Biocompatibility of chitosan-sodium\collagen nanometer fiber scaffold with co-cultured bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: The structure of nanometer chitosan-sodium/collagen (nano-CS/COL) is similar to that of the extracellular matrix (ECM) in the nanometer level. Whether this can promote the adhesion and growth of bone marrow mesenchymal stem cells (MSCs) and the calcification?OBJECTIVE: To investigate the in vitro histocompatibility of nano-CS/COL. DESIGN: Single sample observation.SETTING: Department of Orthopaedics, First Hospital, Jinan University. MATERIALS This study was performed at the Experimental Center, First Hospital Affiliated to Jinan University between March 2007 and July 2007. Ten 4-week-old female SD rats, of SPF grade, weighing 200 g, were provided by the Guangdong Provincial Laboratory Center [Permission No. SCXK (yue) 2003-0002]. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Nano-CS/COL METHODS: Bone marrow MSCs were isolated from SD rats and cultured. Cell surface antigen was detected by loss cellanalyticalmethod.Nano-CS/COLscaffold waspreparedbypolyelectrolyte confocallaser-scanning microscopy. The well-grown cells of the third passage were co-cultured in vitro on the nano-CS/COL scaffold. Taking simple nano-CS/COL scaffold material as control, the histocompatibility of scaffold material and cells were comprehensively evaluated by cell adherence rate, growth curve, cell activity and cycle, and scanning electron microscope observation.MAIN OUTCOME MEASURES: ① Identification of cell surface antigen marker after isolation and culture of bone marrow MSCs. ②The histocompatibility of nano-CS/COL material and bone marrow MSCs 2, 4 and 8 days after nano-CS/COL material compounded with cells. ③Determination of adherence rate of cells to nano-CS/COL material. ? Cell circle and activity detected 5 days after nano-CS/COL material compounding with cells. RESULTS: ① Detection results of cell surface antigen marker The expression of CD29, CD106, CD44, CD34 and CD45 was 90.86%, 73.38%, 82.61%, 0.76% and 0.60%, respectively. ②Histocompatibility of bone marrow MSCs and nano-CS/COL material: It was shown under the scanning electron microscope that nano-CS/COL scaffold presented porous three-dimensional structure, and different sizes of macropoles and interconnected small pores. The interval porosity determined by quality assay was 85%-90%, and aperture averaged 150 μm (range 50 - 300u m). Two days after bone marrow MSCs compounded to nano-CS/COL scaffold, bone marrow MSCs presented globular shape and were scattered; Four days later, bone marrow MSCs presented shuttle shape, extended and anchored on the surface of nano-CS/COL by pseudopods; Eight days later, bone marrow MSCs proliferated and fused each other, and they secreted a lot of extracellular matrix, then which covered most material particles. ③ The adherence rate of bone marrow MSCs to nano-CS/COL Bone marrow MSCs and nano-CS/COL were co-cultured 2 and 6 hours separately. The adherence rate of bone marrow MSCs was higher to nano-CS/COL scaffold than to simple chitosan scaffold. ④ Comparison of cells and cell cycle between on nano-CS/COL scaffold and on the chitosan scaffold On the nano-CS/COL scaffold, cell activity was 96.67%, cell cycle at G0-G1 was 90.81%, at G2-M was 0.52% and at S was 8.66%. G2/G1 was 1.81. On the simple chitosan scaffold, cell activity was 95.27%, cell cycle at G0-G1 was 87.14%, at G2-M was 9.69%, and at S was 4.16%. G2/G1 was 1.80.CONCLUSION: Nano-CS/COL scaffold can be used as tissue engineering biomaterials because bone marrow MSCs can well grow on it.