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.
ABSTRACT
BACKGROUND: The skill to culture osteoblasts primarily has been well developed. However, trypsinase can affect membrane protein of osteoblasts if the time of digestion is long. Therefore, it is of great significance to select an ideal method to avoid the damage from trypsinase to cells as possible when culturing osteoblasts.OBJECTIVE: To explore a novel method to isolate and culture SD rat osteoblasts in vitro, and identify the functions of the cells.DESIGN: Observational study.SETTING: Department of Orthopaedics, First Affiliated Hospital of Jinan University.MATERIALS: This experiment was carried out in the Department of Orthopaedics, First Affiliated Hospital of Jinan University from March to May in 2007. Eight SPF 24-hour old SD rats were used in the experiment. The rats, irrespective of gender, were provided by the Experimental Animal Center of Nanfang Medical University. The experimental animals were disposed according to ethical criteria. The main reagents were detailed as follows: collagenase Ⅱ (Sigma Company);trypsin (Sigma Company); alkaline phosphatase (ALP) kit (Nanjing Jiancheng Biological Products Company); SABC-1021(Wuhan Boster Biotechnology Company).METHODS: 24-hour old SD rats were chosen for experiment. The newly born SD rats were sacrificed by anesthesia and the cranial bones of the rats were obtained cleanly, erased completely of the periosteum and cut to blocks of I mm3. The cranial bones were digested by 0.25 % trypsinase for 20 minutes, then by 0. 1% type Ⅱ collagenase for 60 minutes. The digestive time of trypsinase was controlled in the process of digestion to avoid to harm the cells. The liquid was gathered and centrifuged. The cells were cultured in culture flask and were purified by many times adhered.MAIN OUTCOME MEASURES: Morphology observations under the inverted phase contrast microscope, transmission electron microscope, and scanning electron microscope were performed. The phenotype, calcium tuberculation and the expression of alkaline phosphatase were studied with alizarin red staining and modified Gomori Ca-Co assays respectively.The cells were also evaluated with collage Ⅰ immunohistochemical staining.RESULTS: The cultured cells had active proliferation ability. Cells showed multi-angle or fusiform shape. Nucleus was immature and organell was plentiful. Therefore, they had typical morphological characters of osteoblasts. Moreover, they showed the osteoblastic phenotypes such as their synthesis of alkaline phosphatase, collage Ⅰ and formation of calcium tuberculations.CONCLUSION: The cells cultured by our modified enzymatic digestion method had typical morphological and biological characteristics of osteoblasts.
