In vitro biocompatibility of three chitosan/polycation composite materials for nerve regeneration

Zhenhuan Zheng; Yujun Wei; Gan Wang; Yandao Gong; Xiufang Zhang

摘要：BACKGROUND: It has been reported that chitosan nerve conduits could support axon elongation and improve relevant function during in vivo nerve regeneration.OBJECTIVE: To investigate in vitro biocompatibility of three novel, chitosan/polycation composite materials for nerve regeneration in cultured mouse Schwann cells and PC 12 cells.DESIGN, TIME AND SETTING: The observational, control experiments for nerve tissue engineering were performed at the Department of Biological Sciences and Biotechnology of Tsinghua University from August 2007 to January 2008.MATERIALS: Mouse Schwann cells were isolated from the sciatic nerve of 5-7-day-old BALB/C mice.PC12 cells were purchased from the American Type Culture Collection (ATCC, USA). Chitosan was purchased from Tsiugdao Haishcng Co., China. Poly-L-lysine hydrochloride (PLL), polyethyleneimine (PEI),poly-L-ornithinc hydrobromide (POR), and S-100 antibody was purchased from Sigma Chemical Co., USA.Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Chemical Co., Japan.METHODS: Three chitosan/polycation composite materials for nerve regeneration (PLL-0.25, PEI-0.25,and POR-0.25) were produced by blending chitosan with 0.25% (w/w) poly-L-lysine, polyethyleneimine, and poly-L-ornithine. Pure chitosan was utilized as the control. After 3 days of culture, the morphology of mouse Schwann and PC12 cells cultured on all substrates was observed with an inverted phase contrast microscope.Mouse Schwann cells were stained by immunofluorescence labeling S-100 protein and nuclei, followed by identification with a confocal laser-scanning microscope. The amount of proliferating mouse Sehwann and PC12 cells was determined by CCK-8 after 1, 3, and 5 days in culture. The level of PC12 cell differentiation on all substrates was assessed by measuring neurite length at 1, 3, and 5 days after seeding.MAIN OUTCOME MEASURES: Morphology and amount of proliferation of mouse Schwann cells and PC12 cells cultured on chitnsan and three polycation-modified materials, as well as amount of differentiation in PC12 cells on these substrates.RESULTS: (1) Morphology of mouse Schwann cells and PC 12 cells on all substrates: after 3 days in culture,on three different chitosan/polycation composite substrates, Schwann cells were connected to each other and exhibited greater proliferation, compared to the chitosan control. In particular, on PLL-0.25 and POR-0.25substrates, some cells congregated and nearly reached confluence. The PC12 cells on chitosan substrate, after 3 days in culture, maintained a round shape; few exhibited a bipolar shape and began to form neurite extensions. However, on PLL-0.25 and POR-0.25 substrates, most PC12 cells displayed a bipolar shape with obvious neurite outgrowth, and almost grew as an adherent, spreading monolayer. (2) Proliferation of mouse Schwann cells and PC12 cells on all substrates: on the first day, Sehwann cell proliferation on the three composite substrates was significantly greater than the cells on chitosan control (P < 0.01). After 3 and 5days in culture, PLL-0.25 and POR-0.25 substrates resulted in greater cell proliferation when compared to pure chitosan (P < 0.01). On the third and fifth day in culture PC 12 cell proliferation on PLL-0.25 and POR-0.25 was significantly greater than on chitosan substrate (P < 0.01). (3) Differentiation of PCl 2 cells on all substrates: at all time points, the average neurite length of cells cultured on composite materials was significantly longer than on chitosan control (P < 0.05-0.01). Cells on PLL-0.25 exhibited the longest average ncuritc length at days 3 and 5.CONCLUSION: Mouse Schwann cells and PC12 cells exhibit in vitro biocompatibility with poly-L-lysineand poly-L-ornithine-modified substrates, which indicates that these substrates could serve as suitable substrates for peripheral nerve regeneration.