An Injectable Recombinant Collagen I Peptide–Based Macroporous Microcarrier Allows Superior Expansion of C2C12 and Human Bone Marrow-Derived Mesenchymal Stromal Cells and Supports Deposition of Mineralized Matrix^y>

摘要

The development of scaffold formulations based on extracellular matrix (ECM)-inspired synthetic materials constitutes an important resource for the advance of cell-based therapies in bone tissue engineering approaches, where both cell and scaffold implantation are often needed. Culturing cells on porous microcarriers (MCs) allows cell expansion in a three-dimensional microenvironment and constitutes a possible solution for minimally invasive cell and scaffold simultaneous delivery, but the reduced pore dimension and pore interconnection diameter of several commercially available MCs limits de facto cell ingrowth, and ultimately their suitability for in vivo cell delivery. In this study we investigated the potential of a new macroporous MC based on a collagen I-based recombinant peptide (Cellnest?) for C2C12 cells and human bone marrow-derived mesenchymal stromal cells (hBMSCs) expansion and we analyzed the influence of dehydrothermal (DHT), hexamethylene diisocyanate (HMDIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking strategies on cell vitality, proliferation, and hBMSCs differentiation. We established a double emulsification protocol for the manufacturing of MCs characterized by external pores of 20–40?μm diameter, 73% porosity, and 20?±?3?μm pore interconnection diameter supporting cell ingrowth and proliferation into the MC. MCs cross-linked with DHT and HMDIC supported higher cell proliferation comparing to a commercially available equivalent over the course of 7 days and resulted in higher cell yield by day 28. Moreover, while hBMSCs expansion on Cellnest-MCs did not lead to a significant upregulation of the early markers of osteogenic differentiation Col1a1 and Runx2, their differentiation potential into the osteogenic lineage was preserved when cultured in differentiation medium, as confirmed by mineralized ECM deposition. We believe that Cellnest-MCs will help in reaching clinically relevant cell quantities and ultimately help in accelerating the translation of cell-based therapies for bone tissue engineering in the clinical practice.