Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets

Abstract

Cell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets’ chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets’ initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets’ characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.

Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage

“…), increasing biocompatibility concerns and disrupting direct communication between the transplanted cells and host tissue [ 19 , 27 , 137 , 138 ]. Limited unassisted in vivo tissue engraftment is often attributed to chondrogenic constructs’ inadequate endogenous expression of surface adhesion molecules [ 12 , 80 , 107 , 123 , 124 , 129 , 139 ]. Poor in vivo tissue site engraftment leads to construct delamination, loss of transplanted cell viability, mechanical instability, and decreased integration with host tissue, common precursors for fibrocartilage tissue formation [ 26 ] and suboptimal pre-clinical in vivo outcomes [ 8 , 27 , 31 , 140 , 141 ].…”

“…As cells are seeded and grown under adherent 2D conditions, this abrupt temperature-mediated detachment prompts established cytoskeletal filaments and retained ECM to naturally contract when released from culture surfaces [ 165 , 166 ]. This post-detachment cell sheet contraction spontaneously yields 3D, multi-nuclei thick, scaffold-free cell sheet structures [ 12 , 161 ]. Cell sheet three-dimensionality can be further controlled by cell sheet layering to produce tissues of specified thicknesses and cellular densities, even combining cell sheets from different cell sources [ 157 , 167 , 168 , 169 , 170 ].…”

“…Emerging cell sheet approaches prepare in vitro chondrogenically differentiated MSC sheets that are directly transplantable in vivo, which should support more rapid hyaline cartilage replacement at defect sites for future in vivo regenerative therapies. Reliable fabrication of 3D MSC sheets increases cell–cell interactions, promotes hyaline-like chondrogenesis, and retains construct adhesion capabilities [ 12 ], all of which are essential to support robust and direct replacement of damaged or missing hyaline cartilage. Sheet-enhanced 3D cellular interactions specifically benefit MSC chondrogenesis in vitro, resulting in stable hyaline-like phenotypes and delayed hypertrophic transitions compared to standard pellet cultures [ 12 ].…”

“…However, MSC therapies are often limited by poor survival, engraftment, and control of MSC chondrogenic differentiation fate in vivo [ 7 , 11 ]. Therefore, one unique method of advanced cartilage regeneration aims to prepare MSC-derived cartilage constructs that express hyaline-like characteristics at the time of transplantation with the goal of more rapidly and reliably replacing damaged hyaline articular cartilage [ 12 ].…”

“…As a result, MSC cartilage tissue engineering research has increasingly trended toward developing scaffold-free platforms that not only offer superior in vitro chondrogenic differentiation and optimized control 3D cellular interactions, but also support direct, unassisted delivery for robust engraftment with improved surgical versatility. Of these approaches, cell sheet tissue engineering specifically presents a unique scaffold-free platform that retains endogenous 3D cellular interactions and tissue-like organization for promoting stable in vitro hyaline-like chondrogenesis, while preserving intact adhesion molecules along the transplantation surface for direct in vivo transplantation [ 12 , 24 , 25 ]. The goal of this review is to discuss current and future directions in the development of tissue-engineered 3D MSC-derived hyaline cartilage, emphasizing cell sheet tissue engineering, with specific focus on controlled chondrogenic differentiation through 3D cellular interactions and post-differentiation engraftment capabilities.…”