IDG-SW3 Osteocyte Differentiation and Bone Extracellular Matrix Deposition Are Enhanced in a 3D Matrix Metalloproteinase-Sensitive Hydrogel

Abstract

Osteocytes reside within a heavily mineralized matrix making them difficult to study <i>in vivo</i> and to extract for studies <i>in vitro</i>. IDG-SW3 cells are capable of producing mineralized collagen matrix and transitioning from osteoblasts to mature osteocytes, thus offering an alternative to study osteoblast to late osteocyte differentiation <i>in vitro</i>. The goal for this work was to develop a 3D degradable hydrogel to support IDG-SW3 differentiation and deposition of bone ECM. In 2D, the genes <i>Mmp2</i> and <i>Mmp13</i> increased during IDG-SW3 differentiation and were used as targets to create a MMP-sensitive poly(ethylene glycol) hydrogel containing the peptide crosslink GCGPLG-LWARCG and RGD to promote cell attachment. IDG-SW3 differentiation in the MMP-sensitive hydrogels improved over non-degradable hydrogels and standard 2D culture. Alkaline phosphatase activity at day 14 was higher, <i>Dmp1</i> and <i>Phex</i> were 8.1-fold and 3.8-fold higher, respectively, and DMP1 protein expression was more pronounced in the MMP-sensitive hydrogels compared to non-degradable hydrogels. Cell-encapsulation density (cells/ml precursor) influenced formation of dendrite-like cellular process and mineral and collagen deposition with 80×10⁶ performing better than 2×10⁶ or 20×10⁶, while connexin 43 was not affected by cell density. The cell density effects were more pronounced in the MMP-sensitive hydrogels over non-degradable hydrogels. This study identified that high cell encapsulation density and a hydrogel susceptible to cell-mediated degradation enhanced mineralized collagen matrix and osteocyte differentiation. Overall, a promising hydrogel is presented that supports IDG-SW3 cell maturation from osteoblasts to osteocytes in 3D.