Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment

Abstract

BackgroundBone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank.MethodsExpression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-Cnu/nu mice to verify the safety of the MSCs from these sources.ResultsCultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects.ConclusionsSF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.

A review of decellurization methods caused by an urgent need for quality control of cell‐free extracellular matrix' scaffolds and their role in regenerative medicine

“…The ECM components (i.e., growth factors and bioactive peptides, such as fibronectin that forms integrin‐based focal adhesions) play an important role in defining the microenvironmental niche within which cells function in both normal homeostasis and in response to injury. These gears may enable cell‐free ECM scaffolds to create a niche that is sufficient enough to recruit stem/progenitor cells and support their differentiation into functional tissues . These characteristics of the ECM could be demonstrated as essential reasons for the clinical application of cell‐free ECM scaffolds in regenerative medicine.…”

“…A single cycle of freezing and thawing may, however, reduce adverse immune reactions, including leukocyte infiltration within the ECM . To achieve a more effective process, a number of immediately repeated, consecutive cycles of freezing and thawing must be used to remove cells from both tissues or organs . Despite this fact, these cycles may increase the loss of protein and other adverse structural changes, as well as the overall quality decline of the ECM.…”

“…Enzymes are among the most commonly used agents in the methodology of cell removal.133 Many authors used trypsin (pancreatic endopeptidase) as the main enzyme in the decellularization methodology due to its lack of specificity toward native collagen . Trypsin is a serine endopeptidase formed from trypsinogen in the pancreas.…”

“…These gears may enable cell-free ECM scaffolds to create a niche that is sufficient enough to recruit stem/progenitor cells and support their differentiation into functional tissues. 19,47,[85][86][87][88][89] These characteristics of the ECM could be demonstrated as essential reasons for the clinical application of cell-free ECM scaffolds in regenerative medicine.…”

Histopathological Evaluation and Expression of the Pluripotent Mesenchymal Stem Cell-Like Markers Cd105 and Cd44 in the Synovial Membrane of Patients with Primary versus Secondary Hip Osteoarthritis

“…In some of the cases, free floating intra-articular osteochondral bodies were also seen. Intensity of CD44 expression in the joint components, including the synovial membrane, is associated with the severity of changes in OA 25 26…”