Abstract
Summary Statement
Hypoxia enhances chondrocyte phenotype of cells migrating from cartilage fragments, thus supporting the use of chondral fragment as a potential cell source for one-stage cartilage repair
Introduction
Minced cartilage fragments are a viable cell source for one stage cartilage repair, as shown in both in preclinical and clinical studies. However, the joint microenvironment, in which the repair process takes place, is hypoxic and no evidences are present in literature regarding the behaviour of cartilage fragments in a hypoxic environment. Aim of the study is to verify if hypoxia could influence chondrocyte outgrowth from cartilage fragments into a Hyaluronic-Acid/fibrin scaffold and evaluate its effects on migrating chondrocyte behaviour, compared to normoxic condition. This could be significant in the perspective of a wide clinical application of human chondral fragments for single stage repair.
Materials and methods
Constructs were prepared with minced cartilage fragments harvested from the trochlear region of 20 human young donors during ACL reconstruction, loaded onto a non-woven esterified Hyaluronic-Acid-derivative felt (Hyaff-11) and retained with a coating of fibrin glue (Tisseel). Constructs were cultured either in normoxic or in hypoxic (10% O2) condition. The growth medium contained DMEM-high-glucose (4500mg/l)with 10% fetal-bovine-serum. After 1 month, construct sections were stained with haematoxylin/eosin and Safranin-O and examined for cell counting. Expression of chondrocyte markers (SOX9, collagen-II, collagen-I), hypoxic markers(HIFs) and proliferative markers (beta-catenin, PCNA) was assessed using immunofluorescence.
Results
Migrating cells predominantly showed a spindle-like shape when close to the fragments and a more roundish shape when embedded into the scaffold. A slight decrease in chondrocyte migration and proliferation was observed in hypoxic cultures, albeit not statistically significant. Conversely, an increase in the expression of SOX9, beta-catenin, HIFs, collagen-II (p<0.05) in migrating chondrocytes from hypoxic cultures was shown by immunofluorescence.
Discussion/Conclusion
Hypoxia seems to improve the chondrocyte phenotype/behaviour of cell outgrowing from cartilage fragments onto a HA/fibrin scaffold. Moreover, hypoxic condition did not hamper the ability and the mechanisms by which chondrocytes migrate from cartilage fragments and proliferate into the surrounding environment. This is clinically relevant in order to validate one-step repair techniques by means of human cartilage fragments loaded into composite scaffolds.
