Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering

Abstract

3D-manufactured hydrogels with precise contours and biological adhesion motifs are interesting candidates in the regenerative medicine field for the culture and differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). 3D-bioprinting is a powerful technique to approach one step closer the native organization of cells. This study investigates the effect of the incorporation of collagen type I in 3D-bioprinted polysaccharide-based hydrogels to the modulation of cell morphology, osteogenic remodeling potential, and mineralization. By combining thermo-responsive agarose hydrogels with collagen type I, the mechanical stiffness and printing contours of printed constructs can be improved compared to pure collagen hydrogels which are typically used as standard materials for MSC osteogenic differentiation. The results presented here show that MSC not only survive the 3D-bioprinting process but also maintain the mesenchymal phenotype, as proved by live/dead staining and immunocytochemistry (vimentin positive, CD34 negative). Increased solids concentrations of collagen in the hydrogel blend induce changes in cell morphology, namely, by enhancing cell spreading, that ultimately contribute to enhanced and directed MSC osteogenic differentiation. 3D-bioprinted agarose-collagen hydrogels with high-collagen ratio are therefore feasible for MSC osteogenic differentiation, contrarily to low-collagen blends, as proved by two-photon microscopy, Alizarin Red staining, and real-time polymerase chain reaction.

Corneal bioprinting utilizing collagen‐based bioinks and primary human keratocytes

“…This is an indicator for unsuccessful integration of CSK inside the bioinks, because CSK are naturally dendritic in native corneal stromal tissue, as shown in Figure a,b. We hypothesized that the reason for this difference in cell morphology is due to used solid concentrations of the bioink (Duarte Campos et al, , ). Lower solids concentrations of the polysaccharide component (0.5% agarose) in the blended bioink allowed for increased cell spreading compared to higher solids concentrations (3.0% sodium alginate), as employed in the study by Isaacson et al…”

“…Using DoD, we bioprinted cultivated CSK‐loaded Type I collagen‐based bioinks suitable for corneal stromal regeneration (Figure ). The bioinks were made of two essential components: (a) Type I collagen for improved functionality, and (b) agarose hydrogel for fast and precise printability (Duarte Campos et al, , ). Working with collagen faces some drawbacks, since collagen alone lacks the stability needed to support a corneal construct, which makes further stabilization steps such as cross‐linking necessary (Duan & Sheardown, ).…”

Translating Periosteum's Regenerative Power: Insights From Quantitative Analysis of Tissue Genesis With a Periosteum Substitute Implant

“…While the authors of this study conjecture that collagen containing membranes may be used clinically to promote “formation of a thin osteoblastic cell layer to eventually enhance bone regeneration,” side‐by‐side comparison of bone regeneration in vivo would be needed to prove this [59]. In addition, recent in vitro studies indicated a positive effect of incorporating collagen type I in hydrogels designed to guide stem cell differentiation toward osteogenesis [60]. Such wide variances in in vitro results using different formulations of collagen of varying bioavailability underscore the need for controlled in vivo comparative models using a common critical‐sized bone defect model as well as the side‐by‐side comparison of periosteum substitutes incorporating endogenous ECM structural proteins.…”

Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications

“…Collagen has also been used as part of mixed polymer systems such as collagen/alginate/gelatin for the proliferation of human corneal epithelial cells [257]; collagen with sodium alginate for the proliferation and gene expression of chondrocytes for cartilage tissue engineering [299]; collagen microfibers within a gelatin methacrylate (GelMA) matrix as well as collagen/agarose for the viability, spreading, and differentiation into osteocytes of bone mesenchymal stem cells [300][301][302]; and collagen/hyaluronic acid for 3D liver microenvironments containing primary human hepatocytes and liver stellate cells [209].…”