Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1

Abstract

The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage.

Progenitor Cells Activated by Platelet Lysate in Human Articular Cartilage as a Tool for Future Cartilage Engineering and Reparative Strategies

“…In this study, the proliferative trend demonstrated by doubling number and cyclin D1 protein levels were markedly higher in ACs-PL compared to ACs-FBS and similar between ACs-PL and CPCs-PL. These results are in accordance with previous works, where PL treatment on partially growth-arrested chondrocytes is reported as responsible for their cell cycle re-entry and strong mitogenic response [14,30]. Moreover, we further showed that the increase in proliferation is concomitant with a senescence attenuation in ACs-PL, at a similar level to that of CPCs-PL.…”

“…Due to the high content in mitogenic growth factors, platelet products exert a strong proliferative stimulus on several in vitro cell cultures, as previously reported [14,15,30]. In this study, the proliferative trend demonstrated by doubling number and cyclin D1 protein levels were markedly higher in ACs-PL compared to ACs-FBS and similar between ACs-PL and CPCs-PL.…”

“…At passage 2, confluent monolayers of ACs-FBS, ACs-PL and CPCs-PL were washed with phosphate-buffered saline 1X (PBS) and scraped in cold radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris (pH 7.5), 150 mM sodium chloride, 1% deoxycholic acid, 1% triton X-100, 0.1% SDS, 0.2% sodium azide and proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein extract concentration was quantified by Bradford assay (Serva Electrophoresis GmbH, Heidelberg, Germany) and Western blot was performed according to Nguyen et al [30]. Equal amounts of total proteins (10 µg) were loaded on 4-12% NuPAGE Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA), and electrophoresis was performed.…”

MGF E peptide improves anterior cruciate ligament repair by inhibiting hypoxia‐induced cell apoptosis and accelerating angiogenesis

“…Subsequently, HIF-1α regulates mitochrodrial apoptotic pathway-relevant proapoptotic proteins (Bcl-2 and Bcl-xl) expression and thereby activates caspase family to induce cell apoptosis (Liu, Ai, Feng, Li, & Liu, 2016). In addition, HIF-1α also exhibits a tight link with the cell proliferation (Nguyen, Cancedda, & Descalzi, 2017). As shown in the current study (Supporting Information Figures S1, 1, and 2), HIF-1α expression levels and cell apoptosis of ACL fibroblasts were significantly increased by severe hypoxic environmental conditions, which would lead to the reduction of the number of ACL fibroblasts.…”

Inhibition of Hypertrophy and Improving Chondrocyte Differentiation by MMP-13 Inhibitor Small Molecule-Encapsulated in Alginate-Chondroitin Sulfate-Platelet Lysate Hydrogel

“…Moreover, it was demonstrated chondroitin sulfate, an important molecule in cartilage extracellular matrix, improved chondrogenesis of MSCs [43]. Besides, Nguyen et al, demonstrated that PL induces reprograming of quiescent cartilage cells resulted in new cartilage formation [91] The expression of Coll X along with MMP-13 is typically expressed in the hypertrophy pathway.…”