Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells

Abstract

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as β-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

Silk-Based Materials for Hard Tissue Engineering

“…In another approach, tyrosinase-crosslinked silk fibroin/gelatine hydrogel blends were cell loaded and 3D printed to gain functional constructs [ 122 ]. Silk was functionalized with gelatine and calcium chloride for sustained release of calcium ions from the scaffold, similar to the extracellular release of calcium by osteoclasts during bone modeling ( Figure 5 ).…”

3D Printing and Bioprinting to Model Bone Cancer: The Role of Materials and Nanoscale Cues in Directing Cell Behavior

“…Based on these considerations, in 3D bone bioprinting, natural polymers are preferred, such as alginate [73,[98][99][100][101], gelatin [98,100,102], and gelatin methacrylate (GelMA) [103][104][105][106], silk fibroin [107,108], chitosan [75,109,110], hyaluronic acid [76,111], fibrin [86,112], and collagen [31,109,113]. Considering biomimicry, collagen and its denatured counterpart, gelatin, are the most promising, although alginate is often preferred due to the easiness of printing.…”

“…As already reported, there are two ways to include a mineralized fraction in 3D-printed and bioprinted scaffold: directly by adding micro/nanoparticles in the osteomimetic ink [30,31,116,117] or by grafting on the surface as a coating [117,118], the latter being more diffused in 3D printing technology [119]. Based on these considerations, in 3D bone bioprinting, natural polymers are preferred, such as alginate [73,[98][99][100][101], gelatin [98,100,102], and gelatin methacrylate (GelMA) [103][104][105][106], silk fibroin [107,108], chitosan [75,109,110], hyaluronic acid [76,111], fibrin [86,112], and collagen [31,109,113]. Considering biomimicry, collagen and its denatured counterpart, gelatin, are the most promising, although alginate is often preferred due to the easiness of printing.…”

Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications

“…Porcine chondrocytes showed good attachment, viability, and proliferation over 14 days [340]. For composites, a silk-gelatin bioink encapsulating calcium ions was used to print a scaffold that showed gradual calcium release that promoted osteogenic differentiation of hMSCs and enhancing mineralization processes [344]. Silk-gelatin bioinks have also been used to encapsulate hMSCs [345] and human mesenchymal progenitor cells [346].…”

“…Inclusion of alginate [240,[319][320][321], Laponite [322], nanoclays [323], Pluronic F-127 [208], PCL [324]; Feature size 500 µm [245] [ [313][314][315][316]; Feature size 300 µm (SLA), Compressive E: 0.5-18 MPa [328] Mouse planta dermis [319]; dental pulp stem cells [320]; hMSCs and amniotic epithelial cells [240]; chondrocytes [214,327] Silk ( Mouse articular chondrocytes [342]; human fibroblasts [338]; porcine chondrocytes [340]; hMSCs [344,345]; human mesenchymal progenitor cells [346]…”