Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels

Abstract

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.

Progress in biomechanical stimuli on the cell-encapsulated hydrogels for cartilage tissue regeneration

“…The microgels displayed distinct metabolomic profiles from the uncompressed and monolayer controls after dynamic compression (Fig. 3C (3)) [17].…”

“…The extracellular matrix (ECM) and pericellular matrix (PCM) are the two principal components of articular cartilage. PCM aids in shielding cartilage chondrocytes from mechanical pressures, which is weak in osteoarthritis patients [17]. It is necessary to apply biomechanical stimulation of sufficient magnitude to develop and maintain healthy articular cartilage phenotypes [18].…”

“…B The setup of the in vitro experiment and perfusion chamber, 1: Extraction and amplification of chondrocyte followed by seeding cells in fibrin hydrogel for 3 weeks in both static (well plate) and perfusion bioreactor while being exposed to BMP-2, insulin, and T3 (BIT), 2: Top and side views of macroscopic images displaying the cartilage gel centered in the human osteochondral block, reproduced content[133]. C 1: Schematic of the production process of alginate microgels, 2: Mixing concentrated alginate microgels with agarose hydrogel after 9 days culture, 3: Appling cyclic strain (5 ± 2%) to the agarose constructs containing alginate microgels in the custom-made bioreactor, reproduced content[17]. D 1-4: Osteochondral harvesting and defect creations, 5: osteochondral defect models are stimulated using a joint bioreactor with joint-specific biomechanical stimuli, reproduced content[132].…”

Biomimetic Pericellular Microniche Inspired by Chondron enhances the Cartilage Matrix Accumulation

“…Another study reported that chondrocyte was encapsulated into sodium alginate microgel to mimic the in vivo microenvironment of chondrocyte, which showed the formation of PCM could be promoted. [ 26 ] However, the size of microgel prepared by the emulsion method was dispersed and the cell in situ polymerization had an impact on cell activity. Microfluidics provides charming solution for the construction of biomimetic PCM, which has been demonstrated to show no effect on cellular activity.…”

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels

“…[21][22][23] Our group has previously demonstrated a novel method of encapsulating single primary human chondrocytes (PHCs) in 3D alginate microgels that are ≈100 μm in diameter, enabling the cells to maintain their chondrocyte phenotype and form a collagen VI-rich PCM. [14] While our previous studies analyzed differences in metabolomic profiles, they did not investigate the mechanical similarity between the in vitro developed PCM in microgels and the in vivo formed PCM. Since the PCM directly transfers extracellular mechanical loads to chondrocytes, it is crucial to understand the biomechanical properties of in vitro developed PCM.…”