Calcium Phosphate Ceramic (CPC) scaffolds, and among them hydroxyapatite (HA), are broadly used in regenerative medicine, noticeably showing good results in treating bony defects. Unfortunately, used alone these scaffolds do not meet all the challenges of engineered bone reconstruction. Indeed, the main pitfall is the limited ingrowth of host bone tissue into the biomaterial1.The regeneration of bone tissue is limited to small volumes, the colonization inside the implant hardly extends over than 1 cm from the bone apposition. This is possibly due to a lack of vascularization, limiting the supply of nutrients, oxygen and growth factors, affecting cell survival and bone healing¹. Numerous strategies are currently being applied to improve CPC biomaterials biological properties² including surface functionalization by growth factors and autologous mesenchymal stem cells (MSCs, bone cell precursors)³, to obtain, in the latter case « hybrid biomaterials ». These two approaches showed improved host tissue ingrowth but, due to a lack of neovascularization, still did not solve the problem of large bone defects regeneration. More recently functionalization of CPC scaffolds with endothelial cells associated to osteoblasts has been advocated to overcome this problem and showed promising results⁴. The modulation of 3D scaffold architecture is another way to improve bioceramics biological properties.

In line with this, taking advantage of the bioprinting technology, we sought to develop an hybrid biomaterial constituted by a natural cell association, using stem and precursor cells naturally present in the human bone marrow, to a HA ceramic shaped by additive manufacturing.

Bone marrow cells were extracted from surgical wastes with ethical agreements from the Limoges University Hospital’s committee of ethics and the cells were registered at the Biological Resource Center in compliance with the regulatory requirements, CODECOH declaration n°DC-2008-604. After having assessed the overall cell viability, the respective proportions of precursor cells from distinct lineages (mesenchymal stem cells, hematopoietic stem cells and endothelial precursor cells) were characterized by flow cytometry in order to assess the representativity of native bone marrow niche stem/ precursor cells subpopulation.

An alginate-based bio-ink is being formulated in order to concentrate the cells and promote cell-cell interactions and 3D organization. Its biological properties were characterized using human bone-marrow MSC at 24h, 4J, 7J and 21J by conventional methods of cell and molecular biology, i.e. live-dead staining and detection of specific markers allowing monitoring of the cell fate. Additionally, to fit the needs raised by evaluating the biological properties of scaffolds and to counter the time consuming and high costs of samplings and reagents used in classical biological assays, methodological approaches based on label-free imaging mainly by multiplex Coherent Scattering Raman Scattering microspectroscopy (CARS) were implemented.