The Effect of Two- and Three-Dimensional Cell Culture on the Chondrogenic Potential of Human Adipose-Derived Mesenchymal Stem Cells after Subcutaneous Transplantation with an Injectable Hydrogel

Abstract

Articular cartilage is an avascular tissue composed of chondrocytes, a unique cell type responsible for abundant matrix synthesis and maintenance. When damaged, it never heals spontaneously under physiological circumstances. Therefore, the delivery of mesenchymal stem cells using hydrogel has been considered for cartilage repair. This study aims at investigating the influence of in vitro chondrogenic differentiation of human adipose tissue-derived stem cells (hATSCs) on in vivo cartilage formation when associated with a cellulose-based self-setting hydrogel (Si-HPMC). hATSCs were characterized for their proliferation, surface marker expression, and multipotency. The in vitro chondrogenic potential of hATSCs cultured within Si-HPMC in control or chondrogenic medium was evaluated by measuring COL2A1, ACAN, SOX9, and COMP expression by real-time PCR. Alcian blue and type II collagen staining were also performed. To determine whether in vitro chondrogenically differentiated hATSCs may give rise to cartilage in vivo, cells differentiated as a monolayer or in pellets were finally associated with Si-HPMC and implanted subcutaneously into nude mice. Cartilage formation was assessed histologically by alcian blue and type II collagen staining. Our data demonstrate that hATSCs exhibited proliferation and self-renewal. hATSCs also expressed typical stem cell surface markers and were able to differentiate towards the adipogenic, osteogenic, and chondrogenic lineages. Real-time PCR and histological analysis indicated that Si-HPMC enabled chondrogenic differentiation of hATSCs in inductive medium, as demonstrated by increased expression of chondrogenic markers. In addition, histological analysis of implants showed that chondrogenically differentiated hATSCs (monolayers or pellets) have the ability to form cartilaginous tissue, as indicated by the presence of sulphated glycosaminoglycans and type II collagen. This study therefore suggests that an in vitro induction of hATSCs in 2D was sufficient to obtain cartilaginous tissue formation in vivo. Si-HPMC associated with autologous hATSCs could thus be a significant tool for regenerative medicine in the context of cartilage damage.

The potential of chondrogenic pre-differentiation of adipose-derived mesenchymal stem cells for regeneration in harsh nucleus pulposus microenvironment

“…27,28 Besides, it is reported that the expression of chondrogenic markers increased when grown in three-dimensional (3D) structure compared to monolayer. 29 Type I collagen is another component of collagen fibril in discs. The result of RT-PCR showed similar expression between the control and pre-differentiated ADSCs, partially consistent with previous studies.…”

Isolation and characterization of multipotent postnatal stem/progenitor cells from human alveolar bone proper

“…The media were renewed three times a week. Chondrogenic differentiation was evaluated at day 35 by staining of glycosaminoglycans with Alcian blue (SigmaeAldrich) (Dominici et al, 2006) while the expression of Aggrecan (ACAN) was assessed by real time PCR (Merceron et al, 2011).…”

Cell density and cell size dynamics during in vitro tissue growth experiments: Implications for mathematical models of collective cell behaviour

“…While some experiments are reported in the form of visual snapshots and qualitative comparisons [14,15], others provide more detailed cell density estimates [2,5]. A even more detailed approach is to report the precise numbers and location of individual cells [3].…”