Bone formation performance by autograft of rabbit mesenchymal stem cells co-cultured with nano basal hydroxyapatite materials

Jianzhong ZHU; Zongning MIAO; Hanguang QIAN; Jidong ZHAO

ABSTRACT

BACKGROUND:

Bone bone bone marrow derived mesenchymal stem cells (MSCs) has the capacities of self-renewal and multi-directional differentiation, which can differentiate into osteoblasts after induction. Combined with basal hydroxyapatite materials, MSCs can repair the bone defect with satisfactory shape and function.

OBJECTIVE:

To observe the bone formation performance of segmental bone defect repaired by autologous transplantation after combination of MSCs and basal hydroxyapatite materials.

DESIGN:

Open experiment.

SETTING:

Department of Cells, Third People' s Hospital of Wuxi.MATERIALS The experiment was conducted in the Department of Cells,Third People's Hospital of Wuxi between May 2004 and March 2005.Eighteen New Zealand rabbits of clean grade, aged 6 months were selected and randomly divided into cytoskeleton group, simple scaffold group and blank control group with 6 rabbits in each group. Nano-collagen basal hydroxyapatite bone were provided by the Department of Material, Tsinghua University, which is characteristic of natural-bone microstructure. Composition about 57% were hydroxyapatite,and 30% were collagen and 13%were polylactic acid.

METHODS:

①bone bone marrow derived MSCs of rabbits were isolated and cultured. Well-grown cells of the second generation were inspected of the expression of cell surface antigen (CSA) with EPICS-ALTRA flow cytometry. ②Scaffold materials were cut into blocks with the size of 6 mm ×6 mm×4 mm, sterilized by 60Co irradiance and infiltrated with DMEM-LG before using. Cells of the 2nd and 3rd generations were collected, cultured,the concentration of which was regulated to 109 L-1. Cells were co-cultured with nano-collagen basal hydroxyapatite in vitro. ③Model of segmental defects in radial of rabbits were established in all groups. The prepared compounds in the cytoskeleton compound group were embedded in the bone defect according to autograft principle, and blank scaffold materials were embedded in the bone defects of simple scaffold group. Nothing was transplanted in the blank control group. Rabbits in all groups received no internal or external fixation, whose soft tissues and skins were closely sutured.④The surface structure of materials and cell adherence in all groups were observed under scanning electron microscope (SEM).⑤Bone formation was studied by general observation, histological analysis and X-rays at 4, 8 and 16 weeks after operation respectively.MAIN OUTCOME

MEASURES:

①The isolated culture and identification of MSCs.②Observation of all groups under SEM.③Results of radiological inspection of all groups. ④General morphous of cells in each group after operation.⑤Results of histological inspection of all groups after operation.

RESULTS:

A total of 18 enrolled New Zealand rabbits were involved in the analysis of results.①There were adherent cells at 4 hours after primary culture, which were like cloned at 48 hours after static culture. Cells were in irregular scale-shape with large cell body and the nucleus was in middle. Inspection with flow cytometer showed that CD29,CD44, CD-105 were positive, and the ratios of three positive cells were 97.4% ,98.1% and 86.2% respectively. ②The surface of simple scaffold group was irregular with many ventages. There were cells adherent to the surface of materials as well as the inside of ventage in the cytoskeleton compound group. ③The absorbance of X-rays at 4, 8 and 16 weeks after operation were higher in the cytoskeleton compound group than blank control group and simple scaffold group, moreover, bone union could be seen at the 16th week in the cytoskeleton compound group. ④The implants was embedded in the osteotylus at 16 weeks after operation in the cytoskeleton compound group,while parts of the middle segment of implants in the simple scaffold group was not covered by bone tissues. No porosis was found in the blank control group.⑤The ossifying capacity at each time point after operation was better in the cytoskeleton compound group than simple scaffold group, moreover,there was bone trabecula formed at the 16th week, and the osteoblasts were arranged in order. The bone tissues in the simple scaffold group mainly concentrated on the surface of materials, which sucked little. There were some bony tissues formed in the proximal end of defects in the blank control group, while none was seen in the middle parts.

CONCLUSION:

Rabbit MSCs can greatly proliferate in vitro, which has strong osteogenesis by being co-cultured with nano-collagen basal hydroxyapatite. It is a good kind of seed cells in tissue engineering.