cells Article Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro Graziana Monaco 1,2 , Yann D. Ladner 1,3 , Alicia J. El Haj 4 , Nicholas R. Forsyth 2 , Mauro Alini 1 and Martin J. Stoddart 1,2, *   Citation: Monaco, G.; Ladner, Y.D.; El Haj, A.J.; Forsyth, N.R.; Alini, M.; Stoddart, M.J. Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro. Cells 2021, 10, 2165. https:// doi.org/10.3390/cells10082165 Academic Editors: Karina Theresa Wright and Charlotte Hulme Received: 9 June 2021 Accepted: 18 August 2021 Published: 22 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 AO Research Institute Davos, Regenerative Orthopaedics Program, 7270 Davos Platz, Switzerland; graziana.monaco@aofoundation.org (G.M.); yann.ladner@aofoundation.org (Y.D.L.); mauro.alini@aofoundation.org (M.A.) 2 Guy Hilton Research Centre, School of Pharmacy and Bioengineering, Keele University, Stoke-on-Trent, Staffordshire ST4 7QB, UK; n.r.forsyth@keele.ac.uk 3 Institute for Biomechanics, ETH Zurich, Lengghalde 5, CH-8008 Zurich, Switzerland 4 Healthcare Technology Institute, Institute of Translational Medicine, University of Birmingham, Birmingham B15 2TT, UK; A.ElHaj@Bham.ac.uk * Correspondence: martin.stoddart@aofoundation.org Abstract: In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFβ1 or BMP2, as well as low (2% O 2 ) or high (20% O 2 ) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O 2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O 2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O 2 ; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O 2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population. Keywords: osteogenesis; chondrogenesis; donor comparison; osteochondral constructs 1. Introduction Cartilage and bone are unique and complex tissues, in physical proximity to each other but very different in structure, function, cell phenotype, microenvironment and mechanical stiffness. Bone and cartilage together form the osteochondral unit, which provides the structure and support necessary for load-bearing and movement. Damage to either of these tissues, caused by trauma or diseases such as osteoarthritis, reduces function, eventually leading to joint immobility and severe pain [1,2]. Depending on the nature, size and location of the osteochondral lesion, invasive surgical treatments are often required [3,4], including osteochondral grafts and mosaicplasty [5,6]. As an alternative, cell-based regenerative approaches such as autologous chondrocyte implantation (ACI) [7] Cells 2021, 10, 2165. https://doi.org/10.3390/cells10082165 https://www.mdpi.com/journal/cells