Objective Engineering cartilage requires that a clinically relevant cell type be situated within a 3D environment that supports cell viability, the production and retention of cartilage-specific ECM, and eventually, the establishment of mechanical properties that approach that of the native tissue. In this study, we investigated the ability of bone marrow derived mesenchymal stem cells (MSCs) to undergo chondrogenesis in crosslinked methacrylated hyaluronic acid (HA) hydrogels (MeHA) of different macromer concentrations (1, 2, and 5%). Design Over a six week culture period under pro-chondrogenic conditions, we evaluated cartilage-specific gene expression, ECM deposition within constructs and released to the culture media, and mechanical properties in both compression and tension. Further, we examined early matrix assembly and long term histological features of the forming tissues, as well as the ability of macromolecules to diffuse within hydrogels as a function of MeHA macromer concentration. Results Findings from this study show that variations in macromer density influence MSC chondrogenesis in distinct ways. Increasing HA macromer density promoted chondrogenesis and matrix formation and retention, but yielded functionally inferior constructs due to limited matrix distribution throughout the construct expanse. In 1% MeHA constructs, the equilibrium compressive modulus reached 0.12 MPa and s-GAG content reached nearly 3% of the wet weight, values that matched or exceeded those of control agarose constructs and that are 25% and 50% of native tissue levels, respectively. Conclusions These data provide new insight into how early matrix deposition regulates long term construct development, and defines new parameters for optimizing the formation of functional MSC-based engineered articular cartilage using HA hydrogels.
Background Cartilage degeneration is common in the aged, and aged chondrocytes are inferior to juvenile chondrocytes in producing cartilage-specific extracellular matrix. Mesenchymal stem cells (MSCs) are an alternative cell type that can differentiate toward the chondrocyte phenotype. Aging may influence MSC chondrogenesis but remains less well studied, particularly in the bovine system. Questions/purposes The objectives of this study were (1) to confirm age-related changes in bovine articular cartilage, establish how age affects chondrogenesis in cultured pellets for (2) chondrocytes and (3) MSCs, and (4) determine age-related changes in the biochemical and biomechanical development of clinically relevant MSC-seeded hydrogels. Methods Native bovine articular cartilage from fetal (n = 3 donors), juvenile (n = 3 donors), and adult (n = 3 donors) animals was analyzed for mechanical and biochemical properties (n = 3-5 per donor). Chondrocyte and MSC pellets (n = 3 donors per age) were cultured for 6 weeks before analysis of biochemical content (n = 3 per donor). Bone marrow-derived MSCs of each age were also cultured within hyaluronic acid hydrogels for 3 weeks and analyzed for matrix deposition and mechanical properties (n = 4 per age). Results Articular cartilage mechanical properties and collagen content increased with age. We observed robust matrix accumulation in three-dimensional pellet culture by fetal chondrocytes with diminished collagen-forming capacity in adult chondrocytes. Chondrogenic induction of MSCs was greater in fetal and juvenile cell pellets. Likewise, fetal and juvenile MSCs in hydrogels imparted greater matrix and mechanical properties. Conclusions Donor age and biochemical microenvironment were major determinants of both bovine chondrocyte and MSC functional capacity. Clinical Relevance In vitro model systems should be evaluated in the context of age-related changes and should be benchmarked against human MSC data.
How ion channels localize and distribute on the cell membrane remains incompletely understood. We show that interventions that vary cell adhesion proteins and cell size also affect the membrane current density of inward-rectifier K + channels (K ir 2.1; encoded by KCNJ2) and profoundly alter the action potential shape of excitable cells. By using micropatterning to manipulate the localization and size of focal adhesions (FAs) in single HEK293 cells engineered to stably express K ir 2.1 channels or in neonatal rat cardiomyocytes, we establish a robust linear correlation between FA coverage and the amplitude of K ir 2.1 current at both the local and whole-cell levels. Confocal microscopy showed that K ir 2.1 channels accumulate in membrane proximal to FAs. Selective pharmacological inhibition of key mediators of protein trafficking and the spatially dependent alterations in the dynamics of K ir 2.1 fluorescent recovery after photobleaching revealed that the K ir 2.1 channels are transported to the cell membrane uniformly, but are preferentially internalized by endocytosis at sites that are distal from FAs. Based on these results, we propose adhesion-regulated membrane localization of ion channels as a fundamental mechanism of controlling cellular electrophysiology via mechanochemical signals, independent of the direct ion channel mechanogating.
Articular cartilage pathology is common in the aged population. Numerous studies have shown that aged chondrocytes (CHs) are inferior to juvenile CHs in their ability to proliferate and produce cartilage-specific extracellular matrix proteins, potentially limiting their use in tissue engineering applications for cartilage restoration [1,2]. Mesenchymal stem cells (MSCs) are an alternative cell type that can be expanded in vitro while maintaining their ability to differentiate into cell types comparable to articular chondrocytes. However, organismal aging also influences human MSC proliferation [3,4] and multi-potential differentiation [5], though for chondrogenesis these findings are mixed, with some suggesting that aged progenitor cells retain their chondrogenic capacity [6]. The objective of this study was to assess age related differences in donor-matched CH and MSC potential for chondrogenic repair. In addition, the effects of the chondrogenic growth factor TGF-β3 on CHs and MSCs were evaluated.
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