Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index

Abstract

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

Intra-Articular Injection of 2 Different Dosages of Autologous and Allogeneic Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cells Triggers a Variable Inflammatory Response of the Fetlock Joint on 12 Sound Experimental Horses

“…Sternal bone marrow was taken from each of the 12 horses after sedation (detomidine 0.01 mg/kg, IV; butorphanol 0.02 mg/kg, IV) and local skin anesthesia, as previously described [ 25–27 ] . Briefly, 30 mL of bone marrow was drawn by suction using an 11G Jamshidi biopsy needle in syringes preloaded with heparin.…”

“…MSC isolation and culture were performed in the same manner and using the same culture medium as previously described in in vitro studies [26, 27, 29]. To ensure the safety of isolated cells, bacteriological and virological analyses were carried out targeting nine viral genera, eight bacterial genera, and two protozoa (see Supplementary 7) by an external laboratory according to their internal protocols (Labéo Frank Duncombe, Saint-Contest, France).…”

Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy

“…Unspecific binding sites of the membrane were blocked with 10% non-fat milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h. Then, membranes were incubated overnight at 4 °C with rabbit anti-human type I collagen (Novotec, Bron, France), rabbit anti-human type II collagen (Novotec, Bron, France), rabbit anti-human type II B collagen (Covalab, Villeurbanne, France), rabbit anti-human type X collagen (Abcam, Cambridge, UK), rabbit anti-human HtrA1 (Merck Millipore, Billerica, MA, USA), or rabbit anti-human β-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA). Concerning the anti-human type IIB collagen, its generation was made by Covalab using exactly the same strategy as the one used by Aubert-Foucher et al [ 61 , 62 ]. This antibody detects the pro α1(II)B and pN α1(II)B isoforms of type II collagen, according to the epitope targeted.…”

Therapeutic Potential of Dental Pulp Stem Cells and Leukocyte- and Platelet-Rich Fibrin for Osteoarthritis

“…The absence of aggrecan in differentiated DPSC pellets in our study might be ascribed to several factors. One of these factors might be the differentiation time since an improved chondrocyte phenotype is reported upon prolonged culture times [42]. After a differentiation period of 6 weeks, aggrecan expression was reported in human DPSCs by Mata and colleagues [43].…”