Neural‐differentiated mesenchymal stem cells incorporated into muscle stuffed vein scaffold forms a stable living nerve conduit

Abstract

Autologous nerve grafts to bridge nerve gaps have donor site morbidity and possible neuroma formation resulting in development of various methods of bridging nerve gaps without using autologous nerve grafts. We have fabricated an acellular muscle stuffed vein seeded with differentiated mesenchymal stem cells (MSCs) as a substitute for nerve autografts. Human vein and muscle were both decellularized by liquid nitrogen immersion with subsequent hydrolysis in hydrochloric acid. Human MSCs were subjected to a series of treatments with a reducing agent, retinoic acid, and a combination of trophic factors. The differentiated MSCs were seeded on the surface of acellular muscle tissue and then stuffed into the vein. Our study showed that 35-75% of the cells expressed neural markers such as S100b, glial fibrillary acidic protein (GFAP), p75 NGF receptor, and Nestin after differentiation. Histological and ultra structural analyses of muscle stuffed veins showed attachment of cells onto the surface of the acellular muscle and penetration of the cells into the hydrolyzed fraction of muscle fibers. We implanted these muscle stuffed veins into athymic mice and at 8 weeks postimplantation, the acellular muscle tissue had fully degraded and replaced with new matrix produced by the seeded cells. The vein was still intact and no inflammatory reactions were observed proving the biocompatibility and biodegradability of the conduit. In conclusion, we have successfully formed a stable living nerve conduit which may serve as a substitute for autologous nerves. ß

Collagen-Coated Polylactic-Glycolic Acid (PLGA) Seeded with Neural-Differentiated Human Mesenchymal Stem Cells as a Potential Nerve Conduit*

“…Undifferentiated MSCs displayed a flat fibroblast-like morphology: a spindle shape with short processes whereas differentiated cells appear in star-shape with elongated processes. Flow cytometry analysis also showed that human MSCs isolated from human bone marrow in our lab have tested positive for mesenchymal cell surface markers such as CD 90, CD105, CD 10, CD 29, and CD 44 but negative for hematopoietic cell markers, CD34 and CD 45 [15]. To examine the efficiency of MSC differentiation as an alternative source of SCs, the cells were subjected to a series of treatments with a reducing agent, retinoic acid, and a combination of trophic factors.…”

“…Previous studies have shown that PLGA-coated collagen is superior to vein graft in nerve regeneration [20,21]. Furthermore, the ability of human MSCs to differentiate into neuronal cells and facilitate the neuronal growth that has been studied previously [15,22]. Thus, we undertook the study to evaluate the survival and propagation of human nMSC in collagen-coated PLGA tube.…”

“…Bone marrow was obtained from pedicle blood during the insertion of pedicle screws in spine surgery, intramedullary nailing procedures and amputations of the limbs. These samples were cultured and differentiated as described earlier [15]. Briefly, the bone marrow was cultured in α-minimal essential medium (α-MEM) (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA), and incubated at 37°C and 5% CO 2 .…”

“…Both undifferentiated and differentiated MSC cultures from a total of 6 patients were evaluated by a flow cytometry analysis as described previously [15].…”

“…Previously, we have successfully differentiated the human mesenchymal stem cells into neural-differentiated human mesenchymal stem cells (nMSC) [15,16]. Phase-contrast microscopic observation showed morphological changes of human MSCs before and after induction.…”