Bone Marrow MSC Secretome Increases Equine Articular Chondrocyte Collagen Accumulation and Their Migratory Capacities.

Equine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to the cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternatives to overcome those limitations while preserving MSC restorative properties. The effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) was analyzed with indirect co-culture and/or MSC-conditioned media (CM). The expression of healthy cartilage/OA and proliferation markers was evaluated in eACs (monolayers or organoids). In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. The presence of nanosized EVs in MSC-CM was appraised with nanoparticle tracking assay and transmission electron microscopy. Our results demonstrated that the MSC secretome influences eAC phenotype by increasing cartilage functionality markers and cell migration in a greater way than MSCs, which could delay OA final outcomes. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, the MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects.

Marine Collagen Hydrolysates Downregulate the Synthesis of Pro-Catabolic and Pro-Inflammatory Markers of Osteoarthritis and Favor Collagen Production and Metabolic Activity in Equine Articular Chondrocyte Organoids.

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1ß. In some instances, alone or in the presence of IL-1ß, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression.

Marine Collagen Hydrolysates Promote Collagen Synthesis, Viability and Proliferation While Downregulating the Synthesis of Pro-Catabolic Markers in Human Articular Chondrocytes.

Cartilage is a non-innervated and non-vascularized tissue. It is composed of one main cell type, the chondrocyte, which governs homeostasis within the cartilage tissue, but has low metabolic activity. Articular cartilage undergoes substantial stresses that lead to chondral defects, and inevitably osteoarthritis (OA) due to the low intrinsic repair capacity of cartilage. OA remains an incurable degenerative disease. In this context, several dietary supplements have shown promising results, notably in the relief of OA symptoms. In this study, we investigated the effects of collagen hydrolysates derived from fish skin (Promerim®30 and Promerim®60) and fish cartilage (Promerim®40) on the phenotype and metabolism of human articular chondrocytes (HACs). First, we demonstrated the safety of Promerim® hydrolysates on HACs cultured in monolayers. Then we showed that, Promerim® hydrolysates can increase the HAC viability and proliferation, while decreasing HAC SA-ß-galactosidase activity. To evaluate the effect of Promerim® on a more relevant model of culture, HAC were cultured as organoids in the presence of Promerim® hydrolysates with or without IL-1ß to mimic an OA environment. In such conditions, Promerim® hydrolysates led to a decrease in the transcript levels of some proteases that play a major role in the development of OA, such as Htra1 and metalloproteinase-1. Promerim® hydrolysates downregulated HtrA1 protein expression. In contrast, the treatment of cartilage organoids with Promerim® hydrolysates increased the neosynthesis of type I collagen (Promerim®30, 40 and 60) and type II collagen isoforms (Promerim®30 and 40), the latter being the major characteristic component of the cartilage extracellular matrix. Altogether, our results demonstrate that the use of Promerim® hydrolysates hold promise as complementary dietary supplements in combination with the current classical treatments or as a preventive therapy to delay the occurrence of OA in humans.

RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.

As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

Publisher Correction: Differences in the intrinsic chondrogenic potential of equine umbilical cord matrix and cord blood mesenchymal stromal/stem cells for cartilage regeneration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differences in the intrinsic chondrogenic potential of equine umbilical cord matrix and cord blood mesenchymal stromal/stem cells for cartilage regeneration.

Umbilical cord blood mesenchymal stromal/stem cells (UCB-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) have chondrogenic potential and are alternative sources to standard surgically derived bone marrow or adipose tissue collection for cartilage engineering. However, the majority of comparative studies explore neonatal MSCs potential only on ISCT benchmark assays accounting for some bias in the reproducibility between in vitro and in clinical studies. Therefore, we characterized equine UCB-MSCs and UCM-MSCs and investigated with particular attention their chondrogenesis potential in 3D culture with BMP-2 + TGF-ß1 in normoxia or hypoxia. We carried out an exhaustive characterization of the extracellular matrix generated by both these two types of MSCs after the induction of chondrogenesis through evaluation of hyaline cartilage, hypertrophic and osteogenic markers (mRNA, protein and histology levels). Some differences in hypoxia sensitivity and chondrogenesis were observed. UCB-MSCs differentiated into chondrocytes express an abundant, dense and a hyaline-like cartilage matrix. By contrast, despite their expression of cartilage markers, UCM-MSCs failed to express a relevant cartilage matrix after chondrogenic induction. Both MSCs types also displayed intrinsic differences at their undifferentiated basal status, UCB-MSCs expressing higher levels of chondrogenic markers whereas UCM-MSCs synthesizing higher amounts of osteogenic markers. Our results suggest that UCB-MSCs should be preferred for ex-vivo horse cartilage engineering. How those results should be translated to in vivo direct cartilage regeneration remains to be determined through dedicated study.