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.

Long-Term Safety and Efficacy of Single or Repeated Intra-Articular Injection of Allogeneic Neonatal Mesenchymal Stromal Cells for Managing Pain and Lameness in Moderate to Severe Canine Osteoarthritis Without Anti-inflammatory Pharmacological Support: Pilot Clinical Study.

Objective: To explore the long-term safety and efficacy of canine allogeneic mesenchymal stromal cells (MSC) administered intra-articularly as single or repeated injections in appendicular joints of dogs affected by moderate to severe refractory osteoarthritis. Study Design: 22 pet dogs were recruited into a non-randomized, open and monocentric study initially administering one cellular injection. A second injection was offered after 6 months to owners if the first injection did not produce expected results. Materials and Methods: Anti-inflammatory treatment (if prescribed) was discontinued at last one week before the onset of treatment. Each injection consisted of at least 10 million viable neonatal allogeneic mesenchymal stromal cells obtained from fetal adnexa. Medical data was collected from veterinary clinical evaluations of joints up to 6 months post-injection and owner's assessment of their dog's mobility and well-being followed for a further 2 years when possible. Results: Mild, immediate self-limiting inflammatory joint reactions were observed in 5/22 joints after the first injection, and in almost all dogs having a subsequent injection. No other MSC-related adverse medical events were reported, neither during the 6 months follow up visits, nor during the long-term (2-years) safety follow up. Veterinary clinical evaluation showed a significant and durable clinical improvement (up to 6 months) following MSC administration. Eight dogs (11 joints) were re-injected 6 months apart, sustaining clinical benefits up to 1 year. Owner's global satisfaction reached 75% at 2 years post-treatment Conclusion: Our data suggest that a single or repeated intra-articular administration of neonatal MSC in dogs with moderate to severe OA is a safe procedure and confer clinical benefits over a 24-month period. When humoral response against MSC is investigated by flow cytometry, a positive mild and transient signal was detected in only one dog from the studied cohort, this dog having had a positive clinical outcome.

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.

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

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.

Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1.

Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-ß1) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-ß1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model.

Canine placenta: A promising potential source of highly proliferative and immunomodulatory mesenchymal stromal cells?

In veterinary medicine, therapeutic mesenchymal stromal cells (MSC) have been traditionally isolated from adult bone marrow or adipose tissue. Neonatal tissues, normally discarded at birth from all species have become an alternative source of cells for regenerative medicine in the human clinic. These cells have been described as being more primitive, proliferative and immunosuppressive than their adult counterparts. Our objective was to examine if this phenomena holds true in dogs. Little information exists regarding canine neonatal MSC characterisation. In this study, we were able to both isolate, phenotype and assess the differentiation and immunomodulatory properties of MSC from canine foetal adnexa allowing us to compare their characteristics to their more well-known bone marrow (BM) cousins. Neonatal tissues, including amnion (AM), placenta (PL), and umbilical cord matrix (UCM) were collected from 6 canine caesarean sections. Primary cells were expanded in vitro for 5 consecutive passages and their proliferation measured. BM-MSC were isolated from 5 control dogs euthanised from other studies and grown in vitro using an identical protocol. All MSC lines were systematically evaluated for their ability to differentiate into 3 mesodermal lineages (adipocyte, osteocyte and chondrocyte) and phenotyped by cytometry and qPCR. In addition, the enzymatic activity of the key immunomodulatory marker indoleamine 2,3-dioxygenase (IDO) was evaluated for each MSC line. MSC displaying a fibroblastic appearance were successfully grown from all neonatal tissues. PL-MSC exhibited significantly higher proliferation rates than AM- and UCM-MSC (p=0.05). Cytometric analysis showed that all MSC express CD90, CD29, and CD44, while no expression of CD45, CD34 and MHC2 was detected. Molecular profiling showed expression of CD105 and CD73 in all MSC. Low levels of SOX2 mRNA was observed in all MSC, while neither NANOG, nor OCT4 were detected. All MSC differentiate into 3 mesodermal lineages. Following inflammatory stimulation, the activity of the immunomodulatory enzyme IDO was significantly higher in neonatal MSC compared to BM-MSC (p=0.009). Our results show that canine foetal adnexa cells share very similar properties to their adult equivalents but upon stimulation show significantly higher IDO immunomodulatory activity. Further studies will be needed to confirm the potential therapeutic benefits of these cells.