Biology subjects

Su, A. W.

Publications and source records attributed to Su, A. W..

3 recordsLinked to original sources

bioRxiv2026

Bioorthogonal Tuning of Hydrogel Stiffness Promotes Zonal Redifferentiation of Passaged Chondrocytes

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

bioengineering↗

bioRxiv2026

Fibronectin Coating of Tissue Culture Polystyrene to Improve Superficial Zone Chondrocyte Expansion

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

cell biology↗

bioRxiv2025

The Effects of Hypothermic Storage on Passaged Chondrocyte Viability and Redifferentiation Potential

Cell-based transplantation therapies, such as autologous chondrocyte implantation (ACI), are used to treat focal cartilage defects caused by trauma or degeneration. In ACI, chondrocytes are isolated from non-load-bearing regions of healthy cartilage regions and then sent to a cell manufacturing laboratory, where they are expanded for cell number in monolayer culture. Once a large number of cells are obtained, they are transported to the clinic for reimplantation into the defect site. The storage and transport conditions from cell manufacturing to implantation may be a critical time that could influence cell viability and redifferentiation potential. Although hypothermic storage at sub-physiological temperatures is commonly used to preserve cell viability, long-term storage of cartilage under hypothermic conditions can impair chondrocyte viability and function. However, the impact of short-term, acute hypothermic storage on passaged chondrocytes remains largely unknown. We tested the hypothesis that acute hypothermic storage negatively impacts passaged chondrocyte viability and reduces the capacity for redifferentiation. Passaged chondrocytes were stored either in monolayer culture or in suspension at 36, 19 or 8{degrees}C. In monolayer culture, hypothermic temperatures preserved cell viability with no difference compared to storage at 36{degrees}C for up to three days. Additionally, hypothermic temperatures promoted cell rounding, reduced proliferative capacity, depolymerized filamentous actin, and led to a slight reduction in the mRNA levels of specific matrix molecules compared to 36{degrees}C. Intriguingly, the effects of hypothermia were context-dependent. Exposure of passaged cells in suspension to hypothermia promoted the maintenance of cell viability and reduced aggregation compared to 36{degrees}C. When stored at 8{degrees}C in suspension, passaged cells exhibited enhanced expression of specific matrix molecule mRNA levels compared to cells at 36{degrees}C in suspension. Subsequently, when passaged cells in suspension at 8{degrees}C were seeded in 3D within adherent agarose molds, there was an increase in aggrecan expression 10 days after seeding. The tissues formed by cells stored in suspension at 9{degrees}C were thicker and stained more intensely for aggrecan. Therefore, in contrast to our hypothesis, we found that hypothermic storage did not have a negative impact; when stored for 1 day in suspension, it had lasting effects on matrix deposition. The storage of passaged chondrocytes under hypothermic conditions may be beneficial for ACI, warranting further investigations of cell hypothermic storage for in vivo repair.

bioengineering↗