Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy

Abstract

High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI). The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS) and Stemulate™ (a commercial source of human platelet lysate), on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM) vs. Stemulate™, 13.10 ± 2.57 d (SEM)]. Sulphated glycosaminoglycans (sGAG), total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype) and, hence, for cell therapies (including ACI).

Human Platelet Lysate Promotes Proliferation but Fails to Maintain Chondrogenic Markers of Chondrocytes

“…With both supplements, the cells appeared flattened and elongated. Pereira et al (2013) and Sykes et al (2018) reported a similar observation. They found that the phenotype of monolayer chondrocytes cultured with HPL changed from polygonal to spindle-shaped.…”

“…Cultures supplemented with HPL could yield up to 4-fold more cells. Several researchers have reported the ability of HPL to promote the proliferation of cultured chondrocytes (Hildner et al 2015;Sykes et al 2018). Interestingly, the cell clustering and overlapping in culture did not affect the cell viability, which was >90%.…”

“…Similarly, Hildner et al (2013) showed that chondrocytes expanded with HPL have higher Col II and GAG protein production when the cells were cultured in 3D (Hildner et al 2015). In contrast, Kaps et al (2002) and Sykes et al (2018) found that HPL-expanded chondrocytes cultured in 3D secreted less GAG protein. The discrepancy in the results from these studies and ours might be due to the differences in HPL, as every study uses HPL prepared using different methods.…”

“…Similarly, Hildner et al (2015) also reported higher SOX 9 expression in adipose-derived mesenchymal stem cells cultured with HPL compared to that cultured with FBS. SOX 9 is a transcriptional factor that regulates chondrocyte proliferation, cartilage formation and the suppression of chondrocyte hypertrophic changes (Sykes et al 2018). Dedifferentiated chondrocytes with higher SOX 9 expression are more responsive to chondrogenic signals and produce more cartilage ECM (Hardingham et al 2006;Li et al 2004;Tew et al 2005).…”

“…Mistry et al (2017) proved that ACI is not only cost-effective but also leads to long-term improvement. Scientists and clinicians have come to a consensus that ACI should be performed as early as possible and as the first surgical intervention to yield the best results (Sykes et al 2018). All these findings suggest that ACI has the potential to be the definitive treatment for OA in the near future.…”