In vitro and in vivo characterization of a novel tricalcium silicate-based ink for bone regeneration using laser-assisted bioprinting

Abstract

Grafts aside, current strategies employed to overcome bone loss still fail to reproduce native tissue physiology. Among the emerging bioprinting strategies, Laser-Assisted Bioprinting (LAB) offers very high resolution, allowing designing micrometric patterns in a contactless manner, providing a reproducible tool to test ink formulation. To this date, no LAB associated ink succeeded to provide a reproducible ad integrum bone regeneration on a murine calvaria critical size defect model. Using the CE approved BioRoot RCS® as a mineral addition to a collagen-enriched ink compatible with LAB, the present study describes the process of the development of a solidifying tricalcium silicate-based ink as a new bone repair promoting substrates in a LAB model. This ink formulation was mechanically characterized by rheology to adjust it for LAB. Printed aside Stromal Cells from Apical Papilla (SCAPs), this ink demonstrated a great cytocompatibility, with significant in vitro positive impact upon cell motility, and an early osteogenic differentiation response in the absence of another stimulus. Results indicated that the in vivo application of this new ink formulation to regenerate critical size bone defect tends to promote the formation of bone volume fraction without affecting the vascularization of the neo-formed tissue. The use of LAB techniques with this ink failed to demonstrate a complete bone repair, whether SCAPs were printed or not of at its direct proximity. The relevance of the properties of this specific ink formulation would therefore rely on the quantity applied in situ as a defect filler rather than its cell modulation properties observed in vitro. For the first time, a tricalcium silicate-based printed ink, based on rheological analysis, was characterized in vitro and in vivo, giving valuable information to reach complete bone regeneration through formulation updates. This LAB-based process could be generalized to normalize the characterization of candidate ink for bone regeneration.

In Vivo Application of Silica-Derived Inks for Bone Tissue Engineering: A 10-Year Systematic Review

“…Within this minority, inkjet-associated strategies were the most representative [ 37 , 38 , 39 ] (6% of total), while digital light processing [ 40 , 41 ] and manual deposit or injection [ 42 , 43 ] were both 4%. Electrospinning [ 44 ], laser-assisted bioprinting [ 45 ] and selective laser melting [ 46 ] shared equally the 6% remaining (2% each). The trend of the studies assessing silicon-based inks was here to obtain an extrudable paste, mostly mixed with polyvinyl alcohol (PVA), that went through a sintering process after the printing step.…”

“…Within articles including Si as a native part of the material, 21.6% of the total (11 out of 51) articles placed silicon as a primary element of focus. The compositions of the investigated materials were variable with monocalcium-based inks [ 47 , 48 ], tricalcium-based inks [ 45 , 55 ]; glasses also involved [ 53 , 54 ]; elements such as magnesium [ 28 , 29 , 30 ], strontium [ 52 ] or lithium [ 56 ] were also core components of calcium–silicate materials. Controls were usually either an empty defect, β-TCP or titanium scaffolds.…”

“…Controls were usually either an empty defect, β-TCP or titanium scaffolds. Only Touya et al [ 45 ], using laser-assisted bioprinting in a mouse calvaria defect model, reported an absence of enhanced tissue mineralization despite encouraging in vitro results, whether associated with cells or not. Overall, the outcome was the improvement in bone repair compared to the control.…”

“…Moreover, due to the low viscosity nature of most materials investigated here and the inherent constraints of ink deposition this low viscosity triggered, extrusion was the most relevant tool to perform an efficient, reproducible printing process. Overall, a very limited number of studies investigated the association of cells [ 45 , 62 , 74 ] to the Si-derived material as part of the engineered final product to implant, and most of the publications relied on the host response rather than an enhanced biological activity due to additive cells. Only Wang et al [ 74 ] found the addition of cells to their material to significantly improve bone formation.…”

“…In spite of aiming for a single purpose, bone repair, this review highlighted the plurality in strategies: from the scaffold/ink preparation process to the delivery of the material. Most studies involved processes with several intermediary steps such as soaking, drying, sintering, etc., to generate a scaffold, while few were able to deliver a final product by one-step assembling and in situ printing [ 43 , 45 ]. Although the tissue engineering definition [ 122 ] depicted a trinity involving cells, adjuvants and support materials, almost none of the articles here investigated focused on a strategy relying on the use of cells, and most used an inorganic material-associated approach.…”