<i>In Situ</i> Bioprinting Embryonic-Derived Stem Cells to Repair Human <i>Ex Vivo</i> Chondral Defects.

Abstract

Successful bioprinting requires an appropriate combination of bioinks, cells, and a delivery platform. To demonstrate the potential of <i>in situ</i> bioprinting for regeneration of cartilage lesions we combined clinically relevant embryonic-derived mesenchymal stem cells (ES-MSCs) with a fibrin-based bioink that was delivered into chondral defects created in human <i>ex vivo</i> osteoarthritic (OA) tissue using a bioprinting platform. We used an integrated multitool, 6-axis bioprinting system to laser scan and map the surface of chondral defects and bioprint within the cartilage defects <i>in vitro</i> and <i>ex vivo</i>. For cartilage neotissue generation, clinically relevant ES-MSCs were encapsulated at 20 × 10⁶ cells per mL in chondro-inductive bioinks composed of fibrinogen mixed with nanocellulose or fibrinogen mixed with nanocellulose and hyaluronic acid. After bioprinting as free-standing constructs or <i>in situ</i> within chondral defects, gels were cross-linked in thrombin and cultured for up to 8 weeks in chondrogenic medium. Print fidelity was assessed in the free-standing printed constructs after cross-linking and culture. <i>In situ</i> bioprinted constructs were evaluated for cell viability, mechanical properties, histology (Safranin O and collagen type II immunostaining), and gene expression of chondrogenic genes. Adding nanocellulose to fibrinogen significantly improved print fidelity. ES-MSCs in the fibrinogen-based bioink formulations generated cartilage-like neotissues with positive Safranin O and collagen type II staining. Chondrogenic genes (COLA2A1, ACAN, COMP, and SOX9) were significantly upregulated with negligible expression of hypertrophic markers (COL10A1 and RUNX2). The mechanical properties of the printed constructs increased from 30 to 50 kPa after 3 weeks to ∼150 kPa after 8 weeks in culture. We demonstrated the feasibility of combining clinically relevant ES-MSCs with printable fibrin-based hydrogel bioinks and an integrated bioprinting platform for <i>in situ</i> bioprinting that promoted neocartilage tissue generation and repair of <i>ex vivo</i> lesions in human OA tissues.