Abstract
Postmenopausal females display a chronic inflammatory phenotype with higher levels of circulating pro-inflammatory cytokines. Furthermore, the inflammatory response to injury may be altered under estrogen-deficiency, because it was shown previously that estrogen-deficient mice displayed increased levels of the inflammatory cytokines Midkine (Mdk) and Interleukin-6 (IL-6) in the early fracture hematoma. Because a balanced immune response to fracture is required for successful bone regeneration, this might contribute to the delayed fracture healing frequently observed in osteoporotic, postmenopausal fracture patients. In this study, we aimed to investigate whether further cytokines in addition to Mdk and IL-6 might be affected by estrogen-deficiency after fracture in mice and whether these cytokines are also relevant during human fracture healing. Additionally, we aimed to investigate whether serum from male vs. female fracture patients affects osteogenic differentiation of human mesenchymal stem cells (MSCs). To address these questions, female mice were either sham-operated or ovariectomized (OVX) and subjected to standardized femur osteotomy. A broad panel of pro- and anti-inflammatory cytokines was determined systemically and locally in the fracture hematoma. In a translational approach, serum was collected from healthy controls and patients with an isolated fracture. Mdk and IL-6 serum levels were determined at day 0, day 14 and day 42 after fracture. Subgroup analysis was performed to investigate differences between male and female fracture patients after menopause. In an in vitro approach, human MSCs were cultured with the collected patient serum and osteogenic differentiation was assessed by qPCR and alkaline-phosphatase staining. Our results suggest an important role for the pro-inflammatory cytokines Mdk and IL-6 in the response to fracture in estrogen-deficient mice among all of the measured inflammatory mediators. Notably, both cytokines were also significantly increased in the serum of patients after fracture. However, only Mdk serum levels differed significantly between male and female fracture patients after menopause. MSCs cultivated with serum from female fracture patients displayed significantly reduced osteogenic differentiation, which was attenuated by Mdk-antibody treatment. In conclusion, our study demonstrated increased Mdk levels after fracture in OVX mice and female fracture patients after menopause. Because Mdk is a negative regulator of bone formation, this might contribute to impaired osteoporotic fracture healing.
Highlights
Chronic inflammatory conditions, including rheumatoid arthritis, diabetes mellitus and inflammatory bowel disease, which are associated with bone loss, corroborate an intense coupling of the immune and skeletal systems [1,2,3,4]
The first aim of this study was to conduct multiplex cytokine analysis in blood and fracture hematoma of sham- and OVX-mice to investigate whether further cytokines in addition to Mdk and IL-6 are affected by estrogen-deficiency
Physiological concentrations of IL-13 and Monocyte chemoattractant protein-1 (MCP-1) were detectable in both groups, the concentrations did not increase after fracture and did not change between both groups at the investigated time points
Summary
Chronic inflammatory conditions, including rheumatoid arthritis, diabetes mellitus and inflammatory bowel disease, which are associated with bone loss, corroborate an intense coupling of the immune and skeletal systems [1,2,3,4]. Postmenopausal osteoporotic females frequently display elevated levels of pro-inflammatory cytokines and alterations in immune cell populations, which were shown to negatively affect bone turnover and quality [6,7,8,9]. The process of bone repair starts with a local inflammatory response at the fracture site [14,15]. This inflammatory reaction is marked by blood vessel disruption, tissue and cell damage and the formation of a fracture hematoma, leading to the recruitment of immune cells and mediators. The inflammatory response was shown to have an osteogenic potential by recruiting mesenchymal stem cells (MSCs) to the injury site and inducing the subsequent repair phase [14]. Once the fracture gap is bridged with bone, there is sufficient mechanical stability and the remodeling of the bony fracture callus is initiated [16,17]
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