Evaluation of differentiation capacity of osteoclast precursors on Gellan Gum-based spongy-like hydrogels

Hydrogels have been attracting a great of attention in tissue engineering and regenerative medicine. In fact, they can resemble in some aspects the extracellular matrix, as the high water content. Previous studies of our group allow to engineer cell-compatible gellan gum (GG) spongy-like hydrogels [1]. These hydrogels had the ability to entrap cells upon rehydration with a cell suspension and showed that cells were able to adhere and proliferate, without the need of any backbone modification. Taking advantage of these appealing characteristics for bone tissue regeneration strategies, GG spongy-like hydrogels were combined with hydroxyapatite (HAp). Besides adequate mechanical properties, a successful biomaterial for bone replacement should provide support to both osteoblasts and osteoclasts. In fact, bone is a dynamic and complex tissue, which renews through a process that involves not only osteoblasts for tissue formation and osteoclasts for tissue resorption. Nevertheless, osteoclast differentiation is many times forgotten. Taking this in consideration, this study is focused on the ability of GG and HAp reinforced GG (GG-HAp) spongy-like hydrogelsto support osteoclastogenesis in vitro. First, mechanical properties of developed spongy-like hydrogels were evaluated. Then, osteoclast precursor’s cells were isolated and cultured in the spongy-like hydrogels. To promote osteoclasts differentiation, cell cultures were treated with 1,25-dihydroxyvitamin D3 (Vitamin D3). Cultures without Vitamin D3 were used as control, as well as standard 2D cultures. After 7 days, tartrate-resistant acid phosphatase (TRAP) staining was performed on GG and GG-HAp spongy-like hydrogels to observe cell differentiation. Alamar Blue and Live/Dead assays were carried out to provide information on cells viability. In order to confirm cell differentiation along the osteoclastogenesis gene expression was analyzed. The results have shown that cells cultured in the spongy-like composite hydrogels were viable and metabolically active. Additionally, the GG-HAp spongy-like hydrogels supported the differentiation of osteoclasts precursor’s cells into pre-osteoclasts, as suggested by the presence of aggregates of TRAP-stained cells and the expression of the genes encoding DC-Stamp and Cathepsin K, necessary for cell fusion and resorption capacity, respectively. Overall, the present study demonstrates that GG-HAp spongy-like hydrogels support osteoclastogenesis, an important feature when establishing new biomaterials for bone tissue regeneration scaffolding.