Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo

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Abstract
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 α1(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. Conclusion 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.