Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects.
OBJECTIVE
Combining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions.


METHODS
MSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy.


RESULTS
Effective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (< or = 42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC.


CONCLUSIONS
MSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.
Stiehler
Maik
M
Department of Orthopaedics, University Hospital Carl Gustav Carus, Dresden, Germany. maik.stiehler@uniklinikum-dresden.de
Seib
F Philipp
FP
Rauh
Juliane
J
Goedecke
Anja
A
Werner
Carsten
C
Bornhäuser
Martin
M
Günther
Klaus-Peter
KP
Bernstein
Peter
P
D015415:Biomarkers / Q000378:metabolism; D001847:Bone Diseases / Q000473:pathology / Q000628:therapy*; D001861:Bone Regeneration; D016025:Bone Transplantation; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology* / Q000601:surgery; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology; D010012:Osteogenesis; D011785:Quality Assurance, Health Care; D017154:Stromal Cells / Q000473:pathology / Q000637:transplantation; D014184:Transplantation, Homologous
D015415:Biomarkers