Mechanical forces transduced to the nucleus that affect intranuclear organization are critical regulators for directing the differentiation of mesenchymal stem cells (MSCs). The supraphysiological dose of a mechanical cue, which is from the stiff surface of the substrate like standard tissue culture polystyrene, biases the MSCs toward osteogenic differentiation with irreversible nuclear retention of an osteogenic master transcription factor, RUNX2. To study the mechanism underlying the loss of lineage plasticity and robustness of the irreversible osteogenic differentiation in human MSCs due to the mechanotransduction, we investigated how RUNX2 was retained in the nucleus focusing on the dynamic shuttling of a mechanotransducer, yes-associated protein (YAP), intranuclear chromatin organization, and their regulation by the actin cytoskeleton using pharmaceutical inhibitors to perturb the mechanical properties of chromatin and actin cytoskeleton. YAP and RUNX2 were localized in the nucleus of MSCs during the expansion culture on the polystyrene surfaces followed by 24 hours culture on the glass surface, at the time point just before the inhibitor treatments. The nuclear RUNX2 was diffused into the cytoplasm by chromatin decondensation through the inhibition of histone deacetylase. In contrast, RUNX2 localization was unaffected from the perturbations to the actin organization through the inhibition of actin polymerization and that of Rho kinase activity under the condition that chromatin condensation was maintained, although YAP was released from the nucleus to the cytoplasm. These findings suggest that RUNX2 is stably retained in the nucleus by its engagement with specific DNA and/or nucleoskeletal structures coexisting with the robustly condensed chromatin, in the MSCs biased toward an irreversible osteogenic differentiation.

Keywords

Mesenchymal stem cells, Osteogenic differentiation, Chromatin, Actin cytoskeleton, Histone deacetylase

Paper information

Masashi YAMAZAKI, Hiromichi FUJIE, Hiromi MIYOSHI, “Chromatin condensation retains the osteogenic transcription factor, RUNX2, in the nucleus of human mesenchymal stem cells”, Journal of Biomechanical Science and Engineering, Vol.15, No.2 (2020), p.20-00083. doi:10.1299/jbse.20-00083. Final Version Released on July 15, 2020, Advance Publication Released on May 28, 2020.

In the present study, an image correlation method was used to determine the site-dependent strain in the porcine anterior cruciate ligament (ACL). In particular, the strain of the ACL in the femoral and tibial attachment areas was quantified when the eight knees were subjected to a maximum of 50 N anterior tibial load that was applied using a 6-DOF robotic system. The ACL strains in the anterior, central, and posterior bundles of the medial, middle and lateral layers were determined as a function of the applied anterior load. In addition, the surface of the ACL was observed using a light microscope under no loading condition. Results revealed that the strain in the medial layer of the ACL increased gradually and almost linearly with the increase of anterior load in the midsubstance area. In contrast, the strain in the femoral and tibial attachment areas of the medial layer of the ACL increased rapidly at the beginning of anterior loading and gradually thereafter with significant differences in slope of strain-anterior load curve found in the anterior and posterior bundles. The largest strain in response to 50 N of anterior force was found in the tibial attachment area in medial and middle layers, while the maximum strain was found in the femoral attachment area in the lateral layer. Microscopic observation indicated that crimp structure was more clearly observed in the femoral and tibial attachment areas than in the mid-substance. These microstructural features of the ACL were may be attributable to the higher and load-dependent, nonlinear strain observed in the attachment areas. Our study suggested that the strain in the ACL at full extension is site- and load-dependent in a non-linear manner at the femoral and tibial attachment areas.

Keywords

Anterior cruciate ligament, Strain, Knee joint, Robotic system, Image correlation method

Paper information

Satoshi YAMAKAWA, Richard E. DEBSKI, Hiromichi FUJIE, “Strain distribution in the anterior cruciate ligament in response to anterior drawer force to the knee”, Journal of Biomechanical Science and Engineering, Vol.12, No.1 (2017), p.16-00582. doi:10.1299/jbse.16-00582. Final Version Released on March 31, 2017, Advance Publication Released on February 21, 2017.

We have been developing a new tissue engineering technique for cartilage repair using a scaffold-free tissue engineered construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). A round-shaped chondral defect of 8.5 mm in diameter and 2.0 mm in depth created on the medial condyle of immature (4-month-old) and mature (12-month-old) porcine femur was filled with the TEC. Six months after surgery, a cylindrically shaped specimen of 4 mm in diameter and 4-5 mm in depth was extracted. Stiffness measurements were carried out on the specimen using an AFM after the surface image of the specimen was obtained. The TEC-treated tissues exhibited more irregular surface as compared with normal cartilage, regardless of animal maturity. The stiffness of the superficial layer of the TEC-treated tissue was significantly lower than those of the normal cartilage, indicating 6.8±2.3 mN/m in the immature group and 8.8±2.3 mN/m in the mature group. Histological observation indicated that the defects were repaired with a hyaline cartilage-like tissue with positive Safranine O staining, regardless of animal maturity. However, the superficial layer of the repaired tissues was negatively stained with Safranine O. The present study suggests that the recovery of the superficial layer is delayed in both immature and mature animals although the treatment with TEC enhances the repair of partial chondral defects.

Keywords

Cartilage Repair, Mesenchymal Stem Cell, Tissue Engineered Construct (TEC), Scaffold Free, Atomic Force Microscope (AFM), Stiffness, Surface Morphology

Paper information

Ryosuke NANSAI, Takuya SUZUKI, Kazunori SHIMOMURA, Wataru ANDO, Norimasa NAKAMURA and Hiromichi FUJIE, “Surface Morphology and Stiffness of Cartilage-Like Tissue Repaired with a Scaffold-Free Tissue Engineered Construct”, Journal of Biomechanical Science and Engineering, Vol. 6, No. 1 (2011), pp.40-48 . doi:10.1299/jbse.6.40