[In vitro osteogenesis of the compound of chitosan and recombinant human bone morphogenetic protein 2].
OBJECTIVE
To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2).


METHODS
Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freeze-drying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at the density of 2 x 10(4)/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semi-quantitative RT-PCR with housekeeping gene beta-tubulin as internal-standard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected to the detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition and was treated with the same as group A. Besides these 2 groups, another 2 rhBMP-2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex.


RESULTS
SEM showed that cells attached closely to the complex and grew well. In group A, the expression level (1.28+/-0.17) of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56+/-0.09) of the control group A, being statistically significantly different (P< 0. 05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells.


CONCLUSIONS
The complex made of rhBMP-2 and chitosan-gelatin scaffolds has strong osteogenesis ability in vitro.
Liang
Dongchun
D
Institute of Endocrinology, Tianjin Medical Univesity, Tianjin, 300070, P. R. China. liangdongchun1225@263.net
Zuo
Aijun
A
Wang
Baoli
B
Zhang
Jingyu
J
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D001861:Bone Regeneration / Q000187:drug effects; D018786:Bone Substitutes; D002460:Cell Line; D048271:Chitosan; D005780:Gelatin; D048091:Guided Tissue Regeneration; D006801:Humans; D051379:Mice; D032446:Myoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology*; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D018786:Bone Substitutes; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin; D005780:Gelatin; D048271:Chitosan