Covalent Stem Cell-Combining Injectable Materials with Enhanced Stemness and Controlled Differentiation In Vivo

Abstract

Biohybrid materials, which are defined as engineered functional materials combining living components with nonliving synthetic materials, are considered promising bioactive materials for applications in in vivo tissue engineering. However, the rational design of biohybrid materials applicable to in vivo tissue engineering faces major challenges associated with techniques for combining living cells with nonliving synthetic materials and cell sources. Here, we report injectable covalent stem cell-combing biohybrid materials prepared via a bio-orthogonal click cross-linking reaction of azide-modified adipose-derived stem cells (N3[+]ADSCs), one of the most promising cell sources utilized clinically, with alkyne-modified biocompatible alginate polymers. The mechanical properties of the covalent stem cell-combining biohybrid materials can be adapted to the mechanical properties of the surrounding environment in which they are transplanted by alternating the number of N3[+]ADSCs, the concentration of alkyne-modified alginate, and the number of alkyne groups. Importantly, ADSCs in the covalent biohybrid materials expressed a high level of CD-105, a marker for undifferentiated mesenchymal stem cells, in the body in the absence of differentiation signals, whereas very little CD-105 was expressed in the control physical cell-loading materials, demonstrating that this covalent stem cell-combining approach results in enhanced retention of the material’s “stemness” and controlled differentiation in the body. We assessed the potential utility of the covalent stem cell-combining biohybrid materials for in vivo tissue engineering using a murine severe skeletal muscle defect-healing model. Importantly, all of the tissues regenerated by the covalent biohybrid material treatment expressed MYH3, a myogenic marker protein, whereas no expression of MYH3 was detected in the tissues reconstructed by treatment with control physical stem cell-loading materials and Matrigel, indicating that this covalent stem cell-combining approach results in controlled differentiation in the body. Our data demonstrate the potential utility of covalent stem cell-combining biohybrid materials with host tissue-integrative and controlled differentiation capabilities available for in vivo tissue engineering.

Advances in Cell‐Rich Inks for Biofabricating Living Architectures

“…[78] Despite not being yet applied for bioprinting purposes, the injectability of such systems shows great potential, resembling interesting living inks to be explored for HCD bioprinting. [79,80]…”

Cell Surface Engineering Tools for Programming Living Assemblies

“…[69] Through rationally designing the cell surface, such functionalized cellular building blocks can be spatiotemporally molded and processed for the establishment of robust and complex 3D bioarchitectures with living features. The following section provides an outlook of <30 min (synthetic polycation-PLL) [80] Relatively prolonged exposure (>7 days, natural biopolymer) [61] [ [ 76,77] Up to 7 days (liposome fusion) [10] [ Up to 7 days [178] [71, 155, 179]…”

“…[21] Surface-engineered cells have already been used for macro-scale muscle repair and for promoting a faster generation of functional liver or cardiac tissues when compared to assemblies generated via native cellular adhesions of pristine cells. [32,178,186] Advances in biofabrication technologies (i.e., digital light processing, volumetric-, aspiration-or microfluidic-based bioprinting) may further enable researchers to process functionalized cells into macro-scale living materials with higher spatiotemporal control over tissue organization. This could unlock the so-desired fabrication of cell-dense tissues in a more reproducible and controlled mode.…”

Integrating Metabolic Oligosaccharide Engineering and SPAAC Click Chemistry for Constructing Fibrinolytic Cell Surfaces

“…Since the pioneering work by Bertozzi et al, who introduced exogenous oligosaccharides into the glycocalyx of cell membranes, the emergence of metabolic oligosaccharide engineering strategies has provided additional avenues for “cell membrane surface modification”. , Metabolic oligosaccharide engineering involves the conversion of nonnatural oligosaccharides into activated nucleotide sugars through cellular biosynthetic pathways, followed by the delivery of these monosaccharides bearing unique chemical functional groups to cell surface glycan structures via intracellular metabolic mechanisms. Subsequently, by employing bioorthogonal chemistry, functional chemical moieties with biological activities are introduced onto the cell surface. − In recent years, the strain-promoted azide–alkyne cycloaddition (SPAAC) “click” reaction, which does not require metal catalysts and occurs rapidly and efficiently under physiological conditions, has rapidly developed in the field of “cell membrane surface modification”. − Gibson et al utilized metabolic oligosaccharide engineering to introduce the azide-functionalized nonnatural oligosaccharide tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz) into the cell membrane surface of modified human Caucasian lung carcinoma cells (A549 cells). Subsequently, the modified cells were further reacted with functional molecules containing dibenzocyclooctyne (DBCO) groups (DBCO-pHEA-Biotin) via the SPAAC reaction, ultimately introducing biotin molecules onto the cell membrane surface of A549 cells to achieve further coupling. , Moreover, they also applied this bioorthogonal reaction to the “engineering cells to capture polymers” strategy, where Ac4ManNAz was used to label three types of cancer cells (A549 cells, human Caucasian Dukes’ type B colorectal adenocarcinoma cells (SW480 cells), and human Caucasian breast adenocarcinoma derived from metastatic sites (MCF-7 cells)).…”