The Effects of Epigallocatechin-3-Gallate and Mechanical Stimulation on Osteogenic Differentiation of Human Mesenchymal Stem Cells: Individual or Synergistic Effects

This study aims to investigate the roles and effects of EGCG (epigallocatechin-3-gallate) during the osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. Recent studies have shown that proper mechanical stimuli can induce osteogenic differentiation of hMSCs apart from biochemical factors. In this study, the hMSC cultures were subjected to: (1) 25 uM EGCG alone or (2) 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days) or (3) in combination with 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days). The two factors were applied to the cell cultures separately and in combination to investigate the individual and synergistic effect of both mechanical stimulation and ECGC in the osteogenic differentiation of hMSCs. Utilizing real time PCR, we measured various osteogenic markers and even those related to intracellular signalings. Further investigation of mitochondria was performed that mitochondria biogenesis, antioxidant capacity, and morphological related markers were measured. hMSCs were to be osteogenic or myogenic differentiated when they were under 3% stretching only. However, when EGCG was applied along with stretching they were to be osteogenic differentiated rather than to be myogenic differentiated. This was supported by evaluating intracellular signalings: BMP-2 and VEGF. Therefore, the synergistical effects of simultaneous employment of stretching and EGCG on osteogenic differentiation were confirmed. Moreover, simultaneous employment was found positive in mitochondria biogenesis, antioxidant capacity, and morphological changes. Through this study, we came into the conclusion that the combination of proper mechanical stretching, 3% in this study, and EGCG promote osteogenic differentiation. Reflecting that EGCG can be obtained from plants not from the chemical syntheses, it is worth to be studied further either by animal tests or long-term experiments for clinical applications.

PCL/β-TCP Composite Scaffolds Exhibit Positive Osteogenic Differentiation with Mechanical Stimulation

We investigated the use of Polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) composites with applied mechanical stimulation as scaffold for bone tissue engineering. PCL-based three-dimensional (3D) structures were fabricated in a solvent-free process using a 3D-printing technique. The mass fraction of β-TCP was varied in the range 0–30%, and the structure and compressive modulus of the specimens was characterized. The shape and interconnectivity of the pores was found to be satisfactory, and the compressive modulus of the specimens was comparable with that of human trabecular bone. Human mesenchymal stem cells were seeded on the composites, and various biological evaluations were performed over 9 days. With a mass fraction of β-TCP of 30%, differentiation began earlier; however, the cell proliferation rate was lower. Through the use of mechanical stimulation, however, the proliferation rate recovered, and was comparable with that of the other groups. This stimulation effect was also observed in ECM generation and other biological assays. With mechanical stimulation, expression of osteogenic markers was lower on samples with a β-TCP content of 10 wt% than without β-TCP; however, with mechanical stimulation, the sample with a β-TCP content of 30 wt% exhibited significantly greater expression of those markers than the other samples. We found that mechanical stimulation and the addition of β-TCP interacted closely, and that a mass fraction of β-TCP of 30% was particularly useful as a bone tissue scaffold when accompanied by mechanical stimulation.

Distinguishing Tendon and Ligament Fibroblasts Based on ¹H Nuclear Magnetic Resonance Spectroscopy

Tendon and ligament (T/L) have been known to be obviously different from each other in tissue level. However, due to the overlapping gene markers, distinction in cellular level has not been clearly verified yet. Recently, the use of nuclear magnetic resonance (NMR) spectroscopy has shown the potential to detect biological markers in cellular level. Therefore, in this study we applied a non-invasive technique based on NMR spectroscopy to establish biomarkers to distinguish between T/L fibroblasts. In addition the cellular morphologies and gene expression patterns were also investigated for comparison through optical microscopy and real-time polymerase chain reaction (PCR). No difference was observed from morphology and real-time PCR results, either as expected. However, we found clear differences in their metabolomic spectra using ¹H NMR spectroscopy. The calculated integral values of fatty acids (with chemical shifts at ~0.9, 1.26, 1.59, 2.05, 2.25, and 2.81 ppm), lactate (~1.33 ppm), and leucine (~2.72 ppm) were significantly different between the two types of fibroblasts. To be specific tendon group exhibited higher level of the metabolite than ligament group. In conclusion, in-cell metabolomic evaluation by NMR technique used in this study is believed to provide a promising tool in distinguishing cell types, especially T/L cells, which cannot be classified by conventional biological assays.

The Effects of Substrates and Hydrostatic Pressure on Differentiation of Mesenchymal Stem Cell / 대한정형외과연구학회지

PURPOSE: This study investigated the potential of dual differentiation of stem cells into osteo- and chodrogenesis depending on scaffold type even in the same environment. MATERIALS AND METHODS: For the part of the cartilage tissue section, MSCs were suspended in alginate solution and bead droplets were made using 23G syringe. For the bone tissue section, PCL/HA scaffolds were made using the bio-plotting system followed by seeding mesenchymal stem cells (MSCs) onto the scaffolds. Scaffolds with MSCs were cultured in cocktail media containing osteogenic and chondrogenic growth factors for up to 21 days. To provide mechanical environments which articular cartilage experiences in-vivo, intermittent hydrostatic pressure (IHP) was engaged. Various cellular responses were assessed: the quantitative analysis of DNA contents, GAG contents, ALP activities and immunofluorescence. RESULTS: We found that IHP promoted MSCs differentiation into the targeted cell types. That is, MSCs in alginate scaffolds were able to be differentiated into chondrocytes, while those onto PCL/HA scaffolds were able to be differentiated into osteoblasts. CONCLUSION: Depending on the scaffold characteristics MSCs can be differentiated into bone cells or chondrocytes. This technique can provide a cue for the treatment of osteochondral defects utilizing tissue engineering.

The Effects of Substrates and Hydrostatic Pressure on Differentiation of Mesenchymal Stem Cell / 대한정형외과연구학회지

PURPOSE: This study investigated the potential of dual differentiation of stem cells into osteo- and chodrogenesis depending on scaffold type even in the same environment. MATERIALS AND METHODS: For the part of the cartilage tissue section, MSCs were suspended in alginate solution and bead droplets were made using 23G syringe. For the bone tissue section, PCL/HA scaffolds were made using the bio-plotting system followed by seeding mesenchymal stem cells (MSCs) onto the scaffolds. Scaffolds with MSCs were cultured in cocktail media containing osteogenic and chondrogenic growth factors for up to 21 days. To provide mechanical environments which articular cartilage experiences in-vivo, intermittent hydrostatic pressure (IHP) was engaged. Various cellular responses were assessed: the quantitative analysis of DNA contents, GAG contents, ALP activities and immunofluorescence. RESULTS: We found that IHP promoted MSCs differentiation into the targeted cell types. That is, MSCs in alginate scaffolds were able to be differentiated into chondrocytes, while those onto PCL/HA scaffolds were able to be differentiated into osteoblasts. CONCLUSION: Depending on the scaffold characteristics MSCs can be differentiated into bone cells or chondrocytes. This technique can provide a cue for the treatment of osteochondral defects utilizing tissue engineering.