Synthetic biodegradable microporous hydrogels for in vitro 3D culture of functional human bone cell networks.

Generating 3D bone cell networks in vitro that mimic the dynamic process during early bone formation remains challenging. Here, we report a synthetic biodegradable microporous hydrogel for efficient formation of 3D networks from human primary cells, analysis of cell-secreted extracellular matrix (ECM) and microfluidic integration. Using polymerization-induced phase separation, we demonstrate dynamic in situ formation of microporosity (5-20 µm) within matrix metalloproteinase-degradable polyethylene glycol hydrogels in the presence of living cells. Pore formation is triggered by thiol-Michael-addition crosslinking of a viscous precursor solution supplemented with hyaluronic acid and dextran. The resulting microporous architecture can be fine-tuned by adjusting the concentration and molecular weight of dextran. After encapsulation in microporous hydrogels, human mesenchymal stromal cells and osteoblasts spread rapidly and form 3D networks within 24 hours. We demonstrate that matrix degradability controls cell-matrix remodeling, osteogenic differentiation, and deposition of ECM proteins such as collagen. Finally, we report microfluidic integration and proof-of-concept osteogenic differentiation of 3D cell networks under perfusion on chip. Altogether, this work introduces a synthetic microporous hydrogel to efficiently differentiate 3D human bone cell networks, facilitating future in vitro studies on early bone development.

Interpenetrating network hydrogels for studying the role of matrix viscoelasticity in 3D osteocyte morphogenesis.

During bone formation, osteoblasts are embedded in a collagen-rich osteoid tissue and differentiate into an extensive 3D osteocyte network throughout the mineralizing matrix. However, how these cells dynamically remodel the matrix and undergo 3D morphogenesis remains poorly understood. Although previous reports investigated the impact of matrix stiffness in osteocyte morphogenesis, the role of matrix viscoelasticity is often overlooked. Here, we report a viscoelastic alginate-collagen interpenetrating network (IPN) hydrogel for 3D culture of murine osteocyte-like IDG-SW3 cells. The IPN hydrogels consist of an ionically crosslinked alginate network to tune stress relaxation as well as a permissive collagen network to promote cell adhesion and matrix remodeling. Two IPN hydrogels were developed with comparable stiffnesses (4.4-4.7 kPa) but varying stress relaxation times (t1/2, 1.5 s and 14.4 s). IDG-SW3 cells were pre-differentiated in 2D under osteogenic conditions for 14 days to drive osteoblast-to-osteocyte transition. Cellular mechanosensitivity to fluid shear stress (2 Pa) was confirmed by live-cell calcium imaging. After embedding in the IPN hydrogels, cells remained highly viable following 7 days of 3D culture. After 24 h, osteocytes in the fast-relaxing hydrogels showed the largest cell area and long dendritic processes. However, a significantly larger increase of some osteogenic markers (ALP, Dmp1, hydroxyapatite) as well as intercellular connections via gap junctions were observed in slow-relaxing hydrogels on day 14. Our results imply that fast-relaxing IPN hydrogels promote early cell spreading, whereas slow relaxation favors osteogenic differentiation. These findings may advance the development of 3D in vivo-like osteocyte models to better understand bone mechanobiology.