[Experimental comparative study on osteogenic activity between freeze-dried tissue engineered bone and tissue engineered bone].
OBJECTIVE
Tissue engineered bone (TEB) lacks of an effective and feasible method of storage and transportation. To evaluate the activity of osteogenesis and capability of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB.


METHODS
Human bone marrow mesenchymal stem cells (hBMSCs) and decalcified bone matrix (DBM) were harvested from bone marrow and bone tissue of the healthy donators. TEB was fabricated with the 3rd passage hBMSCs and DBM, and they were frozen and dried at extremely low temperatures after 3, 5, 7, 9, 12, and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone (FTEB). TEB and FTEB were observed by gross view and scanning electron microscope (SEM). Western blot was used to detect the changes of relative osteogenic cytokines, including bone morphogenetic protein 2 (BMP-2), transforming growth factor beta1 (TGF-beta1), and insulin-like growth factor 1 (IGF-1) between TEB and FTEB. The ectopic osteogenesis was evaluated by the methods of X-ray, CT score, and HE staining after TEB and FTEB were transplanted into hypodermis space in athymic mouse.


RESULTS
SEM showed that the cells had normal shape in TEB, and secretion of extracellular matrix increased with culture time; in FTEB, seeding cells were killed by the freeze-dried process, and considerable extracellular matrix were formed in the pore of DBM scaffold. The osteogenic cytokines (BMP-2, TGF-beta1, and IGF-1) in TEB were not decreased after freeze-dried procedure, showing no significant difference between TEB and FTEB (P > 0.05) except TGF-beta1 15 days after culture (P < 0.05). The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation, there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score. On the contrary, there was no ectopic osteogenesis in group DBM 12 weeks after operation. HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation.


CONCLUSIONS
The osteogenic activity of TEB and FTEB is similar, which provides a new strategy to preserve and transport TEB.
Hou
Tianyong
T
Tissue Engineering Research and Development Institute of Third Military Medical University, Chongqing, 400038, P R China.
Luo
Fei
F
Liu
Jie
J
Bian
Baluobu
B
Xu
Jianzhong
J
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D001857:Bone Matrix / Q000166:cytology; D018786:Bone Substitutes; D016025:Bone Transplantation; D001842:Bone and Bones / Q000166:cytology; D002478:Cells, Cultured; D005612:Freeze Drying; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010012:Osteogenesis; D023822:Tissue Engineering / Q000379:methods*
D018786:Bone Substitutes