Tissue Engineering of Cartilage Using a Random Positioning Machine

Abstract

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.

Engineered Human Meniscus in Modeling Sex Differences of Knee Osteoarthritis in Vitro

“…As expected, the wet weight of engineered constructs in the CHP group was the highest among the three treatment groups since the GAG is a major water-binding component of cartilage (KIANI et al, 2002). The ratio of COL2A1 to COL1A2 has also been used to evaluate chondrogenic capacity (Wuest et al, 2018;Wehland et al, 2020), and in our results, CHP substantially increased the COL2A1/ COL1A2 ratio compared to SMG groups. Although upregulation of type II collagen deposition and COL2A1 expression was observed in the SMG groups compared to static controls, the magnitude of upregulation was not less than in the CHP groups.…”

“…On the contrary, prolonged mechanical unloading by space flight has been shown to accelerate cartilage degeneration (Herranz et al, 2013;Fitzgerald, 2017;Fitzgerald et al, 2019). Although several studies suggested that SMG was beneficial for preserving a chondrogenic phenotype through induction of 3D aggregates from monolayer cultures (Aleshcheva et al, 2015;Wuest et al, 2018;Wehland et al, 2020) and promoted cartilaginous components deposition for scaffold cultures (Freed and Vunjak-Novakovic, 1997), the degree of hypertrophic TABLE 3 | Key enriched KEGG pathways and associated differentially expressed genes (DEGs) in CHP and SMG groups as compared to static controls within female and male donor cohorts. *q < 0.05, **q < 0.01, ***q < 0.001 represent statistical difference of the two groups in each fold change value.…”

Role of Myostatin in Muscle Degeneration by Random Positioning Machine Exposure: An in vitro Study for the Treatment of Sarcopenia

“…Indeed, prolonged exposure to no load is known to affect regenerative growth and tissue repair by impairing adult stem cell proliferation and differentiation processes (Blaber et al, 2014). The negative impact of microgravity and ground-based simulation conditions on physiological systems has been confirmed by numerous studies, which report skeletal muscle cell atrophy, inhibition of osteoblast differentiation, reduction in osteoblast number, altered immune cell activation, and abnormal chondrocyte formation as the main harmful effects on cellular function (Nabavi et al, 2011;Chatziravdeli et al, 2019;Wehland et al, 2020;Ludtka et al, 2021). Based on this evidence and taking our previous work as a starting point (Tarantino et al, 2020), we have here evaluated the efficacy of anti-MSTN antibodies in preventing muscle degeneration induced by RPM exposure in primary cultures of human satellite cells, reproducing identical experimental conditions and enriching previously obtained data.…”

Microgravity and Space Medicine

“…The authors demonstrated that long-term µg created by RPM-induced 3D growth in human chondrocytes. The tissue-engineered spheroids without any scaffolds showed no signs of apoptosis and the typical cartilage morphology [18].…”

“…The authors focused on the linker of nucleoskeleton and cytoskeleton (LINC) complex and showed that the nuclear shape and differential gene expression are both responsive to s-µg in a LINC-dependent manner [20]. Cytoskeletal changes were also detected in 3D multicellular aggregates when human chondrocytes were exposed to an RPM for 28 d [18]. The authors demonstrated that long-term µg created by RPM-induced 3D growth in human chondrocytes.…”