Pursuing a novel bioactive dressing: stimulation of dermal fibroblasts with calcium-phosphate nanoparticles

摘要

Introduction: Hard-to-heal wounds are a major socio-economic problem in developed countries. In the recent years, bioactive dressings have been developed, mainly based in the addition of biological factors. However, these present significant limitations, such as high cost of production or regulatory challenges. Extracellular calcium could be considered as an alternative biostimulator, since it has been probed to affect the healing process of skin injuries. However, a better understanding of its effect is needed, together with a system to achieve a controlled release. In this study we tested whether the ionic dissolution of calcium-releasing nanoparticles achieved a similar stimulating effect as extracellular calcium on dermal fibroblast in vitro. Materials and Methods: Rat dermal fibroblasts were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5mM Ca2+. Before adding it to the cells, media was incubated with calcium chloride(CaCl2) or with calcium-phosphate nanoparticles (CaP) for 24h at 37°C and filter sterilized. Several relevant aspects in the wound healing process were assessed by different in vitro assays. Metabolic activity was determined with the Alamarblue® kit. Proliferation was measured by DNA quantification. Cell migration was quantified by a wound scratch assay. Total collagen synthesis was determined by means of a commercial kit. Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) levels of activity were analyzed by a zymography assay. Finally, VEGF synthesis was measured with an ELISA. Results and Discussion: The effects stimulated by calcium from media conditioned with CaP did not exactly correlate with the one from CaCl2. This means that the other factors that the CaP are releasing, such as phosphates, are also affecting the cells. Interestingly, 3.5mM from both CaCl2 and CaP enhanced metabolic activity, in vitro wound closure and collagen synthesis. However, CaP decreased MMP activity, proliferation and VEGF synthesis. Conclusions: Even though calcium coming from CaP did not have the same effect as CaCl2 on dermal fibroblasts, the concentration of 3.5mM Ca2+ from CaP was able to enhance metabolic activity, migration, collagen synthesis and to decrease MMP activity. All these effects might promote a faster healing. A better understanding of how other cell types involved in the healing process respond to a range of concentrations of calcium-phosphate particles may lead to a wiser design of novel bioactive dressings for skin wound healing.