CD73/5’-ecto-nucleotidase acts as a regulatory factor in osteo-/chondrogenic differentiation of mechanically stimulated mesenchymal stromal cells

Abstract

Bone regeneration is influenced by mesenchymal stromal cells (MSCs) and mechanical conditions. How healing outcome and mechanical stability are linked on the cellular level, however, remains elusive. Cyclic-compressive loading of MSCs affects the expression of molecules involved in angiogenesis and matrix assembly, but also reduces the expression of CD73, an ecto-5'-nucleotidase, which plays a crucial role in extracellular adenosine generation. Although, for almost 20 years, CD73 has been a major cell surface marker defining MSCs, little is known about its function in these cells. Therefore, the aim of this study was to determine the putative involvement of CD73 in MSC differentiation after cyclic-compressive loading. After cultivation in appropriate differentiation media, chondrogenic differentiation ability was significantly increased in loaded MSCs, hence following current models. Through treatment with the CD73 inhibitor adenosine 5'-(α, β-methylene) diphosphate, chondrogenic matrix deposition was further increased; in contrast, mineral matrix deposition and expression of osteogenic markers was reduced. One major signal transduction pathway, which is activated via CD73-mediated adenosine, is the adenosine receptor pathway. Thus, the adenosine receptor expression pattern was investigated. MSCs expressed the four known adenosine receptors at the mRNA level. After mechanical stimulation of MSCs, Adora2a was down-regulated. These data point towards a role of CD73 in MSC differentiation possibly via A2AR signalling, which is mutually regulated with CD73. In conclusion, the findings of this study suggest that CD73 is another regulatory factor in osteo-/ chondrogenic differentiation of MSCs and may provide a -thus far underestimated -therapeutic target to guide bone regeneration. Keywords IntroductionBone healing is a complex, however well-orchestrated, multistage regenerative process. Bone fracture coincides with disruption of blood vessels resulting in activation of the coagulation cascade and formation of the haematoma, which encloses the fracture area. Inflammatory cells, fibroblasts, and mesenchymal stromal cells (MSCs) are recruited to the site. Once MSCs have reached the bone fracture site they are confronted with a challenging milieu characterised not only by inflammatory cytokines and low oxygen tension (hypoxia) but also by constant mechanical strain (Goodship and Kenwright, 1985;Komatsu and Hadjiargyrou, 2004). This condition is likely to affect MSCs, which are known to be mechanosensitive (Wang and Thampatty, 2008). Thus, detailed knowledge about the influence of mechanical loading on MSCs is pivotal for understanding the physiological processes during bone regeneration in order to develop innovative cell therapy approaches.Recently, we and others provided evidence that mechanical strain leads to reduced expression of the cell surface marker CD73 in vitro (Kang et al., 2011;Ode et al., 2011). In our study, bone marrow (BM)-MSCs underwent cyclic-compressive loading for three days. CD73 protein a...

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“…[14][15][16][17] CD105 has also been suggested in osteogenesis enhancement due to a reduction in TGF-β1/smad2 signaling. 18 CD73 has a regulatory function in controlling adipocyte, chondrogenic and osteogenic differentiation of MSCs. 18,19 Moreover, CD 105 was regulated by cytokines, growth factors, as well as culture conditions e.g.…”

“…18 CD73 has a regulatory function in controlling adipocyte, chondrogenic and osteogenic differentiation of MSCs. 18,19 Moreover, CD 105 was regulated by cytokines, growth factors, as well as culture conditions e.g. hypoxia.…”

“…18 Although CD73 is known to be down-regulated during osteogenic, adipocyte, and chondrogenic differentiation, 18 until recently, there are no information whether CD73 acts as a transient or definite marker. Although CD90, CD105, and CD73 are not tissue specific markers and the expression are slightly reduced during differentiation, 18,19 the combination is often used as defining criteria for MSCs. 2 -4 In our study, hADSCs underwent spontaneous chondrogenic differentiation for Ps-1, Ps-2, and Ps-3 ( Figure 2).…”

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