchymal stem cells (MSC) were purchased from ScienceCell Research Laboratories (Carlsbad CA) as CD73, CD90, CD105-positive cells capable of adipogenic, chondrogenic and osteogenic differentiation. Cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM)

Chondrogenic and osteogenic gene expression

Our gene expression results demonstrate an overall activation of the chondrogenic differentiation of the cells subjected to dynamic unconfined compression (Fig. 1). Fig. 1 shows how both the Sox9 and Aggrecan gene expression is augmented with the application of compressive loading and that, while not statistically significant for the sox-9 gene, this up-regulation is significant for the aggrecan gene expression. Furthermore, upon the treatment of the cells to the ERK1/2 inhibitor, there is a

Discussion

It is known that during the differentiation process of MSCs, one or several intracellular chemical cascades are modified (activated or inhibited) influencing the ultimate commitment of the cells [19]. However, it remains unclear how each individual pathway affects the differentiation program of the cells and how manipulation of these pathways could lead to more efficient differentiation protocols. Similarly, the nature of the differentiating stimulus applied can induce different biochemical

Acknowledgment

The current project was supported by a Veterans Affairs (VA) Merit Review Grant as well as a VA Senior Research Career Scientist Award at the Miami VA Medical Center.

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