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

Abstract

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-β) 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-β 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.

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

“…It is widely accepted that neonatal MSCs are able to differentiate toward the chondrogenic lineage in vitro , but the choice of the most suitable MSCs source for cartilage tissue-engineered remains difficult. In the equine model, different groups reported the production of a cartilage neosubstitute using bone marrow-derived MSCs cultured in 3 dimensional (3D) models 17 – 19 . Nevertheless, only few studies explored the chondrogenic differentiation ability of neonatal equine MSCs.…”

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

“…Data from the literature demonstrated that TGF-β1 is able to induce HtrA1 [ 60 ], and that HtrA1 can interfere with BMP-2 signalling [ 61 ] . Nevertheless, our current studies carried out on equine bone marrow MSCs show that the concomitant BMP-2/TGF-β1 treatment during chondrogenic differentiation induces a strong inhibition of HtrA1 protein synthesis compared with untreated MSCs [ 62 ], such as BMP-2 in the eAC of the present investigation.…”

Osteochondritis dissecans (OCD) in Horses – Molecular Background of its Pathogenesis and Perspectives for Progenitor Stem Cell Therapy

“…Since bone and joint disorders are the main limiting factors affecting the horse industry through loss of breeding potential and reduced market value, it would therefore be important to first add further data on the pathology and the underlying risk factors, as well as to develop possible new strategies to enhance the management of OCD in horses [ 12 ]. Thus, as a part of the development of innovative therapies for the management and treatment of various orthopaedic conditions, the horse seems to represent an experimental model of choice, mainly because of the high similarity of its cartilage to that of human beings, from a structure, thickness and cellularity point of view, and offers great opportunities for advancing research in terms of understanding and developing new preventive and curative therapies [ 13 ].…”