Covalent Stem Cell-Combining Injectable Materials with Enhanced Stemness and Controlled Differentiation In Vivo

Biohybrid materials, which are defined as engineered functional materials combining living components with nonliving synthetic materials, are considered promising bioactive materials for applications in in vivo tissue engineering. However, the rational design of biohybrid materials applicable to in vivo tissue engineering faces major challenges associated with techniques for combining living cells with nonliving synthetic materials and cell sources. Here, we report injectable covalent stem cell-combing biohybrid materials prepared via a bio-orthogonal click cross-linking reaction of azide-modified adipose-derived stem cells (N3[+]ADSCs), one of the most promising cell sources utilized clinically, with alkyne-modified biocompatible alginate polymers. The mechanical properties of the covalent stem cell-combining biohybrid materials can be adapted to the mechanical properties of the surrounding environment in which they are transplanted by alternating the number of N3[+]ADSCs, the concentration of alkyne-modified alginate, and the number of alkyne groups. Importantly, ADSCs in the covalent biohybrid materials expressed a high level of CD-105, a marker for undifferentiated mesenchymal stem cells, in the body in the absence of differentiation signals, whereas very little CD-105 was expressed in the control physical cell-loading materials, demonstrating that this covalent stem cell-combining approach results in enhanced retention of the material's "stemness" and controlled differentiation in the body. We assessed the potential utility of the covalent stem cell-combining biohybrid materials for in vivo tissue engineering using a murine severe skeletal muscle defect-healing model. Importantly, all of the tissues regenerated by the covalent biohybrid material treatment expressed MYH3, a myogenic marker protein, whereas no expression of MYH3 was detected in the tissues reconstructed by treatment with control physical stem cell-loading materials and Matrigel, indicating that this covalent stem cell-combining approach results in controlled differentiation in the body. Our data demonstrate the potential utility of covalent stem cell-combining biohybrid materials with host tissue-integrative and controlled differentiation capabilities available for in vivo tissue engineering.