Three-D Models for Megakaryopoiesis and Platelet Production

Access to human platelets is fundamental in the quest for understanding the mechanisms of platelet-related diseases and the clinical need for platelet transfusions for patients. Effective solutions for producing platelets ex vivo are essential to obtaining this goal. Circulating platelets are highly specialised cells produced by megakaryocytes through the extension of unique pseudopods called pro-platelets. Leading studies point to the bone marrow niche as the core of blood cell production, revealing interesting and complex environmental factors for consideration. Megakaryocytes take cues from the physic-chemical 3D environment in the bone marrow, which includes contact with extracellular matrix components, interaction with other cell types, particularly endothelial cells, and the shear stress generated by the blood circulation into the sinusoid lumen. The technical impossibility of obtaining an intact bone marrow organ prompted the investigation of potential models to extrapolate ex vivo structures and functions to gain new insight on its activity. Thus, future advancements in the study of megakaryopoiesis depend on the evolution of bioengineering techniques for developing physiologically relevant models that reproduce the conditions in the bone marrow niche environment. Silk fibroin, derived from Bombyx mori silkworm cocoons,¹ is one of the most promising biomaterials for bone marrow tissue engineering due to its tunable architecture and mechanical properties. Silk is a naturally derived, biocompatible biomaterial, which can be prepared in a range of material formats and processed entirely in aqueous systems allowing the incorporation of labile compounds without loss of bioactivity (e.g., extracellular matrix components). Exploiting innovative silk biomaterials, we have developed a series of 3D scalable systems that reproduce structure and composition of the human bone marrow, supporting natural platelet release from megakaryocytes.^2-4 Data demonstrate that 1) silk supports platelet production maintaining their function without premature activation; 2) silk surface topography and stiffness direct megakaryocyte behaviour by activating mechanosensors and calcium signalling; 3) bloodstream impact on megakaryocyte function can be mimicked by perfusing the silk models at different shear rate and flows; 4) silk functionalisation with fibronectin and/or endothelial cells enhances megakaryocyte function; 5) silk models can be used to reproduce and study the primary megakaryocyte related disorders such as Myeloproliferative Neoplasms and Inherited Thrombocytopenias; 6) the same silk models can be exploited to test drug efficacy and/or toxicology on a per patient basis. Our models provide valuable and unique technology to reproduce the complexity of human bone marrow and study megakaryocytes and platelet formation. The silk platform has potentially limitless applications for basic research, clinic, pharmaceutical industry and transfusion medicine.

1. Omenetto FG and Kaplan DL. New opportunities for an ancient material. Science. 2010; 329: 528.

2. Di Buduo CA, Wray LS, Tozzi L, et al. Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood. 2015; 125: 2254.

3. Di Buduo CA, Soprano PM, Tozzi L, et al. Modular flow chamber for engineering bone marrow architecture and function. Biomaterials. 2017; 146: 60.

4. Abbonante V, Di Buduo CA, Gruppi C, et al. A new path to platelet production through matrix sensing. Haematologica. 2017; 102: 1150.