Research Of Silk Fibroin/Collagen Scaffold Combined With A Co-Culture Of Schwann Cells And Adipose-Derived Stem Cells For Sciatic Nerve Repair In Rats

Objectives:As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an ideal regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body's natural regenerative microenvironment closely, we co-cultured Schwann cells(SCs) and adipose-derived stem cells(ADSCs) as seed cells and introduced them into a silk fibroin(SF)/collagen scaffold to construct a tissue-engineered nerve conduit(TENC). Then they were transplanted to bridge 10-mm long sciatic nerve defects in rats and a series of electrophysiological examinations and morphological analyses were performed twelve weeks after surgery to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration.Methods:1.To isolate and culture of Schwann cells, and were identified by morphological observation and immunocytochemical labeling of the S100 protein. To isolate and culture of adipose-derived stem cells, and were determined by flow cytometry analysis(passage 3) with cell surface antigens of CD29?CD34?CD45?CD73?CD90and CD105. SCs and ADSCs(passage 3) were co-cultured at a ratio of 2?1 and observed the cells morphological changes under inverted phase contrast microscope.Immunofluorescence were adopted to investigate the expression differences for microtubule-associated protein 2(MAP2), neuronal nuclei protein(Neu N), and glial fibrillary acidic protein(GFAP) of SCs and ADSCs at 14 days after co-cultured.2.Preparation of silk fibroin/collagen scaffold for tissue engineering: To prepare silk protein and collagen with silkworm cocoon and beef tendon, blend the two solution at a quality ratio of 2?1 and used freeze-drying technique to make composite silk fibroin/collagen scaffold.3. Construction and characterization testing of tissue-engineered nerve:introduced the co-cultured system of Schwann cells and adipose-derived stem cells into silk fibroin /collagen scaffold to construct a tissue-engineered nerve conduit(TENC). Scanning electron microscopy was performed to observe the structure of theSF/collagen scaffold and the cells' growth. The mechanical properties were examined using a universal testing machine(Instron5865), and Young's modulus was calculated.4. Sciatic nerve defect model construction and transplanted with different grafts:those bridged with plain SF/collagen scaffolds(Scaffold group), those bridged with TENCs(TENC group), those bridged with autografts(Autograft group) and those unoperated side(Normal group).5. General observations, immunocytochemical labeling, electrophysiological analysis, and morphological econometric analysis were performed to evaluate the effect of regenerated nerves.Results:1. SCs was successfully separated and purificated. After cultured three passages showed high purity without impurities. The ADSCs(passages 3) were positive for CD29(99.38%), CD90(96.21%), CD73(99.80%), and CD105(97.97%) but negative for CD34(0.57%) and CD45(0.67%). We could see ADSCs showed contraction of nucleus, increasing of soma refraction, and several long and thick protrusions of cell body after SCs-ADSCs co-cultured 14 days. Schwann cell volume increases, and the surrounding projection became thicker at the same time. Immunofluorescence results also showed the expressions of MAP2, Neu N, and GFAP.2. The plain SF/collagen scaffold shows appropriate pore size and good intercommunicating of holes. The cells were tightly attached to and partly coiled about the scaffold and exhibited either a spindle or a spherical shape. The results of the mechanical measurement revealed that the maximum and average Young's moduli of the SF/collagen scaffold were 10.8 ± 0.3 MPa and 8.14 ± 0.2 MPa, respectively.The mechanical properties ensure that the scaffold could resist muscular contraction and maintain its shape unchanged for a considerable period of time after grafting.3. All animals in each group have achieved nerve defect regeneration in varying degrees. But in terms of the effect of the repaired nerve,those treated with TENC were similar to those with autologous nerve grafts but superior to those with plain SF/collagen scaffolds.Conclusions:1. The co-culture system of SCs and ADSCs not only could grow together, but also could obviously induce ADSCs neural transdifferentiation.2. Silk fibroin and collagen as two natural and biodegradable materials were blend at a suitable quality ratio, and possessed adequate mechanical properties, good biocompatibility and suitable degradation. Silk fibroin/collagen scaffold may providean ideal carrier for the tissue-engineered nerve.3.The tissue-engineered nerve conduit(TENC) that combined silk fibroin/collagen scaffold with a co-culture of Schwann cells and adipose-derived stem cells had normal nerve-like structure, and could bridge sciatic nerve defect and promote nerve growth.