Investigation of Cell Adhesion and Cell Viability of the Endothelial and Fibroblast Cells on Electrospun PCL, PLGA and Coaxial Scaffolds for Production of Tissue Engineered Blood Vessel.

Endothelialization of artificial scaffolds is considered an effective strategy for increasing the efficiency of vascular transplantation. This study aimed to compare the biophysical/biocompatible properties of three different biodegradable fibrous scaffolds: Poly (ɛ-caprolactone) (PCL) alone, Poly Lactic-co-Glycolic Acid (PLGA) alone (both processed using Spraybase® electrospinning machine), and Coaxial scaffold where the fiber core and sheath was made of PCL and PLGA, respectively. Scaffold structural morphology was assessed by scanning electron microscope and tensile testing was used to investigate the scaffold tension resistance over time. Biocompatibility studies were carried out with human umbilical vein endothelial cells (HUVEC) and human vascular fibroblasts (HVF) for which cell viability (and cell proliferation over a 4-day period) and cell adhesion to the scaffolds were assessed by cytotoxicity assays and confocal microscopy, respectively. Our results showed that all biodegradable polymeric scaffolds are a reliable host to adhere and promote proliferation in HUVEC and HVF cells. In particular, PLGA membranes performed much better adhesion and enhanced cell proliferation compared to control in the absence of polymers. In addition, we demonstrate here that these biodegradable membranes present improved mechanical properties to construct potential tissue-engineered vascular graft.

Fabrication and Characterization of PCL/PLGA Coaxial and Bilayer Fibrous Scaffolds for Tissue Engineering.

Electrospinning is an innovative new fibre technology that aims to design and fabricate membranes suitable for a wide range of tissue engineering (TE) applications including vascular grafts, which is the main objective of this research work. This study dealt with fabricating and characterising bilayer structures comprised of an electrospun sheet made of polycaprolactone (PCL, inner layer) and an outer layer made of poly lactic-co-glycolic acid (PLGA) and a coaxial porous scaffold with a micrometre fibre structure was successfully produced. The membranes' propriety for intended biomedical applications was assessed by evaluating their morphological structure/physical properties and structural integrity when they underwent the degradation process. A scanning electron microscope (SEM) was used to assess changes in the electrospun scaffolds' structural morphology such as in their fibre diameter, pore size (µm) and the porosity of the scaffold surface which was measured with Image J software. During the 12-week degradation process at room temperature, most of the scaffolds showed a similar trend in their degradation rate except the 60 min scaffolds. The coaxial scaffold had significantly less mass loss than the bilayer PCL/PLGA scaffold with 1.348% and 18.3%, respectively. The mechanical properties of the fibrous membranes were measured and the coaxial scaffolds showed greater tensile strength and elongation at break (%) compared to the bilayer scaffolds. According to the results obtained in this study, it can be concluded that a scaffold made with a coaxial needle is more suitable for tissue engineering applications due to the improved quality and functionality of the resulting polymeric membrane compared to the basic electrospinning process. However, whilst fabricating a vascular graft is the main aim of this research work, the biological data will not present in this paper.

Degradation and Characterisation of Electrospun Polycaprolactone (PCL) and Poly(lactic-co-glycolic acid) (PLGA) Scaffolds for Vascular Tissue Engineering.

The current study aimed to evaluate the characteristics and the effects of degradation on the structural properties of Poly(lactic-co-glycolic acid) (PLGA)- and polycaprolactone (PCL)-based nanofibrous scaffolds. Six scaffolds were prepared by electrospinning, three with PCL 15% (w/v) and three with PLGA 10% (w/v), with electrospinning processing times of 30, 60 and 90 min. Both types of scaffolds displayed more robust mechanical properties with increased spinning times. The tensile strength of both scaffolds with 90-min electrospun membranes did not show a significant difference in their strengths, as the PCL and PLGA scaffolds measured at 1.492 MPa ± 0.378 SD and 1.764 MPa ± 0.7982 SD, respectively. All membranes were shown to be hydrophobic under a wettability test. A degradation behaviour study was performed by immersing all scaffolds in phosphate-buffered saline (PBS) solution at room temperature for 12 weeks and for 4 weeks at 37 °C. The effects of degradation were monitored by taking each sample out of the PBS solution every week, and the structural changes were investigated under a scanning electron microscope (SEM). The PCL and PLGA scaffolds showed excellent fibre structure with adequate degradation, and the fibre diameter, measured over time, showed slight increase in size. Therefore, as an example of fibre water intake and progressive degradation, the scaffold's percentage weight loss increased each week, further supporting the porous membrane's degradability. The pore size and the porosity percentage of all scaffolds decreased substantially over the degradation period. The conclusion drawn from this experiment is that PCL and PLGA hold great promise for tissue engineering and regenerative medicine applications.

Comparison of biophysical properties characterized for microtissues cultured using microencapsulation and liquid crystal based 3D cell culture techniques.

Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 µm and 141 ± 70 µm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications.

In Vitro Growth of Human Keratinocytes and Oral Cancer Cells into Microtissues: An Aerosol-Based Microencapsulation Technique.

Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line (ORL-48) based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 µL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 µm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM), fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies.

Effect of transforming growth factor-ß3 on mono and multilayer chondrocytes.

Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-ß), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-ßs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-ß3 in biological regulation of primary chondrocyte was investigated in this work. TGF-ß3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-ß3 were larger than the cells without TGF-ß3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-ß3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-ß3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.

Transforming growth factor beta (TGF-ß) isomers influence cell detachment of MG-63 bone cells.

Bone repair and wound healing are modulated by different stimuli. There is evidence that Transforming Growth Factor-beta (TGF-ß) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules influence cell behaviour. In this study cell surface attachment was examined via a trypsinization assay using various TGF-ß isomers in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. Three TGF-ß isomers (TGF-ß1, 2 and 3), four combined forms (TGF-ß(1+2), TGF-ß(1+3), TGF-ß(2+3) and TGF-ß(1+2+3)) along with four different controls (BSA, HCl, BSA/HCl and negative control) were investigated in this study. The results indicated that treatment with TGF-ß1, 2, 3 and HCl decreased cell attachment, however, this effect was significantly greater in the case of TGF-ß3 (p<0.001) indicating perhaps that TGF-ß3 does not act alone in cell detachment, but instead functions synergistically with signalling pathways that are dependent on the availability of hydrogen ions. Widefield Surface Plasmon Resonance (WSPR) microscope was also used to investigate cell surface interactions.

Tracking traction force changes of single cells on the liquid crystal surface.

Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.

Effects of different transforming growth factor beta (TGF-ß) isomers on wound closure of bone cell monolayers.

This study aimed at determining the role of the transforming growth factor-beta (TGF-ß) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-ß1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-ßs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-ßs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-ßs and their solvent/carrier was investigated in 300 µm ± 10-30 µm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-ß isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-ßs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-ß3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-ß1 (p < 0.001), TGF-ß2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-ß1 and TGF-ß2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-ß combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-ß combinations would be greater than those healing rates following treatments with TGF-ß isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-ß(2 + 3) and TGF-ß(1 + 3) (p < 0.001). Any cell treated with TGF-ß1, TGF-ß(1 + 2) and TGF-ß(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-ß isomers. In terms of proliferation rate, TGF-ß3 and TGF-ß(2 + 3) increased cell numbers more than TGF-ß1, TGF-ß2 and other combinations. TGF-ß1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-ß3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and ß1. The WSPR experiments also indicated that TGF-ßs influenced the distribution of focal contacts. In conclusion, combining TGF-ß3 with any other TGF-ß isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-ß1 in any TGF-ß combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-ß1 has an inhibitory effect on bone wound healing while TGF-ß3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment.

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals.

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and ß1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Interfacial study of cell adhesion to liquid crystals using widefield surface plasmon resonance microscopy.

Widefield surface plasmon resonance (WSPR) microscopy provides high resolution imaging of interfacial interactions. We report the application of the WSPR imaging system in the study of the interaction between keratinocytes and liquid crystals (LC). Imaging of fixed keratinocytes cultured on gold coated surface plasmon substrates functionalized with a thin film of liquid crystals was performed in air using a 1.45NA objective based system. Focal adhesion of the cells adhered to glass and LC were further studied using immunofluorescence staining of the vinculin. The imaging system was also simulated with 2×2 scattering matrix to investigate the optical reflection of the resonant plasmonic wave via the glass/gold/cell and glass/gold/LC/cell layers. WSPR imaging indicated that keratinocytes are less spread and formed distinct topography of cell-liquid crystal couplings when cultured on liquid crystal coated substrates. The simulation indicates that glass/LC shifted the surface plasmon excitation angle to 75.39° as compared to glass/air interface at 44°. The WSPR microcopy reveals that the cells remodelled their topography of adhesion at different interfaces.