3D Silk-Based Bone Marrow Models Identify Autophagy As a Critical Mechanism in Red Blood Cell Production
Authors: Di Buduo Christian Andrea , Careddu Francesca , Metti Samuele , Perotti Cesare , Del Fante Claudia , Braghetta Paola , Minetti Giampaolo , Malcovati Luca , Balduini Alessandra 
Published: December 2024

Bone marrow (BM) models hold great potential for advancing our understanding of the interactions between hematopoietic stem and progenitor cells (HSPCs) and their niche, investigating hematological disorders, and producing blood cells for translational applications. Obtaining mature, enucleated red blood cells (RBCs) ex vivo is challenging due to the lack of models replicating the specialized microenvironment of erythroblastic islands (EBIs), where macrophages and erythroblasts interact. Current methods fail to replicate the 3D structure and function of EBIs, and techniques to construct, modulate, and assess RBC production ex vivo are still underdeveloped.
Our approach to mimicking erythropoiesis in the BM niche was initiated with the evaluation of human and mouse bone marrow biopsies, which revealed that fibronectin fibers are predominantly localized near erythroid nests and interspersed within EBIs. Inspired by this native microenvironment, we developed 3D scaffolds made of silk fibroin biomaterial, a protein from Bombyx mori cocoons, widely used in tissue engineering due to its biocompatibility and ability to retain bioactive substances. Our niche mimic was fabricated using a salt leaching method, incorporating the silk fibroin solution with salt particles and human fibronectin. This process yielded 3D constructs with spatially distinct micro-niches and uniformly distributed pores, exhibiting high interconnectivity with fibronectin distributed at their surface to facilitate cell adhesion. The scaffold efficiently supported the rapid soaking and homogeneous distribution of human CD34+ HSPCs and CD68+ macrophages to replicate natural cell-cell interactions critical for EBI formation. Efficient hydraulic conductivity facilitated the perfusion of the system into customized chambers to provide a flow-through mimicking the natural bloodstream.
Our simplified culture regimen, with minimal erythropoietin and no stem cell-associated cytokines, prioritized physiological HSPC differentiation over three weeks. Approximately 150 cellular nests/mm³ resembling EBI-like clusters formed. Here, small enucleated CD235⁺ RBCs were primarily localized at the periphery, typically around co-cultured macrophages. The silk BM niche significantly increased the volume of EBI-like clusters at the final differentiation stages, as shown by automated image segmentation analysis. EBI formation was impaired without fibronectin, highlighting its role in niche development. Cells within foci of erythropoiesis remodeled during culture, transitioning from stemness to lineage-specific markers such as CD36, CD71, and CD235a. Ultrastructural analysis revealed a significant reduction in cell size, fewer mitochondria, and increased glycogen deposits, indicating a shift from oxidative metabolism to glycolysis as RBCs matured. This was accompanied by increased hemoglobin synthesis and decreased expression of α4β1 integrin, responsible for binding to fibronectin. Indeed, the perfusion of the system into a specialized bioreactor chamber enabled the detachment and collection of this mature enucleated population, while EBIs maintained their structural integrity and adhesion to the scaffolds.
Mechanistically, high-resolution imaging and volumetric reconstruction revealed autophagic activation in cells at the erythroblastic stage within EBIs, while a reduced signal was quantified in reticulocytes and small enucleated RBCs. Pharmacological inhibition of autophagy impaired EBI formation and RBC maturation, trapping cells at the immature erythroblastic stage and preventing final morphological and functional transformations.
In summary, by recognizing that fibronectin fiber-like structures are primarily located near erythroid nests in vivo, we engineered a BM model made of silk as the structural component and fibronectin as a biochemical guide enhancing EBI cohesion by binding to α4β1 integrin, crucial for RBC maturation. By replicating the physiological attributes of the BM niche, the silk scaffold could activate autophagy, promote ex vivo RBC maturation, and significantly enhance enucleation. These advancements provide a physiologically accurate tissue model for replicating human erythropoiesis ex vivo and underscore the benefits of tailored tissue models in advancing our understanding of the mechanisms underlying human erythropoiesis.