Characterization of Maturation of Tissue Engineered Skeletal Muscle Bundles in Rheumatoid Arthritis

Abstract

Rheumatoid Arthritis (RA) is a chronic inflammatory auto-immune disease typically involving the joints, mainly the diarthrodial joint and generally starts between the age of 30 and 60 in women and somewhat later in life in men. It is the most common inflammatory arthritis and about one percent of the population is affected by RA. A complex interaction between various genetic and environmental factors lead to the development of the disease, though the specific cause of RA is not known. The goal of this study is to characterize the maturation of skeletal muscle bundles made with myoblasts isolated from RA patients and compare it with maturation of age-matched controls. Moreover, the engineered myobundles were treated with pro-inflammatory cytokines to assess their effect on the bundle maturation and to replicate the pro-inflammatory phenotype of RA. Myobundles were prepared with human skeletal muscle (HSkM) samples obtained from young controls, age-matched controls and RA patients through biopsy of vastus lateralis muscle (biopsy of hamstring muscle was taken for young controls). We measured nuclei count, cross-sectional area, Myogenin count, Sarcomeric alpha-actinin (SAA) positive area and the myofiber diameter for each time course studies and cytokine treated bundles.Contrary to our expectations, the time course study did not indicate significant reduction in fiber formation. This may be due to the effect of medications taken by the RA patient which might be helping the muscle function. Another possible reason might be that the cells could have regained their normal function once they were taken out from the inflammatory environment induced by the pro-inflammatory cytokines. Yet another possible reason may be that the time course considered may not be enough to access changes in the maturation and a longer time period may be required.We then moved forward to replicate the disease pro-inflammatory phenotype by carrying out cytokine treatments on the engineered myobundles. IFNγ, IFNγ+GMCSF, TNFα+GMCSF and IFNγ+TNFα+GMCSF were chosen for the cytokine treatments. According to our results, the cross-sectional area, nuclei count/CSA, MyoG count/CSA, MyoG/Nuclei count, SAA+ area and the myofiber diameter each decreased with cytokine treatments indicating that the cytokines may indeed affect the regeneration ability of skeletal muscle cells. The results from cytokines treatment studies indicate that cytokines do play a role in disease development and progression. A longer time course study say for up to 10 days or more post differentiation, more patient data regarding the disease severity and medications might also be helpful in further investigation.