[Analysis of hBMSCs spatial distribution and gene expression in biocoral scaffold with different seeding methods].
OBJECTIVE
To compare the effect of two different methods of cell seeding on spatial distribution and gene expression of hBMSCs in biocoral scaffold in vitro cultures.


METHODS
The composite of hBMSCs and biocoral scaffold was prepared by traditional seeding (group A) and fibrin glue seeding (group B). The seeding efficiency was measured after 30 minutes of incubation in group B and after 3 hours in group A. At 2, 7, 14 and 21 days after culture, the samples were harvested and the serial longitudinal sections were cut for each embedded composite. The sections were stained with DAPI and were measured using fluorescence microscope with apostome under serial optical sections. The cell number in every 10 x objective field was automatically measured by AxioVision image analysis software and levels (from seeding surface to bottom L1-L5) or columns (from centre to margin) for comparing cell distribution were set up. The specific osteogenic genes [osteonectin (ON), core binding factor alpha1 (Cbfalpha1), osteocalcin (OC)] expression was measured by RT-PCR.


RESULTS
The seeding efficiency was significantly higher in group B (88.32% +/- 4.2%) than in group A (66.51% +/- 12.33%, P < 0.01). At 2 days after culture, the cell number from L1 to L4 decreased gradully in two groups (P < 0.05); in the cell number of different columns, there was no significant difference in group A (P > 0.05) whereas significant difference in group B (P < 0.05); there was no significant difference in gene expression between two groups (P > 0.05). At 7 days after culture, the cell number was less than that at 2 days in group A and there was significant difference among levels (P < 0.05). The cell number and osteogenic gene expression increased sharply and there appeared uniform cell distribution in group B (P > 0.05). The gene expression of ON and Cbfalpha1 in group B was higher than that in group A (P < 0.05). At 14 days after culture, the cell number in levels or columns in group A decreased sharply and was less than that at 7 days (P < 0.05); whereas the cell number was similar to that at 7 days in group B (P > 0.05). The OC gene expression reached the highest level in group B at 14 days. The gene expression was higher in group B than in group A (P < 0.05). At 21 days after culture, there was significant difference in the cell number among levels and in the gene expression between group A and group B (P < 0.05); there was no significant difference in the cell number among columns in two groups (P > 0.05). In addition, the cell number of most levels and columns in group B was more than that in group A at 7, 14 and 21 days after culture (P < 0.05).


CONCLUSIONS
More uniform cell distribution with rapid proliferation and osteogenic differentiation is available in different levels or columns of scaffold by fibrin glue seeding than by traditional seeding.
Zhu
Huiyong
H
Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou Zhejiang, 310003, P.R. China. zhuhy71@yahoo.com.cn
Sun
Lei
L
Chen
Jianfen
J
Wang
Huiming
H
D001672:Biocompatible Materials / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology*; D002119:Calcium Carbonate; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D017154:Stromal Cells; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D001672:Biocompatible Materials; C434403:calcium carbonate Biocoral; D002119:Calcium Carbonate