Abstract
Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation.
Highlights
Fractures are generally accompanied by soft tissue trauma that is aggravated by subsequent surgical stabilization
Values are mean ± SEM, n = 10 from 3 independent experiments using ASCs obtained from 5 stem cell donors. *Significant effect of the combination hypothermia and hypoxia compared to hypoxia alone; ‡Significant effect compared to controls, p
Hypothermia may modulate the effects of ischaemia and hypoxic conditions that are prevalent in the wound environment, but whether the combined effect of hypothermia and hypoxia is beneficial for the cells responsible for callus formation is unknown
Summary
Fractures are generally accompanied by soft tissue trauma that is aggravated by subsequent surgical stabilization. The interruption of arterial vascular flow causes regional ischaemia and hypoxia, resulting in inflammation [1]. Cryotherapy seems to be a modulator of the posttraumatic inflammatory reaction, but results obtained do not unanimously agree on the way it affects inflammation. Cryotherapy has been reported to reduce posttraumatic microvascular dysfunction, inflammation, and structural impairment in a rodent model [2]. Hypothermia prolongs the inflammatory response systemically and locally in fracture hematomas in a porcine model [3]. Cryotherapy can be applied in the acute recovery phase of musculoskeletal trauma and after orthopaedic surgical interventions, such as knee arthroplasty to prevent pain and inflammation [4]. The clinical effect of cryotherapy is being investigated in postoperative hip fracture patients [5], even though the effect of application of cryotherapy on osteoblast precursor proliferation and differentiation during bone tissue repair has not been clearly established
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