Activated platelet-rich plasma improves adipose-derived stem cell transplantation efficiency in injured articular cartilage

Abstract

Introduction: Adipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency.

Methods: ADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage.

Results: PRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs.

Conclusion: Pretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage.

Figure 1

Adipose-derived stem cell culture and marker confirmation. (A) At 24 hours after seeding, fibroblast-like cells adhered to the surface of the flask, (B) proliferated and reached confluence after 1 week, and (C) became homogeneous after three subcultures. At the third passage, adipose-derived stem cells expressed mesenchymal stem cell-specific markers including (G) CD44, (H) CD90, and (I) CD73, while (D) CD14, (E) CD34, and (F) CD45 were negative.

Figure 2

Adipose-derived stem cell proliferation in experimental groups. Adipose-derived stem cells (ADSCs) maintained the shape in four different media: (A) 10% platelet-rich plasma (PRP10), (B) 15% PRP (PRP15), (C) 20% PRP (PRP20) and (D) 10% fetal bovine serum (FBS10). (E) ADSC proliferation significantly increased in medium containing PRP at 10%, 15%, and 20% compared with that in medium containing 10% FBS. OD, optical density.

Figure 3

Platelet-rich plasma does not change adipose-derived stem cell marker expression but changes chondrocyte-related gene expression. The expression of CD14, CD34, CD44, CD45, CD73, and CD90 was changed in the (A) 10% platelet-rich plasma (PRP10), (B) 15% PRP (PRP15), and (C) 20% PRP (PRP20) groups compared with the 10% fetal bovine serum (FBS10) group (Figure 1). (D) Expression of collagen type II (COL-II), Sox9, and aggrecan was strongly promoted in the PRP10, PRP15, and PRP20 groups compared with that in the FBS10 group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SSC.

Figure 4

Vascular endothelial growth factor-A secretion is reduced in platelet-rich plasma-treated adipose-derived stem cells. Vascular endothelial growth factor (VEGF)-A concentrations were significantly decreased in culture supernatants of the 10% platelet-rich plasma (PRP10), 15% PRP (PRP15), and 20% PRP (PRP20) groups compared with that in the 10% fetal bovine serum (FBS10) group.

Figure 5

Recovery of mouse knee joints. (A) The cartilage layer of 15% platelet-rich plasma (PRP)-cultured adipose-derived stem cell (ADSC)-treated mice was similar to that in normal mice. There was evidence of regenerated cartilage formation at the articular cartilage margin in the treated mice, and the thickness of the cartilage layer of the treated mice compared with (B) before treatment and (C) control. H & E-stained articular cartilage sections of mice that received (D, E) 15% PRP-cultured ADSC transplantation, (F) 10% fetal bovine serum (FBS)-cultured ADSC transplantation, or (C) PBS injections.