skip to main content

This content will become publicly available on March 1, 2023

Title: 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.
; ; ; ; ; ;
Award ID(s):
Publication Date:
Journal Name:
Page Range or eLocation-ID:
Sponsoring Org:
National Science Foundation
More Like this
  1. 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 administrationmore »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.« less
  2. Smad4 is an intracellular effector of the TGFβ family that has been implicated in Myhre syndrome, a skeletal dysplasia characterized by short stature, brachydactyly and stiff joints. The TGFβ pathway also plays a critical role in the development, organization and proliferation of the growth plate, although the exact mechanisms remain unclear. Skeletal phenotypes in Myhre syndrome overlap with processes regulated by the TGFβ pathway, including organization and proliferation of the growth plate and polarity of the chondrocyte. We used in vitro and in vivo models of Smad4 deficiency in chondrocytes to test the hypothesis that deregulated TGFβ signaling leads tomore »aberrant extracellular matrix production and loss of chondrocyte polarity. Specifically, we evaluated growth plate chondrocyte polarity in tibiae of Col2-Cre+/-;Smad4fl/fl mice and in chondrocyte pellet cultures. In vitro and in vivo, Smad4 deficiency decreased aggrecan expression and increased MMP13 expression. Smad4-deficiency disrupts the balance of cartilage matrix synthesis and degradation, even though the sequential expression of growth plate chondrocyte markers was intact. Chondrocytes in Smad4 deficient growth plates also showed evidence of polarity defects, with impaired proliferation and ability to undergo the characteristic changes in shape, size and orientation as they differentiate from resting to hypertrophic chondrocytes. Therefore, we show that Smad4 controls chondrocyte proliferation, orientation, and hypertrophy and is important in regulating the extracellular matrix composition of the growth plate.

    « less
  3. Measuring LTGF-beta activation in live tissues in situ is a major challenge due to the short half-life of activated TGF-beta in cartilage (due to rapid receptor internalization/degradation). As such, activation assessments typically require analysis of downstream events. However, assessments of intracellular TGF-beta signaling molecules (Smad2/3 phosphorylation) yield mostly qualitative measures and reporter cell assays are not compatible with intact cartilage tissues. Alternatively, in the current project, we proposed quantifying LTGF-beta activation in situ through a novel assay that capitalizes on TGF-beta’s robust autoinduction behavior; active TGF-beta activity induces a predictable increase in synthesis of soluble LTGF-beta. The dominant fraction ofmore »newly synthesized LTGF-beta is secreted from the tissue (not retained in ECM) and stable. Accordingly, measurements of LTGF-beta secretion into culture medium allows for quantifications of TGF-beta activity in cartilage. In order to confirm that LTGF-beta secretion enhancements result from TGF-beta activity (and not other load-initiated signaling cascades), a control group can readily be utilized, consisting of TGF-beta activity inhibition from a TGF-beta-receptor specific kinase inhibitor. Using this platform, we performed the first-ever measurement of the activity of TGF-beta in cartilage explants from load-induced activation. Results demonstrate that LTGF-beta secretion rates do indeed increase with cartilage mechanical loading. Upon exposure to a TGF-beta inhibitor, LTGF-beta secretion rates return to basal control levels, thus confirming that LTGF-beta secretion enhancements can be predominantly attributed to TGF-beta activity in the tissue. Upon standard curve conversion, autoinduction assay results demonstrate that mechanical load-induced activation of ECM-bound LTGF-beta gives rise to ~0.15ng/mL of TGF-beta activity in cartilage. Importantly, this measure represents the first quantitative assessment of TGF-beta activity in articular cartilage. While these levels represent the activation of only a small fraction of the total LTGF-beta stores in the cartilage ECM (~300ng/mL), they are indeed capable of giving rise to considerable chondrocyte biosynthesis enhancements in the tissue. As such, these measurements support the mechanobiological role of load-induced LTGF-beta activation in maintaining articular cartilage integrity. The assay platform advanced in this study sets the foundation for considerable advances in our understanding of the mechanistic details and physiologic importance of load-induced LTGF-beta activation in cartilage. In the future, we plan to use this quantitative platform to assess: 1) the influence of varying loading regimens on LTGF-beta activation rates (e.g., physiologic exercise, elevated stresses, high-impact trauma), and 2) changes to load-induced LTGF-beta activation with aging or joint degeneration. An abstract on this work was presented at the 2020 ASME SB3C Conference (virtual meeting) and a full-length manuscript is currently in preparation.« less
  4. Experiments have shown that external mechanical loading plays an important role in bone development and remodeling. In fact, recent research has provided evidence that osteocytes can sense such loading and respond by releasing biochemical signals (mechanotransduction, MT) that initiate bone degradation or growth. Many aspects on MT remain unclear, especially at the cellular level. Because of the extreme hardness of the bone matrix and complexity of the microenvironment that an osteocyte lives in, in vivo studies are difficult; in contrast, modeling and simulation are viable approaches. Although many computational studies have been carried out, the complex geometry that can involvemore »60+ irregular canaliculi is often simplified to a select few straight tubes or channels. In addition, the pericellular matrix (PCM) is usually not considered. To better understand the effects of these frequently neglected aspects, we use the lattice Boltzmann equations to model the fluid flow over an osteocyte in a lacuno-canalicular network in two dimensions. We focus on the influences of the number/geometry of the canaliculi and the effects of the PCM on the fluid wall shear stress (WSS) and normal stress (WNS) on an osteocyte surface. We consider 16, 32, and 64 canaliculi using one randomly generated geometry for each of the 16 and 32 canaliculi cases and three geometries for the 64 canaliculi case. We also consider 0%, 5%, 10%, 20%, and 40% pericellular matrix density. Numerical results on the WSS and WNS distributions and on the velocity field are visualized, compared, and analyzed. Our major results are as follows: (1) the fluid flow generates significantly greater force on the surface of the osteocyte if the model includes the pericellular matrix (PCM); (2) in the absence of PCM, the average magnitudes of the stresses on the osteocyte surface are not significantly altered by the number and geometry of the canaliculi despite some quantitative influence of the latter on overall variation and distribution of those stresses; and (3) the dimensionless stress (stress after non-dimensionalization) on the osteocyte surface scales approximately as the reciprocal of the Reynolds number and increasing PCM density in the canaliculi reduces the range of Reynolds number values for which the scaling law holds.« less
  5. A comprehensive understanding of multiscale and multiphasic intervertebral disc mechanics is crucial for designing advanced tissue engineered structures aiming to recapitulate native tissue behavior. The bovine caudal disc is a commonly used human disc analog due to its availability, large disc height and area, and similarities in biochemical and mechanical properties to the human disc. Because of challenges in directly measuring subtissue-level mechanics, such as in situ fiber mechanics, finite element models have been widely employed in spinal biomechanics research. However, many previous models use homogenization theory and describe each model element as a homogenized combination of fibers and themore »extrafibrillar matrix while ignoring the role of water content or osmotic behavior. Thus, these models are limited in their ability in investigating subtissue-level mechanics and stress-bearing mechanisms through fluid pressure. The objective of this study was to develop and validate a structure-based bovine caudal disc model, and to evaluate multiscale and multiphasic intervertebral disc mechanics under different loading conditions and with degeneration. The structure-based model was developed based on native disc structure, where fibers and matrix in the annulus fibrosus were described as distinct materials occupying separate volumes. Model parameters were directly obtained from experimental studies without calibration. Under the multiscale validation framework, the model was validated across the joint-, tissue-, and subtissue-levels. Our model accurately predicted multiscale disc responses for 15 of 16 cases, emphasizing the accuracy of the model, as well as the effectiveness and robustness of the multiscale structure-based modeling-validation framework. The model also demonstrated the rim as a weak link for disc failure, highlighting the importance of keeping the cartilage endplate intact when evaluating disc failure mechanisms in vitro . Importantly, results from this study elucidated important fluid-based load-bearing mechanisms and fiber-matrix interactions that are important for understanding disease progression and regeneration in intervertebral discs. In conclusion, the methods presented in this study can be used in conjunction with experimental work to simultaneously investigate disc joint-, tissue-, and subtissue-level mechanics with degeneration, disease, and injury.« less