An official website of the United States government
Abstract One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop‐based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten‐fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.
more »
« less
Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels
Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.
more »
« less
- PAR ID:
- 10327658
- Date Published:
- Journal Name:
- Cells
- Volume:
- 11
- Issue:
- 5
- ISSN:
- 2073-4409
- Page Range / eLocation ID:
- 900
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract To elucidate the mechanisms of cellular mechanotransduction, it is necessary to employ biomaterials that effectively merge biofunctionality with appropriate mechanical characteristics. Agarose and collagen separately are common biopolymers used in cartilage mechanobiology and mechanotransduction studies but lack features that make them ideal for functional engineered cartilage. In this study, agarose is blended with collagen type I to create hydrogels with final concentrations of 4% w/v or 2% w/v agarose with 2 mg/mL collagen. We hypothesized that the addition of collagen into a high-concentration agarose hydrogel does not diminish mechanical properties. Acellular and cell-laden studies were completed to assess rheologic and compressive properties, contraction, and structural homogeneity in addition to cell proliferation and sulfated glycosaminoglycan production. Over 21 days in culture, cellular 4% agarose–2 mg/mL collagen I hydrogels seeded with primary murine chondrocytes displayed structural and bulk mechanical behaviors that did not significantly alter from 4% agarose-only hydrogels, cell proliferation, and continual glycosaminoglycan production, indicating promise toward the development of an effective hydrogel for chondrocyte mechanotransduction and mechanobiology studies.more » « less
-
Abstract Tissue‐engineered cartilage has shown promising results in the repair of focal cartilage defects. However, current clinical techniques rely on an extra surgical procedure to biopsy healthy cartilage to obtain human chondrocytes. Alternatively, induced pluripotent stem cells (iPSCs) have the ability to differentiate into chondrocytes and produce cartilaginous matrix without the need to biopsy healthy cartilage. However, the mechanical properties of tissue‐engineered cartilage with iPSCs are unknown and might be critical to long‐term tissue function and health. This study used confined compression, cartilage on glass tribology, and shear testing on a confocal microscope to assess the macroscale and microscale mechanical properties of two constructs seeded with either chondrocyte‐derived iPSCs (Ch‐iPSCs) or native human chondrocytes. Macroscale properties of Ch‐iPSC constructs provided similar or better mechanical properties than chondrocyte constructs. Under compression, Ch‐iPSC constructs had an aggregate modulus that was two times larger than chondrocyte constructs and was closer to native tissue. No differences in the shear modulus and friction coefficients were observed between Ch‐iPSC and chondrocyte constructs. On the microscale, Ch‐iPSC and chondrocyte constructs had different depth‐dependent mechanical properties, neither of which matches native tissue. These observed depth‐dependent differences may be important to the function of the implant. Overall, this comparison of multiple mechanical properties of Ch‐iPSC and chondrocyte constructs shows that using Ch‐iPSCs can produce equivalent or better global mechanical properties to chondrocytes. Therefore, iPSC‐seeded cartilage constructs could be a promising solution to repair focal cartilage defects. The chondrocyte constructs used in this study have been implanted into humans for clinical trials. Therefore, Ch‐iPSC constructs could also be used clinically in place of the current chondrocyte construct.more » « less
-
Abstract Articular joints facilitate motion and transfer loads to underlying bone through a combination of cartilage tissue and synovial fluid, which together generate a low‐friction contact surface. Traumatic injury delivered to cartilage and the surrounding joint capsule causes secretion of proinflammatory cytokines by chondrocytes and the synovium, triggering cartilage matrix breakdown and impairing the ability of synovial fluid to lubricate the joint. Once these inflammatory processes become chronic, posttraumatic osteoarthritis (PTOA) development begins. However, the exact mechanism by which negative alterations to synovial fluid leads to PTOA pathogenesis is not fully understood. We hypothesize that removing the lubricating macromolecules from synovial fluid alters the relationship between mechanical loads and subsequent chondrocyte behavior in injured cartilage. To test this hypothesis, we utilized an ex vivo model of PTOA that involves subjecting cartilage explants to a single rapid impact followed by continuous articulation within a lubricating bath of either healthy synovial fluid, phosphate‐buffered saline (PBS), synovial fluid treated with hyaluronidase, or synovial fluid treated with trypsin. These treatments degrade the main macromolecules attributed with providing synovial fluid with its lubricating properties; hyaluronic acid and lubricin. Explants were then bisected and fluorescently stained to assess global and depth‐dependent cell death, caspase activity, and mitochondrial depolarization. Explants were tested via confocal elastography to determine the local shear strain profile generated in each lubricant. These results show that degrading hyaluronic acid or lubricin in synovial fluid significantly increases middle zone chondrocyte damage and shear strain loading magnitudes, while also altering chondrocyte sensitivity to loading.more » « less
-
Disorders of cartilage homeostasis and chondrocyte apoptosis are major events in the pathogenesis of osteoarthritis (OA). Herein, we sought to assess the chondroprotective effect and underlying mechanisms of a novel chemically modified curcumin, CMC2.24, in modulating extracellular matrix (ECM) homeostasis and inhibiting chondrocyte apoptosis. Rats underwent the anterior cruciate ligament transection and medial menisci resection were treated by intra-articular injection with CMC2.24. In vitro study, rat chondrocytes were pretreated with CMC2.24 before stimulation with sodium nitroprusside (SNP). The effects of CMC2.24 on cartilage homeostasis and chondrocyte apoptosis were observed. The results from in vivo studies demonstrated that the intra-articular administration of CMC2.24 delayed cartilage degeneration and suppressed chondrocyte apoptosis. CMC2.24 ameliorated osteoarthritic cartilage destruction by promoting collagen 2a1 production and inhibited cartilage degradation and apoptosis by suppressing hypoxia-inducible factor-2a (Hif-2α), matrix metalloproteinase-3 (MMP-3), runt-related transcription factor 2 (RUNX2), cleaved caspase-3, vascular endothelial growth factor (VEGF), and the phosphorylation of IκBα and NF-κB p65. The in vitro results revealed that CMC2.24 exhibited a strong inhibitory effect on SNP-induced chondrocyte catabolism and apoptosis. The SNP-enhanced expression of Hif-2α, catabolic and apoptotic factor, decreased after CMC2.24 treatment in a dose-dependent manner. CMC2.24 pretreatment effectively inhibited SNP-induced IκBα and NF-κB p65 phosphorylation in rat chondrocytes, whereas the pretreatment with NF-κB antagonist BMS-345541 significantly enhanced the effects of CMC2.24. Taken together, these results demonstrated that CMC2.24 attenuates OA progression by modulating ECM homeostasis and chondrocyte apoptosis via suppression of the NF-κB/Hif-2α axis, thus providing a new perspective for the therapeutic strategy of OA.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/cells11050900
