Myogenic differentiation of human myoblasts, ADSC, and Schwann cells on PCL-collagen I-nanofibers in the neurotized arteriovenous loop model

2. Abstract 2.1 Background Tissue engineering is a promising alternative treatment for volumetric muscle loss. In tissue engineering, vascularization and innervation play essential roles in the establishment of functional skeletal muscle. Adipogenic mesenchymal stromal cells (ADSC) are capable of differentiating towards the myoblast lineage when co-cultured with myoblasts (Mb). In addition, motor neurons co-cultured with myoblasts also contribute to myogenic differentiation and innervation. Schwann cells (SC), as a component of the peripheral nervous system, have been reported to promote myogenic differentiation of Mb/ADSC co-cultures in vitro. For adequate 3D assembly, biocompatible aligned nanofibers are beneficial as matrices for cell seeding. The aim of this study was to analyse the impact of SC on Mb/ADSC co-cultures seeded on poly-ɛ-caprolacton (PCL)-collagen I-nanofibers, implanted into the neurotized arteriovenous loop model (EPI loop model). 2.2 Material and Methods Mb and ADSC were isolated from human tissue. Human SC was purchased from Innoprot. Mb and SC were characterized by Desmin and S100 staining. Characterization of ADSC was performed by flow cytometry and by their ability to differentiate into multiple lineages. Cells were seeded on PCL-collagen I-nanoscaffolds for Mb/ADSC group at a ratio of 1:1 and Mb/ADSC/SC group at a ratio of 1:1:0.5 based on preliminary in vitro experiments. After one-week of proliferation and one-week of differentiation in vitro, scaffolds were implanted into the EPI-loop model of immunodeficient rats. 22 rats were divided into Mb/ADSC and Mb/ADSC/SC group. Five rats from each group were used for gene expression analysis and other six were used for histological analysis. After four weeks, rats were sacrificed and explantation was performed. Afterwards, the constructs were prepared for micro-CT, immunohistology and evaluated in terms of angiogenesis and immunoreaction. Evaluation of myogenic differentiation and innervation was performed via immunofluorescence and quantitative polymerase chain reaction (qPCR) of gene expression including MYOD (myoblast determination protein 1), MYOG (myogenin), ACTA1 (skeletal muscle actin α), MYH2 (myosin heavy chain 2), MYH3 (myosin heavy chain 3), MBP (myelin basic protein), CHRNB (cholinergic receptor nicotinic beta subunit), and LAMB2 (laminin subunit beta 2). 2.3 Results Desmin staining of Mb showed that all selected isolations yielded high purity of Mb. Flow cytometry revealed characteristic surface markers of ADSC in P3 and P6. ADSC were positive for CD90/73 (99.0 % and 97.6 %, respectively) and CD105 (99.8 % and 98.6 %, respectively) in P3 and P6. ADSC could also be successfully differentiated into the adipogenic, osteogenic, and chondrogenic lineage in both P3 and P6. SC were positive for S100 staining. In terms of gel degradation, there was a tendency towards lower construct weight and height for Mb/ADSC compared to Mb/ADSC/SC. Vascularization of the constructs by the EPI loop was successfully demonstrated in all groups via alpha smooth muscle actin (ASMA) staining and µCT. After four weeks, both Mb/ADSC and Mb/ADSC/SC co-culture systems showed similar amounts and distribution of vascularization. Obvious vascularization was also observed in the vicinity of the implanted motor nerve. With regard to immunological activity, CD68 and CD163 positive macrophages were observed in both groups without statistical difference. Myogenic differentiation could be observed in both groups via histological staining (desmin, myosin heavy chain) and gene expression (MYOD, MYH3, ACTA1) without significant difference between groups. Expression of CHRNB and LAMB2 also implied possible neuromuscular junction formation. 2.4 Conclusion Our study suggested that the addition of SC did not significantly impact myogenesis and innervation in our vascularized and innervated EPI-loop model. The implanted motor nerve branch may have played a more significant role than the presence of SC. The EPI-Loop model in combination with PCL-collagen I-nanoscaffolds may serve as a possible model for skeletal muscle tissue engineering.