Sustained release of bioactive protein from a lyophilized tissue‐engineered construct promotes the osteogenic potential of mesenchymal stem cells

Tissue‐engineered constructs (TECs) seeded with mesenchymal stem cells (MSCs) represent a therapy for large bone defects. However, massive cell death in TECs in the early postimplantation period prompted us to investigate the osteoinductive mechanism of TECs. Previous studies demonstrated that stem cell extracts retained equivalent levels of bioactive proteins and exhibited an osteoinductive nature similar to that of intact cells. These data led us to hypothesize that despite the massive cell death in TECs, devitalized MSC‐derived proteins remain on the scaffolds and are released to improve cell function. Here, TECs were prepared using demineralized bone matrix seeded with human umbilical cord Wharton's jelly‐derived MSCs (hWJMSCs), and the cells seeded in TECs were devitalized by lyophilizing the TECs. Scanning electron microscopy, BCA protein assays, quantitative cytokine array analysis and immunofluorescent staining indicated that approximately 3 mg/cm³ of total protein and 49 types of cytokines derived from hWJMSCs were preserved in the lyophilized TECs (LTECs). The sustainable release of total protein and cytokines from LTECs lasted for more than 2 weeks. The released protein improved the osteogenic behavior of and gene expression in MSCs. Furthermore, the lyophilized hWJMSC‐derived proteins had immunoregulatory properties similar to those of live MSCs in mixed lymphocyte reactions. Collectively, we present a novel perspective on the osteoinductive mechanism of TECs and introduce LTECs as new systems for delivering multiple cytokines to enhance MSC behavior. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:386–394, 2016.