Development of a new method to harvest chondroprogenitor cells from underneath cartilage defects in the knees.
BACKGROUND
Mesenchymal progenitor cells from bone marrow hold great potential as a cell source for cartilage repair. Aspiration from the iliac crest is the most widely used method to harvest bone marrow cells for cartilage repair. The objective of our study was to establish a new method to isolate mesenchymal progenitor cells by direct aspiration of bone marrow from the subchondral spongious bone underneath cartilage defects during microfracture treatment and to confirm the chondrogenic potential of the resulting cell cultures.


METHODS
Bone marrow was aspirated arthroscopically from patients treated for isolated cartilage defects. Adherent stromal cells were isolated, expanded in monolayer cultures, and characterized by flow cytometry. Chondrogenic induction of cells was achieved by combination of spheroid cultures in hanging drops and the concomitant use of transforming growth factor-beta (TGFbeta). Articular chondrocytes established in three-dimensional (3D) cultures were used as positive cartilage-forming units, and skin fibroblasts were used as negative controls. Three-dimensional constructs were stained for immunohistochemical and histological examination, and a real-time polymerase chain reaction (PCR) was performed to quantify the expression of aggrecan, collagen types 1 and 2, and Sox9.


RESULTS
Mesenchymal stem cell-like progenitor cells (MSCs) displaying chondrogenic differentiation capacity were harvested arthroscopically from underneath cartilage lesions on distal femurs using the one-hole technique. Stem cell-related surface antigens analyzed by flow cytometry confirmed the nature of the isolated adherent cells. MSC spheroids stained positive for glycosaminoglycans and collagen type 2. Realtime PCR showed that MSCs in 3D spheroids significantly increased gene expression of collagen type 2, aggrecan, and Sox 9 and down-regulated expression of collagen type 1 when compared to the mRNA levels measured in MSCs monolayers.


CONCLUSIONS
We describe a new technique that may be applied for harvesting bone marrow cells from cartilage defects during arthroscopic intervention of the knee. Cells harvested in this way hold full chondrogenic differentiation potential. Our data imply that MSC storage may be established by using marrow from this approach, bypassing the need for cell aspiration from the iliac crest.
Elvenes
Jan
J
Department of Orthopaedic Surgery, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway.
Knutsen
Gunnar
G
Johansen
Oddmund
O
Moe
Bjørn T
BT
Martinez
Inigo
I
D001182:Arthroscopy / Q000379:methods; D001854:Bone Marrow Cells / Q000166:cytology; D002357:Cartilage Diseases / Q000175:diagnosis / Q000601:surgery*; D002358:Cartilage, Articular / Q000166:cytology; D017690:Cell Transplantation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology; D003470:Culture Media; D005434:Flow Cytometry; D005544:Forecasting; D006801:Humans; D007150:Immunohistochemistry; D007719:Knee Joint / Q000503:physiopathology / Q000601:surgery*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D011336:Probability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018570:Risk Assessment; D033581:Stem Cell Transplantation; D020858:Tissue and Organ Harvesting / Q000379:methods*; D016896:Treatment Outcome
D003470:Culture Media