Genetic marking with the DeltaLNGFR-gene for tracing goat cells in bone tissue engineering.
The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (DeltaLNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40-60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with DeltaLNGFR is applicable to trace goat BMSC's in bone tissue engineering research.
Kruyt
M C
MC
Tissue Engineering Task Force, Department of Orthopaedics, G05.228, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. m.c.kruijt@chir.azu.nl
Stijns
M M C
MM
Fedorovich
N E
NE
De Bruijn
J D
JD
Van Blitterswijk
C A
CA
Verbout
A J
AJ
Rozemuller
H
H
Hagenbeek
A
A
Dhert
W J A
WJ
Martens
A C M
AC
D000818:Animals; D001846:Bone Development; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D016026:Bone Marrow Transplantation; D001842:Bone and Bones / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D006041:Goats; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D020800:Receptor, Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D017154:Stromal Cells / Q000166:cytology / Q000637:transplantation; D023822:Tissue Engineering / Q000379:methods*
D005819:Genetic Markers; D020800:Receptor, Nerve Growth Factor