Evaluation of β1-integrin expression on chondrogenically differentiating human adipose-derived stem cells using atomic force microscopy

Abstract

The expression of β1-integrin on human adipose-derived stem cells, differentiating toward a chondrogenic lineage, is hypothesized to decrease when cells are grown under in vivo-like environments due to sufficient extracellular matrix (ECM) buildup in the engineered tissues. The opposite is true when cells are grown in static cultures such as in pellet or micromass. To probe β1-integrin distribution on cellular surfaces, atomic force microscopy cantilevers modified with anti-β1-integrin antibodies were used. Specific antibody-antigen adhesion forces were identified and indicated the locations of β1-integrins on cells. ECM properties were assessed by estimating the Young's modulus of the matrix. Specific single antibody-antigen interactions averaged 78 ± 10 pN with multiple bindings occurring at approximate multiples of 78 pN. The author's results show that upregulated β1-integrin expression coincided with a less robust ECM as assessed by mechanical properties of tissues. In micromass and pellet cultures, transforming growth factor β3(TGF-β3) elicited a decrease in Young's modulus by 3.7- and 4.4-fold while eliciting an increase in β1-integrin count by 1.1- and 1.3-fold, respectively. β1-integrin counts on cells grown in the presence of TGF-β3 with oscillating hydrostatic pressure decreased by a 1.1-fold while the Young's modulus increased by a 1.9-fold. Collectively, our results suggest that cells in insufficiently robust ECM express more integrin perhaps to facilitate cell-ECM adhesion and compensate for a looser less robust ECM.

Combined effects of oscillating hydrostatic pressure, perfusion and encapsulation in a novel bioreactor for enhancing extracellular matrix synthesis by bovine chondrocytes

“…To further assess culture condition effects on ECM deposition, we analyzed β1 integrin mRNA expression reduction as indicative of a sufficiently robust matrix (Quisenberry et al 2016) and Col I and Col X as indicative of less chondrogenic conditions. Integrins are transmembrane receptors consisting of α and β subunits that are involved in cell adhesion to ECM (Grashoff et al 2003; Knudson and Loeser 2002) and are therefore expected participants in mechanotransduction (Knudson and Loeser 2002).…”

“…Quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify gene expression (Quisenberry et al 2016). Briefly, total RNA was isolated with TRIzol and chloroform was used for phase separation.…”

Characterization of adipose-derived stem cells freshly isolated from liposuction aspirates performed with Prolipostem®

“…It is well known that ADSCs express, among the other superficial markers, β1-integrin (Quisenberry et al 2016;Lu et al 2014). To this end, an immunocytochemical approach was used to demonstrate the expression of β1-integrin on the surface of the ADSCs recovered.…”

A flow perfusion bioreactor with controlled mechanical stimulation: Application in cartilage tissue engineering and beyond

“…The experiment continued for three weeks in total. The results from this experiment were presented in three published articles [105][106][107]. In brief, some of the most important indings are that:…”

“…β1-integrin expression is inversely related to Young's moduli of engineered tissue constructs [107].…”