INTRODUCTION Bone marrow adipocytes (BMAds) have been revealed as important contributor cells in bone homeostasis. These lipid-laden cells primarily arise in close vicinity of trabeculae within bones and dramatically accrue in various types of osteoporosis, such as due to aging, menopause, and type 2 diabetes (T2D). In T2D, skeletal fragility is associated with several changes in bone quality that are incompletely understood. In this pathophysiological context, the phenotype of BMAds is altered and their quantity increases in relationship to poor glycemic control. Yet, whether BMAds can produce a specific extracellular matrix (ECM) which could interfere with the mineralization process of osteoblasts is barely studied. While various techniques exist to assess the bone quality, Raman microspectroscopy has been shown to be the technique of choice for analyzing the quality of the osteoblast-formed mineral. PURPOSE: In complementarity of molecular approaches, we aimed to determine in an in vitro model the quality of the ECMs following BMAd removal and the contribution of these ECMs on mineralization quality in the context of chronic hyperglycemia. METHODS Human Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) were differentiated for 21 days in adipogenic medium containing either a normoglycemic (LG, 5.5 mM) or a high glucose concentration (HG, 25 mM). The ECMs are laid down by BMAds. The BMAds were removed by hypotonic shock. The ECMs were analyzed using Raman microspectroscopy to assess their integrity and basic composition. BM-MSCs were seeded on the BMAd ECMs and differentiated toward osteoblastogenesis in LG and HG conditions for 16 days. The quality of the mineral formed by osteoblasts was evaluated by Raman microspectroscopy through the measurements of the mineral/organic ratio (amount of mineral compared to organic), the type-B carbonate content and the crystallinity (the perfection/length of the crystal). The quality of the organic part was assessed with 2 collagen crosslink ratios. RESULTS: Raman spectroscopy analyses demonstrated that the BMAd ECMs displayed a similar composition compared to the ECM underlying the nondecellularized BMAds, indicating the good integrity. These analyses also showed several differences in the composition of ECM according to the glucose concentration, which further guides the identification of BMAd ECM components. Raman spectrometry revealed that culturing on BMAd matrices specifically prevents type-B carbonate substitution and favors collagen crosslinkings, in contrast to exposure to HG concentration alone. Moreover, the mineral to organic ratio was disrupted according to the presence of BMAd ECM and the glucose concentration used for BMAd or osteoblast culture. HG concentration and BMAd ECM thus led to different defects in mineralization quality. CONCLUSIONS: This finding shed light on the involvement of BMAds, which should be considered in the compromised bone quality of T2D and osteoporosis patients more generally.