Peripheral blood derived mononuclear cells enhance osteoarthritic human chondrocyte migration

Abstract

Introduction: A major problem in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into defects. Cartilage is essentially avascular and therefore its healing is not considered to involve mononuclear cells. Peripheral blood derived mononuclear cells (PBMC) offer a readily available autologous cell source for clinical use and therefore this study was designed to evaluate the effects of PBMCs on chondrocytes and cartilage.

Methods: Human primary chondrocytes and cartilage tissue explants were taken from patients undergoing total knee replacement (n = 17). Peripheral blood samples were obtained from healthy volunteers (n = 12) and mononuclear cells were isolated by density-gradient centrifugation. Cell migration and chemokinetic potential were measured using a scratch assay, xCELLigence and CyQuant assay. PCR array and quantitative PCR was used to evaluate mRNA expression of 87 cell motility and/or chondrogenic genes.

Results: The chondrocyte migration rate was 2.6 times higher at 3 hour time point (p < 0.0001) and total number of migrating chondrocytes was 9.7 times higher (p < 0.0001) after three day indirect PBMC stimulus and 8.2 times higher (p < 0.0001) after three day direct co-culture with PBMCs. A cartilage explant model confirmed that PBMCs also exert a chemokinetic role on ex vivo tissue. PBMC stimulation was found to significantly upregulate the mRNA levels of 2 chondrogenic genes; collagen type II (COL2A1 600-fold, p < 0.0001) and SRY box 9 (SOX9 30-fold, p < 0.0001) and the mRNA levels of 7 genes central in cell motility and migration were differentially regulated by 24h PBMC stimulation.

Conclusion: The results support the concept that PBMC treatment enhances chondrocyte migration without suppressing the chondrogenic phenotype possibly via mechanistic pathways involving MMP9 and IGF1. In the future, peripheral blood mononuclear cells could be used as an autologous point-ofcare treatment to attract native chondrocytes from the diseased tissue to aid in cartilage repair.

Fig. 1

Schematic illustration of migration studies. a Scratch assay, b Boyden chamber with chondrocytes and c Boyden chamber with explants demonstrating the position of cartilage explant and chondrocytes in relation to peripheral blood mononuclear cells (PBMCs)

Fig. 2

Chondrocyte migration experiment results (n = 4). a Scratch assay measuring the distance between the wound migration fronts in a chondrocyte monolayer and b wound closure rate in a chondrocyte monolayer scratch assay at the 3-h time point. c Representative image of xCELLigence cell migration analysis from four wells (technical replicates) with chondrocytes measured for 3 days evaluating the chemokinetic effect of peripheral blood mononuclear cells (PBMC+ in the lower chamber and PBMC# in the same chamber). Error bars represent standard deviation and cell index value is based on impedance measurements that provide quantitative information about cell migration through the pores of the membrane measured continuously with microelectrodes. d Total chondrocyte migration at day 3 in the xCELLigence assay and e xCELLigence assay comparing mesenchymal stroma cell (MSC) and chondrocyte (CHO) migration at day 3 (n = 6). f Total cartilage cell migration from tissue explant at day 28 in the xCELLigence assay. g Cartilage cell migration rate from tissue explant at day 14 in the xCELLigence assay. **p <0.001, ***p <0.0001 and ****p <0.00001

Fig. 3

a Cell proliferation measured with CyQUANT total DNA assay (n = 5) showing non-significant difference between the test groups. b PCR array results showing a change in mRNA levels after 24 h peripheral blood mononuclear cell (PBMC) stimulation with a cutoff value of 4. Heat map visualization of 2log2 fold change of the 84 genes in the human cell motility array (red upregulated and green downregulated). Grey shows the genes that were undetermined (no Ct value with a cutoff value of 35). c Scatterplot shows up- and down regulated genes and core genes with no change (biological n = 5). d Messenger RNA fold change of Collagen type II and Sox9 after 24 h PBMC stimulation (n = 5) p <0.00001. All data normalized to B2M housekeeping gene and unstimulated control. In every mRNA expression study chondrocytes in passage 3 were used