Proinflammatory Mediators Enhance the Osteogenesis of Human Mesenchymal Stem Cells after Lineage Commitment

Abstract

Several inflammatory processes underlie excessive bone formation, including chronic inflammation of the spine, acute infections, or periarticular ossifications after trauma. This suggests that local factors in these conditions have osteogenic properties. Mesenchymal stem cells (MSCs) and their differentiated progeny contribute to bone healing by synthesizing extracellular matrix and inducing mineralization. Due to the variation in experimental designs used in vitro, there is controversy about the osteogenic potential of proinflammatory factors on MSCs. Our goal was to determine the specific conditions allowing the pro-osteogenic effects of distinct inflammatory stimuli. Human bone marrow MSCs were exposed to tumor necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS). Cells were cultured in growth medium or osteogenic differentiation medium. Alternatively, bone morphogenetic protein 2 (BMP-2) was used as osteogenic supplement to simulate the conditions in vivo. Alkaline phosphatase activity and calcium deposition were indicators of osteogenicity. To elucidate lineage commitment-dependent effects, MSCs were pre-differentiated prior treatment. Our results show that TNF-α and LPS do not affect the expression of osteogenic markers by MSCs in the absence of an osteogenic supplement. In osteogenic differentiation medium or together with BMP-2 however, these mediators highly stimulated their alkaline phosphatase activity and subsequent matrix mineralization. In pre-osteoblasts, matrix mineralization was significantly increased by these mediators, but irrespective of the culture conditions. Our study shows that inflammatory factors potently enhance the osteogenic capacity of MSCs. These properties may be harnessed in bone regenerative strategies. Importantly, the commitment of MSCs to the osteogenic lineage greatly enhances their responsiveness to inflammatory signals.

Fig 1. Expression of osteogenic markers by MSC treated with proinflammatory mediators.

MSCs were exposed to TNF-α (5 ng/mL) or LPS (0.5 μg/mL). A. ALP normalized for DNA content was measured at day 10. Bars represent the means ± SD (n = 6). *P<0.05 compared to untreated cells cultured in the same medium. B. Alizarin Red S staining was performed to demonstrate matrix mineralization (representative for 4 donors). Scale bar: 500 μm. See S2A and S2B Fig for all concentrations.

Fig 2. ALP expression by MSCs after short TNF-α or LPS treatment.

Cells were exposed to TNF-α (50 ng/mL) or LPS (5 μg/mL) for 2 days, after which the mediators were withdrawn. At day 10, ALP activity levels were measured and normalized for DNA. Data represent the means ± SD (n = 4). *P<0.05 compared to untreated cells cultured in the same medium. See S2C Fig for all experimental conditions.

Fig 3. Late osteogenic differentiation in pre-osteoblasts.

A. Pre-osteoblasts were exposed to TNF-α (50 ng/mL) or LPS (5 μg/mL). The ALP activity was measured after 8 days and normalized for DNA content. The total calcium deposition was quantified at day 14 after continuous (B) and short (C) stimulation with TNF-α (5 ng/mL) or LPS (0.5 μg/mL). D. Cells were exposed to LPS (0.5 μg/mL) or TNF-α (5 ng/mL) from days 12 to 26 of culture. Immunocytochemical staining for intracellular osteocalcin was performed as a marker of osteogenic differentiation. Osteocalcin and Hoechst are shown in green and blue, respectively (representative for 2 donors). Scale bar: 500 μm. See S4 Fig for a dose response. Data represent the means ± SD (n = 4). *P<0.05/**P<0.005 compared to untreated cells cultured in the same medium.