Induction of bone marrow mesenchymal stem cell chondrogenesis following short‐term suspension culture

Abstract

ABSTRACT:The objective of this study was to evaluate mesenchymal stem cell (MSC) chondrogenesis following incubation in chondrogenic suspension cultures from which single cells were obtained. MSCs were maintained in suspension over a nonadherent surface for 3 days, dissociated into a suspension, and then evaluated for chondrogenesis in agarose in the presence or absence of transforming growth factor beta (TGF␤). In a second experiment, MSCs from suspension culture were returned to monolayer expansion for 2 days prior to testing for chondrogenesis. In both cases, undifferentiated MSCs were evaluated as controls. Suspension culture alone did not stimulate chondrogenesis. Suspension followed by expansion stimulated a four-to ninefold increase in extracellular matrix (ECM) synthesis in TGF␤-free cultures, a finding that was attributed to an increase in viable MSCs that secreted a proteoglycan-rich ECM. Gene expression of aggrecan and type II collagen increased with suspension culture, but decreased with postsuspension expansion. Therefore, stimulation of ECM synthesis without additional TGF␤ exposure could not be attributed to an enhancement of chondrogenesis with monolayer culture. ECM synthesis of suspension/expansion-conditioned MSCs without additional TGF␤ exposure was less than samples maintained in TGF␤ throughout the differentiation culture. Based on these findings, a better understanding of factors associated with early-stage chondrogenesis and MSC differentiation to a highly active phenotype may lead to improved methods for stimulating chondrogenesis during short-term culture. Keywords: chondrogenesis; mesenchymal stem cell; cartilage tissue engineering While the biochemical conditions for stimulating chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs) in vitro have been defined, 1 the potential of MSCs to generate neo-cartilage in vivo remains a topic of ongoing research. Animal studies often utilize MSCs from monolayer expansion culture, 2-6 a state in which MSCs are undifferentiated. Long-term follow-up has shown that transplanted undifferentiated MSCs in an adult equine model 6 and human patients 5 have not produced a lasting, hyaline-like tissue. Therefore, it has been suggested that induction of MSC chondrogenesis prior to implantation would promote superior repair. 6 In support of this hypothesis, small animal models have shown an increase in cartilage repair when MSC-seeded scaffolds were cultured in chondrogenic medium before implantation in experimental defects. 7,8 Pre-implantation culture of cell-seeded scaffolds is a means by which MSC chondrogenesis may be stimulated. However, ex vivo scaffold seeding is a step that would be otherwise unnecessary for carriers such as hydrogels, which as liquids can be mixed with cell suspensions and then crosslinked within defects 9 without the need for additional fixation, and using minimally invasive techniques. 6 In order to preserve this feature of injectable scaffolds, pre-implantation differentiation strategies must induce MSC chondrogene...

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self‐assembling peptide hydrogel

“…Days of chondrogenic preconditioning in KLD hydrogel resulted in high levels of ECM accumulation without additional exposure to TGFb in agarose culture, which was a marked improvement over previous testing of equine MSC preconditioning in scaffold-free suspension cultures. 23 This result is consistent with reports that temporary exposure to TGFb is sufficient to induce MSC chondrogenesis or sustain ECM accumulation. [12][13][14][15][16][17]24 In particular, seven days of TGFb exposure, 13 or constructs preloading with TGFb that persisted for less than a week 17 stimulated GAG accumulation over continuous multiweek TGFb exposure.…”

“…The soft physical properties of the hydrogel enabled rapid isolation of the encapsulated cells, and routine exposure to cell culture enzymes produced an individual cell suspension. Days of chondrogenic preconditioning in KLD hydrogel resulted in high levels of ECM accumulation without additional exposure to TGFβ in agarose culture, which was a marked improvement over previous testing of equine MSC preconditioning in scaffold‐free suspension cultures . This result is consistent with reports that temporary exposure to TGFβ is sufficient to induce MSC chondrogenesis or sustain ECM accumulation .…”

Tissue engineering potential of human dermis-isolated adult stem cells from multiple anatomical locations

“…Following ARC in this study, a 22.8% decrease in hDIAS cell number was also observed. Given that cell death has been reported previously in MSCs undergoing aggregate suspension culture despite the upregulation in COL2 and ACAN [ 53 ], the results seen in this study were not unexpected. Indeed, it may be that the enhancement of chondrogenic functional properties seen here was due to an elimination of cells that are unfit to participate in the chondrogenic self-assembling process.…”

Clinical Efficacy of Arthroscopic Therapy With Infrapatellar Fat Pad Cell Concentrates in Treating Knee Cartilage Lesion: a Randomized Controlled Trial

“…MSCs, such as bone marrow mesenchymal stem cells (BMSCs), synovial-derived mesenchymal stem cells (SDMSCs), and adipose-derived mesenchymal stem cells (ADMSCs), can be extracted from different kinds of tissue, whereas only MSCs derived from bone marrow and adipose can be acquired in a large amount for clinical use [14][15][16][26][27][28][29]. Compared with BMSCs,ADMSCs have more active proliferative capacity, and the acquiring procedure of them causes less damage to the donor zone [24,[30][31][32]. Meanwhile, studies have also showed that MSCs from different kinds of tissue have signi cantly different ability of proliferation and differentiation.…”