The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

Tissue engineering. Part A

PubMedID: 25693425

Schrobback K, Klein TJ, Woodfield TB. The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models. Tissue Eng Part A. 2015;.
Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in pre-cartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, i.e., hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM - MSCs) and articular chondrocytes. Expanded human chondrocytes and BM - MSCs were (re-)differentiated either in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-a glycyrrhetinic acid (18aGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared to pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM - MSC hydrogels. However, differentiation capacity was not abolished completely by 18aGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM - MSC differentiation consistent with the delayed response of BM - MSCs to 18aGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM - MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies which promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.