C. Paggi (Enschede, NL)

University of Twente Developmental bioengineering

Presenter Of 2 Presentations

Podium Presentation Biomechanics

10.4.5 - Cartilage-on-chip: mimicking the complex mechanical stimulation present in articular cartilage

Presentation Number
10.4.5
Presentation Topic
Biomechanics
Lecture Time
14:06 - 14:15
Session Type
Free Papers
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Human cartilage-on-chip models that faithfully mimic the complex mechanical movements of the joint hold great promise for increasing our understanding of joint diseases and development of therapeutic interventions. Current models, however,fail to appropriately replicate the combined compression and shear strain present in articulations. The aim of this study is to develop an innovative organ-on-chip platform mimicking these complex movements in the knee joint.

Methods and Materials

The cartilage-on-a-chip platform was fabricated from polydimethylsiloxane (PDMS) using soft-lithography. The platform comprises three different sections as detailed in Fig. 1.

Compressive strain and shear strain were imposed on the hydrogel by deforming the thin membrane by applying independently pressure to the three chambers.

Human chondrocytes (hCHs) were cultured in an agarose matrix using proliferation medium. Microbeads (15 µm) were used to evaluate hydrogel deformation upon mechanical stimulation. Cell viability was evaluated using a live dead/assay with (800 mbar applied at 1Hz for 1.5hrs per day, starting from day 4) or without mechanical stimulation after 6 days.

Results

We quantified agarose deformation as a function of strain (fig. 2): With higher pressure, higher strain was generated. Importantly, both physiological healthy (5-20%) and pathophysiological (>20%) strains could be applied on the hydrogel. Moreover, shear strain was obtained by single chamber stimulation (not shown).

We next determined the decrease in chondrocytes surface area in a hydrogel subjected to a homogeneous compression (800 mbar at 1Hz for 1.5hrs). Cell volume decrease was inversely correlated with the cell’s proximity to the compression membrane. hCHs could be cultured in our platform for at least 6 days, without a noticeable effect on cell viability.

icrs figure 1.png

icrs figure 2.png

Conclusion

This novel microfluidic platform can be used to explore the impact of various mechanical stimuli on responses of individual cells. We will extent this platform with units mimicking synovium, ligaments, bone and meniscus to engineer a joint-on-chip.

Collapse
Podium Presentation Stem Cells

18.2.5 - Autophagy drives the therapeutic effect of MSCs in single-stage cell-based cartilage regeneration

Presentation Number
18.2.5
Presentation Topic
Stem Cells
Lecture Time
14:51 - 15:00
Session Type
Free Papers
Corresponding Author
Disclosure
No Significant Commercial Relationship

Abstract

Purpose

Combination of chondrocytes with autologous or allogeneic mesenchymal stem cells (MSCs) has enabled the development of single-stage cell-based cartilage regeneration procedures, with proven clinical success. With repeat long-term sampling, the MSCs population dies and disappears. Characterization of the mechanism and role of MSC death in the therapeutic effect of MSCs and chondrocytes is currently unknown and hence the aim of the current study.

Methods and Materials

One human chondrocyte donor was cultured with 3 different human adipose derived MSCs at distinct ratios (100% chondrocytes, 80% aMSCs + 20% chondrocytes, 100% aMSCs) for 7 days in pellets (figure 1a). The cells were cultured under hypoxia (2% oxygen) in chondrocyte proliferation medium. RNA sequencing and bioinformatic analysis was performed followed by western blot and immunofluorescence.

Results

Up and down regulation of specific genes was detected using MORPEUS heat map analysis (figure 1b). Among the 362 significantly up regulated genes, 92 were present in all the 3 co-cultures (figure 1c). These genes were analysed using ClueGo software to determine their GO-pathway. To further asses the interaction between the up regulated genes STRING software was used. Of the 92 genes, 51 had interconnections. Of this subgroup, a literature research showed that 73% of these genes play a role in cell death pathways like autophagy and/or apoptosis (figure 2).

To determine which of the two pathways was involved in cell death, the protein levels of cleaved caspase 3 as marker for apoptosis and P62, LC3I & LC3II as markers for autophagy were analysed. The results showed no support for activation of caspase-dependent cell death at the protein level. Instead markers for autophagy were clearly and substantially upregulated.

figure 1 icrs abstract.pngfigure 2 icrs abstract.png

Conclusion

In single-stage cell-based cartilage regeneration procedures, MSCs disappear by a caspase independent autophagy pathway. We postulate that the self-sacrifice of the MSCs by autophagy generates a chondrocyte stimulatory environment boosting cartilage formation.

Collapse