The Autologous Gelsolin Combined with Exogenous Nucleotides enhance Chondrogenic Differentiation in Equine Adipose Derived Mesenchymal Stromal Cells - An In Vitro Research

Abstract

Background: Nowadays, veterinary practice, involves treatment of disorders associated with cartilage degeneration using advanced therapy, based on mesenchymal stromal cells application. Preparation of cells for the transplantation requires in vitro culture and evaluation of their chondrogenic potential. Purpose: To determine if autologous gelsolin derived from equine serum and exogenous nucleotides may enhance the metabolic activity and chondrogenic differentiation of equine adipose derived mesenchymal stromal cells (EqASCs). Methods: Mesenchymal stem cells were isolated from equine subcutaneous fat tissue. Standard growth medium was supplemented with 1% of gelsolin or/and 0.1 mg/ml of nucleotides. Proliferation activity of the cells was determined basing on results obtained with cytotoxic assay and analysis of microvesicles shedding. Morphology, cytophysiological activity and chondrogenic differentiation potential were evaluated utilizing light, fluorescent and scanning electron microscopy. The effects of chondrogenic stimulation were determined via (i) analysis of gene expression for cytoskeleton and matrix proteins; (ii) proteoglycan histochemistry and (iii) analysis of culture growth pattern and chondro-nodule formation. Results: The enhancement of proliferation activity was noticed in cultures stimulated with exogenous nucleotides. Although the addition of gelsolin did not affect EqASCs proliferation, it contributed to cytoskeleton integrity. Both investigated factors positively influenced on chondrogenic differentiation – gelsolin through stabilization of cytoskeleton proteins expression, while nucleotides by promoting nodules formation. Combination of gelsolin and nucleotides enhanced cells proliferation, what was reflected by higher cellular activity and microvesicles shedding and influenced on chondro nodules formation. Conclusions: Autologous gelsolin and exogenous nucleotides implemented in the in vitro cultures of mesenchymal stromal stem cells isolated from adipose tissue may enhance cellular integrity, proliferation and chondrogenic differentiation of those cells, thus directly might influence on regenerative potential of adipose derived mesenchymal stromal stem cells transplants used for equine osteoarthritis treatment.