# New Insights on Mechanical Stimulation of Mesenchymal Stem Cells for Cartilage Regeneration
## 4 Practical Applications  
To reveal the physiological effect of mechanical stimulation on stem cells, loading machines were designed to be able to apply different types of mechanical forces with different intensity, duration, and frequency. [PERSON] et al. [35] cultivated MSCs in a methylcellulose solution retained within a porous polyurethane matrix to evaluate the suitability of the matrix in supporting mechanically induced chondrogenesis of the cells in the absence of exogenous factors. The composite underwent a combination of compression and shear forces by the use of a bioreactor which applied compressive and rolling movements through a ceramic ball. This ball compressed the scaffold dynamically at 1 Hz, resulting in a strain amplitude of 10-20% of its height, and provided shear stress with oscillations perpendicular to the scaffold axis of \(\pm 25^{\circ}\) at 1 Hz. After 21 days, the samples were analyzed by reverse transcription polymerase chain reaction (RT-PCR), biochemical, histochemical, and immunofluorescence assays, and the authors concluded that the physical stimulation led to the activation of TGF-\(\beta\) and to accumulation in the surrounding matrix of glycosaminoglycans and type II collagen, confirming the chondrogenesis process. Another study on the involvement of mechanical forces in the destiny of adipose-derived mesenchymal stem cells (ADSCs) by [PERSON] et al. [16] highlights the role of dynamic compression in combination with exogenous SOX-9 on chondrogenesis. ADSCs were seeded in 3D porous polylactic-co-glycolic acid (PLGA) scaffolds. Gradual, meaning with a unique structure at each level, and uniform scaffolds were subjected, with the use of a bioreactor, to sequential uniaxial compressions of 5-10% strain amplitude and frequency of 0.1 Hz. The authors analyzed the morphology of the ADSCs and the ECM deposition within the scaffolds by scanning electron microscopy (SEM), noticing that ECM accumulation by ADSCs, on gradual scaffolds and in the presence of SOX-9, was higher than that in uniform scaffolds or in gradual scaffolds without SOX-9. RT-PCR analysis also showed the highest expressions of Proteoglycan 4 (PRG4), Parathyroid hormone-related protein (PTHrP), type II collagen, aggrecan, SOX-9, and Hypoxia-inducible factor 1-alpha (HIF-1\(\alpha\)) in the group of SOX-9 gradual scaffolds. To better understand the single contributions of compression and shear forces in chondrogenic induction, [PERSON] et al. [27] analyzed these two stimuli, both alone and in combination. MSCs were seeded onto polyurethane scaffolds and underwent either compression at 1 Hz, in a strain amplitude of 10-20%, or oscillation of \(\pm 25^{\circ}\) at 1 Hz, or a combination of both loads. Oscillation was imposed through the shifting of a ball perpendicularly to the scaffold axis. Superimposed compression was applied along the axis of the scaffold as well. The results suggested that stimulation by combined  
Figure 2: Types of mechanical stimuli experienced by the cells. Stem cell cultures, used in tissue engineering, undergo biophysical stimulation in order to promote chondrogenesis and recreate the dynamic environment within the joint. strains, instead of the application a single stimulus alone, is the best way to assure a chondrogenic phenotype in the absence of exogenous growth factors. The authors gave evidence of significant upregulation, in comparison to the control group, of the chondrogenesis markers (type II collagen, Aggrecan (AGG), Cartilage oligomeric matrix protein (COMP), SOX-9) only in the samples loaded with both compression and shear. Also, this last group was the only one in which type II collagen immunostaining was detected and that seemed to maintain a constant release of GAG in medium. [PERSON] et al. [36] developed a novel construct made up by platelet-rich fibrin (PRF) membrane, which functions as a growth-factor-rich scaffold for bone-marrow-derived stem cells (BMSCs), for transplantation in cartilage defects.