Articular cartilage defects represent an inciting factor for future osteoarthritis (OA) and degenerative joint disease progression. Despite multiple clinically available therapies that succeed in providing short term pain reduction and restoration of limited mobility, current treatments do not reliably regenerate native hyaline cartilage or halt cartilage degeneration at these defect sites. Novel therapeutics aimed at addressing limitations of current clinical cartilage regeneration therapies increasingly focus on allogeneic cells, specifically mesenchymal stem cells (MSCs), as potent, banked, and available cell sources that express chondrogenic lineage commitment capabilities. Innovative tissue engineering approaches employing allogeneic MSCs aim to develop three-dimensional (3D), chondrogenically differentiated constructs for direct and immediate replacement of hyaline cartilage, improve local site tissue integration, and optimize treatment outcomes. Among emerging tissue engineering technologies, advancements in cell sheet tissue engineering offer promising capabilities for achieving both in vitro hyaline-like differentiation and effective transplantation, based on controlled 3D cellular interactions and retained cellular adhesion molecules. This review focuses on 3D MSC-based tissue engineering approaches for fabricating “ready-to-use” hyaline-like cartilage constructs for future rapid in vivo regenerative cartilage therapies. We highlight current approaches and future directions regarding development of MSC-derived cartilage therapies, emphasizing cell sheet tissue engineering, with specific focus on regulating 3D cellular interactions for controlled chondrogenic differentiation and post-differentiation transplantation capabilities.

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.

Mesenchymal stromal cells (MSCs) represent a promising treatment for immune-related diseases due to their diverse immunomodulatory paracrine functions. However, progress of culture-expanded MSCs is hindered by inconsistent cell function, poor localization, and insufficient retention when administered as suspended cell injections, thus placing spatiotemporal dosing constraints on therapeutic functions. To address these limitations, we introduce the combination of in vitro interferon-gamma (IFN-γ) priming, a key stimulator of MSC immunosuppressive potency, and thermoresponsive cultureware to harvest cultured MSCs as directly transplantable scaffold-free immunosuppressive cell sheets. Here, we demonstrate that MSC sheets produced with IFN-γ priming upregulate expression of immunosuppressive factors indoleamine 2,3-dioxygenase (IDO-1), interleukin-10 (IL-10), programmed death ligand-1 (PD-L1), and prostaglandin E2 (PGE2) in both dose- and duration-dependent manners. In addition, IFN-γ primed MSC sheets showed increased ability to inhibit T-cell proliferation via indirect and direct contact, specifically related to increased IDO-1 and PGE2 concentrations. Furthermore, this study’s use of human clinical-grade single-cell-derived clonal bone marrow-derived MSCs, contributes to the future translatability and clinical relevancy of the produced sheets. Ultimately, these results present the combination of IFN-γ priming and MSC sheets as a new strategy to improve MSC-mediated treatment of localized inflammatory diseases.

Cell-based therapies are increasingly focused on allogeneic stem cell sources because of several advantages in eliminating donor variability (e.g., aging and disease pathophysiology) affecting stem cell quality and in cell-banked sourcing of healthy donors to enable “off-the-shelf” products. However, allogeneic cell therapy is limited by host patient immunologic competence and inconsistent performance due to cell delivery methods. To address allogeneic cell therapy limitations, this study developed a new allogeneic stem cell sheet using human umbilical cord mesenchymal stem cells (hUC-MSC) that present low antigenicity (i.e., major histocompatibility complex, MHC). Optimal conditions including cell density, passage number, and culture time were examined to fabricate reliable hUC-MSC sheets. MHC II antigens correlated to alloimmune rejection were barely expressed in hUC-MSC sheets compared to other comparator MSC sheets (hBMSC and hADSC). hUC-MSC sheets easily graft spontaneously onto subcutaneous tissue in immune-deficient mice within 10 minutes of placement. No sutures are required to secure sheets to tissue because sheet extracellular matrix (ECM) actively facilitates cell-target tissue adhesion. At 10 days post-transplantation, hUC-MSC sheets remain on ectopic target tissue sites and exhibit new blood vessel formation. Furthermore, implanted hUC-MSC sheets secrete human HGF continuously to the murine target tissue. hUC-MSC sheets described here should provide new insights for improving allogenic cell-based therapies.

Knee cartilage does not regenerate spontaneously after injury, and a gold standard regenerative treatment algorithm has not been established. This study demonstrates preclinical safety and efficacy of scaffold-free, human juvenile cartilage-derived-chondrocyte (JCC) sheets produced from routine surgical discards using thermo-responsive cultureware. JCCs exhibit stable and high growth potential in vitro over passage 10, supporting possibilities for scale-up to mass production for commercialization. JCC sheets contain highly viable, densely packed cells, show no anchorage-independent cell growth, express mesenchymal surface markers, and lack MHC II expression. In nude rat focal osteochondral defect models, stable neocartilage formation was observed at 4 weeks by JCC sheet transplantation without abnormal tissue growth over 24 weeks in contrast to the nontreatment group showing no spontaneous cartilage repair. Regenerated cartilage was safranin-O positive, contained type II collagen, aggrecan, and human vimentin, and lacked type I collagen, indicating that the hyaline-like neocartilage formed originates from transplanted JCC sheets rather than host-derived cells. This study demonstrates the safety of JCC sheets and stable hyaline cartilage formation with engineered JCC sheets utilizing a sustainable tissue supply. Cost-benefit and scaling issues for sheet fabrication and use support feasibility of this JCC sheet strategy in clinical cartilage repair.