Yu-Tsang Lee

Effects of Cyclic Mechanical Stretching on the mRNA Expression of Tendon/Ligament-Related and Osteoblast-Specific Genes in Human Mesenchymal Stem Cells

The purpose of this study was to explore the influences of cyclic mechanical stretching on the mRNA expressions of tendon/ligament-related and osteoblast-specific marker genes in human MSCs seeded onto a collagen type I-coated surface. The stretch-induced mRNA expressions of mesenchymal stem cell protein (MSCP), matrix metalloproteinase-3 (MMP-3), and marker genes related to tendon/ligament cells (type I collagen, type III collagen, and tenascin-C) and those typical of osteoblasts (core binding factor alpha 1 (Cbfa1), alkaline phosphatase (ALP), and osteocalcin (OCN)) were analyzed by quantitative real-time PCR. The results revealed significant downregulation of MSCP and upregulation of MMP-3 genes in MSCs subjected to mechanical loading, regardless of the magnitude of the stretching (high or low). Moreover, the typical marker genes of the osteoblast lineage were upregulated by low-magnitude stretching, whereas tendon/ligament-related genes were upregulated by high-magnitude stretching for a long period. Cbfa1 and ALP were upregulated starting as early at 8 hr, followed by a downward trend and no significant change in expression at the other time points. The mRNA expressions of type I collagen, type III collagen, and tenascin-C significantly increased in MSCs subjected to 10% stretching for 48 hr, and this effect still existed after the stretched cells had rested for 48 hr. This study demonstrated the effect of cyclic mechanical stretching on differential transcription of marker genes related to different cell lineages. Low-magnitude stretching increased mRNA expressions of Cbfa1 and ALP and was possibly involved in the early osteoblastic differentiation of MSCs, whereas high-magnitude stretching upregulated the mRNA expressions of tendon/ligament-related genes.

Modulation of gene expression and collagen production of anterior cruciate ligament cells through cell shape changes on polycaprolactone/chitosan blends

Our previous study has illustrated that chitosan could enhance human anterior cruciate ligament (ACL) cells to exhibit a dramatic effect on increasing the gene expression of transforming growth factor beta1 (TGF-beta1), which is a specific gene for wound healing and collagen synthesis. However, human ACL cells could not adhere and proliferate well on chitosan. In order to overcome this drawback, we introduced polycaprolactone (PCL) into chitosan by the method of blending in this study. It was found that the morphology, viability and gene expression of human ACL cells on the chitosan/PCL blends could be effectively regulated. With the increase of PCL content in blends, human ACL cells presented more flatten shape, well-organized cytoskeleton, and higher proliferated ability. Compared to flatten shape, human ACL cells with round shape exhibited higher levels of mRNA expression of TGF-beta1 and collagen type III through 3-day culture period. Furthermore, these blended materials could upregulate protein synthesis of human ACL cells, which corresponded to their gene expressions. Therefore, it is possible to combine the advantages of chitosan and PCL to create a new blended material, which could control cellular morphologies specifically, and further to regulate the gene expression and protein production of cells for specific applications. We expected this concept, controlling the cell shape through biomaterial to modulate the behavior of cells, could provide a new vision for the material selection of ligament tissue engineering.

The phenotypic responses of human anterior cruciate ligament cells cultured on poly(ϵ‐caprolactone) and chitosan

The purpose of this study is to evaluate the phenotypic responses of human anterior cruciate ligament (ACL) cells on two biodegradable materials: poly(epsilon-caprolactone) (PCL) and chitosan. ACL cells cultured on PCL displayed phenotypes that were well spread with a developed cytoskeleton. In comparison, chitosan was not an appropriate substrate to support the attachment and spreading of ACL cells, which was attributed to the low fibronectin (FN) adsorption of chitosan. However, ACL cells cultured on chitosan exhibited a dramatic effect on increasing transcripts of transforming growth factor beta1 (TGF beta1) and collagen III. After coating FN on chitosan surface, cell morphology and the mRNA levels of all tested genes had the similar levels on PCL and FN-coated chitosan. This indicates the expression of TGF beta1 and collagen III mRNA of human ACL cells was seem to correlate closely with the adhesion behavior of human ACL cells and was influenced by the underlying substrate properties. Since an ideal scaffold used in ACL tissue engineering is not only for cell attachment but also for extracellular matrix deposition during ligament regeneration, chitosan may be considered as a scaffold for ACL tissue engineering, which can upregulate the expression of specific genes of matrix production and wound healing in human ACL cells to synthesize more quantity of FN and TGF beta1 proteins.

Response of Human Mesenchymal Stem Cells (hMSCs) to the Topographic Variation of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) (PHBHHx) Films

The influence of the topographic morphology of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) films on human mesenchymal stem cells (hMSCs) was investigated in this study. PHBHHx films with various surface characteristics were prepared by compression-molding, solvent-casting and electrospinning. The adhesion, proliferation and differentiation behaviors of hMSCs were significantly modulated by the surface characteristics of these films. HMSCs could aggregate and form cellular clusters on the cast PHBHHx films, and the time to form cellular aggregates increased as the surface roughness increased. The aggregated hMSCs on the cast films kept their original surface markers and presented much higher viability during the regular culture and lower differentiation ability upon osteogenic induction than the spread cells on the compression-molded films and TCPS. HMSCs spread well and showed a specific orientation on the surface of the random electrospun fibrous films, they were not able to migrate into the interior of electrospun fibrous films, and they revealed the highest viability during the regular culture but a lower differentiation activity upon osteogenic induction. The electrospun fibrous PHBHHx films could serve as a suitable substrate for large quantity culturing of hMSCs when undifferentiated hMSCs are desired.

Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF) in human fibroblast-like synovial cells from advanced-stage osteoarthritis in vitro

Intraarticular injection of hyaluronan (hyaluronic acid; HA) is the common way to treat osteoarthritis (OA) of knees. This treatment cannot only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is unknown. In this study, after human synovial cells were stimulated with HA and Hylan (Synvisc®) for 24 h, real‐time polymerase chain reaction (real‐time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor‐β1 (TGF‐β1), and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis of synovial cells in serum deprivation environment. The gene expressions of TGF‐β1 and VEGF were significantly increased at the concentration of 0.1 mg/mL HA and 0.1 mg/mL Hylan, respectively (α < 0.05). The synovial cells with treatment of 0.1 mg/mL Hylan decreased the CTGF gene expression (0.66‐fold) and VEGF (0.78‐fold) compared to 0.1 mg/mL HA (α < 0.05). We suggested that the profile of CTGF, TGF‐β1, and VEGF gene expressions in our study might provide the rational mechanism for the therapeutic effect of hyaluronan on OA knees.

Effects of the Surface Characteristics of Polyhydroxyalkanoates on the Metabolic Activities and Morphology of Human Mesenchymal Stem Cells

Polyhydroxyalkanoates (PHAs) are a newer family of biomaterials for tissue-engineering applications. The objective of this study is to investigate the behavior of human mesenchymal stem cells (hMSCs) grown on various PHA films. The surface characteristics of PHA co-polymer films were varied by the content of 3-hydroxyvalerate (HV) or 3-hydroxyhexanoate (HHx) and by the film preparation methods such as compression-molding and solvent-casting. Hyaluronic acid (HA) was further applied to modify the surface properties of PHA membranes. As HV content increased, the crystallinity and the hydrophobicity of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) membranes decreased and the metabolic activity of hMSCs raised, although the distribution and morphology of hMSCs did not show significant variation. Hyaluronic acid (HA) coating on PHA membranes could improve the metabolic activity and reduce the death rate of hMSCs. Aggregates and spheroidal clusters of hMSCs were found on the surface of cast poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) membranes. The growth of hMSCs was remarkably influenced by various surface characteristics of the PHA films.

Effects of the Surface Characteristics of Nano-Crystalline and Micro-Particle Calcium Phosphate/Chitosan Composite Films on the Behavior of Human Mesenchymal Stem Cells In Vitro

Calcium phosphate (CaP) compounds, the main inorganic constituent of mammalian bone tissues, are believed to support bone precursor cell growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the present study, different scales of CaP crystals on chitosan membranes were prepared for culture of human mesenchymal stem cells (hMSCs) in vitro. A series of aqueous CaP suspensions with different concentrations were mixed with chitosan solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. With different weight ratios of CaP in chitosan solution, the various surface characteristics of nano-amorphous (C/CaP 0.1), nano-crystalline (C/CaP 0.5) and micro-particle (C/CaP 2) CaP compounds were examined by scanning electron microscopy and electron dispersion spectroscopy. X-ray diffraction on micro-particles of CaP indicated the formation of crystalline hydroxyapatite. The behavior of hMSCs, including proliferation, cell spreading and osteogenic differentiation, was studied on the C/CaP films. In basal culture medium, the incorporation of CaP into chitosan films could promote the proliferation of hMSCs. The C/CaP 0.5 film with connected CaP nano-crystals had better cellular viability. The fluorescence microscope images at 14 days of culture revealed extensive networks of F-actin filaments of hMSCs on chitosan, C/CaP 0.1 and C/CaP 0.5 films. The cellular morphology on C/CaP 2 film with discrete CaP micro-particles was partly restrained. In osteogenic medium, the alkaline phosphatase (ALP) activity of hMSCs increased and showed the process of osteogenic differentiation. The ALP levels on C/CaP 2 film were higher than those on C/CaP 0.1 and C/CaP 0.5 films. These results demonstrated that the crystallinity and topography of CaP on chitosan membranes could modulate the behaviors of cultured hMSCs in vitro.

Chondrogenesis of human bone marrow mesenchymal cells by transforming growth factors β1 through cell shape changes on controlled biomaterials

The phenotypic responses of human bone marrow mesenchymal cells (hBMSCs) on different ratio of chitosan/polycaprolactone (PCL) blends were investigated in this study. The results showed that hBMSCs existed different morphology on chitosan/PCL blends due to the different adhesion characteristic of cell on neat PCL and neat chitosan. Interestingly, comparing to hBMSCs on neat PCL, hBMSCs aggregated to form spheroid and to express ascendant trend of transforming growth factor β1, collagen type II, collagen type X, and Sox9 mRNA on the chitosan/PCL blended substrates with the decrease of PCL content. To confirm chondrogenesis of hBMSCs with spheroid on test substrates, Alcian Blue and Safranin O staining were used to detect the cartilaginous extracellular matrix (ECM). It revealed hBMSCs with spheroid on neat chitosan and 10 wt % PCL did turn to chondrogenic differentiation and synthesize cartilaginous ECM. Therefore, these findings provided new insights into the role of chitosan/PCL blended material could mediate the endogenous gene expression of hBMSCs to alter the phenotypic behavior through mediating the cell shape.

Modulating the Activities of Human Mesenchymal Stem Cells (hMSCs) and C3A/HepG2 Hepatoma Cells by Modifying the Surface Characteristics of Poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx)

The new biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx) has a potential application in tissue engineering. The aim of this study was to present a deeper picture of the relationship between the cellular behavior and the surface characteristics of PHBHHx films. The pristine PHBHHx film was prepared by adopting the compression-molding method, and then the acrylic acid molecules were grafted on PHBHHx membrane surface by UV irradiation. The hydrophilic nature and surface roughness of various PHBHHx films were controlled by adjusting the acrylic acid concentration and the UV irradiation time. Although the surface characteristics of various PHBHHx films could not affect the metabolic activity of hMSCs, the performance of morphology of hMSCs was deeply affected by the hydrophilic nature and the orientation of surface scars. The hydrophilic nature would effectively improve the spread of hMSCs, and the orientation of surface scars would guide the growth direction of cytoskeleton (actin) inside hMSCs. In contrast, the behaviors of C3A/HepG2 hepatoma cells presented an opposite outcomes. Those surface characteristics were obviously associated with the performance of metabolic activity of C3A cells, but not with the morphology of C3A cells. Both hMSCs and C3A cells have unique cellular characteristics; therefore, their responses to environmental stimulations are significantly different.