Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo.
OBJECTIVE
To establish a model and associated molecular markers for monitoring the capacity of in vitro-expanded chondrocytes to generate stable cartilage in vivo.


METHODS
Adult human articular chondrocytes (AHAC) were prepared by collagenase digestion of samples obtained postmortem and were expanded in monolayer. Upon passaging, aliquots of chondrocyte suspensions were either injected intramuscularly into nude mice, cultured in agarose, or used for gene expression analysis. Cartilage formation in vivo was documented by histology, histochemistry, immunofluorescence for type II collagen, and proteoglycan analysis by 35S-sulfate incorporation and molecular sieve chromatography of the radiolabeled macromolecules. In situ hybridization for species-specific genomic repeats was used to discriminate human-derived from mouse-derived cells. Gene expression dynamics were analyzed by semiquantitative reverse transcription-polymerase chain reaction.


RESULTS
Intramuscular injection of freshly isolated AHAC into nude mice resulted in stable cartilage implants that were resistant to mineralization, vascular invasion, and replacement by bone. In vitro expansion of AHAC resulted in the loss of in vivo cartilage formation. This capacity was positively associated with the expression of fibroblast growth factor receptor 3, bone morphogenetic protein 2, and alpha1(II) collagen (COL2A1), and its loss was marked by the up-regulation of activin receptor-like kinase 1 messenger RNA. Anchorage-independent growth and the reexpression of COL2A1 in agarose culture were insufficient to predict cartilage formation in vivo.


CONCLUSIONS
AHAC have a finite capacity to form stable cartilage in vivo; this capacity is lost throughout passaging and can be monitored using a nude mouse model and associated molecular markers. This cartilage-forming ability in vivo may be pivotal for successful cell-based joint surface defect repair protocols.
Dell'Accio
F
F
University Hospitals Katholieke Universiteit Leuven, Belgium.
De Bari
C
C
Luyten
F P
FP
D000199:Actins / Q000235:genetics; D000328:Adult; D000367:Age Factors; D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002358:Cartilage, Articular / Q000166:cytology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D003094:Collagen / Q000235:genetics; D024042:Collagen Type I; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D006818:Hyalin / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D023421:Models, Animal; D011237:Predictive Value of Tests; D011505:Protein-Tyrosine Kinases; D011509:Proteoglycans / Q000378:metabolism; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D012685:Sepharose; D016212:Transforming Growth Factor beta
D000199:Actins; C525718:BMP2 protein, human; D015415:Biomarkers; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D017931:DNA Primers; D011509:Proteoglycans; D017468:Receptors, Fibroblast Growth Factor; D016212:Transforming Growth Factor beta; D003094:Collagen; D012685:Sepharose; C496365:FGFR3 protein, human; C496366:Fgfr3 protein, mouse; D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3; D000469:Alkaline Phosphatase