Efficient characterisation of human cell-bioceramic interactions in vitro and in vivo by using enhanced GFP-labelled mesenchymal stem cells.
Human mesenchymal stem cells (hMSCs) were transfected using four retroviral pseudotypes, amphotropic murine leukemia viruses 4070 (MuLV-10A1), a modification of amphotropic pseudotype 4073 (A71G, Q74K, V139M), gibbon ape leukemia virus (GaLV), or feline endogenous virus (RD114) encoding the neomycin resistance (Neo(r)) gene and enhanced green fluorescent protein (eGFP) as genetic markers. It was observed that the MuLV4073 was the most efficient pseudotype for hMSC transfection. The proliferation and differentiation characteristics of eGFP-labelled hMSCs were not significantly different from control hMSCs. G418 selected eGFP-labelled cells were cultured for 3 weeks on two porous, commercially available calcium phosphate bioceramics, a "synthetic hydroxyapatite" and a "deproteinised bone", before implantation into NOD/SCID mice for up to 4 weeks. The eGFP-labelled hMSCs could be readily visualised by their intense green fluorescence both in vitro and in vivo. In "synthetic hydroxyapatite" implants the cells remained in a monolayer, whereas in "deproteinised bone" implants mineralised tissues were detected by histology, scanning electron microscopy and energy dispersive X-ray spectrometry. From the results, it is concluded that the use of eGFP-labelled hMSCs is an effective tool to trace the fate of hMSCs and evaluate the interactions between cells and ceramics both in vitro and in vivo. This is of great value in prospective assessments of these cell populations for use in tissue engineering applications.
Xia
Zhidao
Z
BotnarResearch Centre , Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford OX3 7LD, UK.
Ye
Hua
H
Locklin
Rachel M
RM
Ferguson
David J P
DJ
Cui
Zhanfeng
Z
Triffitt
James T
JT
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D002516:Ceramics / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D008855:Microscopy, Electron, Scanning; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D013997:Time Factors; D023822:Tissue Engineering; D014162:Transfection; D014961:X-Ray Diffraction
D001672:Biocompatible Materials; D049452:Green Fluorescent Proteins