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The process of electro-spinning was used to obtain nanometer diameter fibres of 12.5% Poly-ï¥-caprolactone (PCL) polymer solution and two types of scaffold were fabricated namely aligned fibres & random fibres. The random fibres were collected directly at the ground collector, whereas a rotating mandrill was used to get the aligned fibres. A total of 6 scaffolds was fabricated (3 of each type) and sterilized using 70% IMS. For this study, MG-63 osteoblast like cells, derived from the human osteosarcoma has been used. The 100% confluent cells were trypsinised and centrifugated to separate it from the medium. Cell count was done in a hemocytometer and the number of cells required were extracted, and resuspended in DMEM - HG medium. 100,000 cells were seeded onto each scaffold and left to adhere in the incubator for 1.5 hours. Images under optical microscope were taken for assessing the change in cell morphology due to fibre orientation. TE buffer with 0.1% Triton X was added, in order to rupture the cell membrane and release DNA. Quantitative analysis to investigate the adhesion activity of MG-63 cells was done using Picogreen assay. The concentration of DNA in each sample type was calculated and the amount of DNA between fibre orientations was compared using statistyical analysis. The results showed a change in cell morphology due to the fibre orientation, but the difference in the number of cells that adhered to the scaffolds was not found to be statistically significant.
Tissue engineering for reconstructive surgery requires a proper cell source, adequate culture conditions and a biodegradable scaffold as the basic elements (H. Yoshimoto et. al, 2003). Recently, synthetic scaffolds, which are capable of replacing autologous and allogenic bone implants, have gathered the interest of researchers. Not only do we require a scaffold that can mimic the structure and function of bone extra cellular matrix (ECM) but, bone - implant interface is also an important issue in bone tissue engineering. (Porter et. al, 2004)
Electrospinning can be defined as a process of applying electric charge to generate forces strong enough to stretch and eject a polymer solution. This results in the drawing by a formed jet to produce nanofibres (Ramakrishna et. al, 2005).
"Electrospun fibres have been studied as a class of promising scaffold for tissue engineering because they can mimic the nanoscale features of the ECM" (Xiaoran li et. al, 2008). Electrospinning is capable of producing continuous fibres from submicron diameter to nanometer diameter. The formation of nano fibres by electrospinning is based on the principle of uniaxial stretching of a viscoelastic solution. It utilises electrostatic forces for stretching the solution as it solidifies. A simple electrospinning set up consists of a polymer reservoir, to which the solution is fed. High power voltage is applied to the solution and as the repulsive forces within the charged solution become greater than the surface tension, a jet erupts from the tip of a needle. This jet soon enters into a bending instability stage, wherein, the solution continues to stretch under the electrostatic forces as the solvent evaporates. A ground collector is used as the resultant target for the fibres and the fibres are deposited as a nonwoven mesh. (W.E. Teo & S. Ramakrishna, 2006)
There are a number of parameters which affect the fibre diameter. Few widely studied parameters are solution viscosity, concentration, applied voltage, working distance, and humidity (W.E. Teo & S. Ramakrishna, 2006). For example, if we reduce the polymer concentration, the resulting electrospun fibre diameter also reduces (Shenoy et al 2005). However, if we take the working distance into consideration, then, reducing the working distance can result in inter-connected fibres being collected at the target (Buchko et al 1999). It is also seen that the fibre diameter is affected by the applied voltage. At low voltages, the fibre diameter decreases as the voltage is increased. But, at high voltages, it is seen that as the voltage increases, the solution jet does not separate out and hence, results in an increase in fibre diameter (J.Y. Park et al, 2008). Increasing the applied voltage has also resulted in bead defects in the electrospun mats (J.M. Deitzel et al, 2001).
Nonwoven, fibrous mats electrospun from a variety of biodegradable polymers like, poly(lactic acid) (PLA),(Badami et. al, 2006) poly(lactic-co-glycolic acid) (PLGA),(Xin et. al, 2007) and poly(Îµ-caprolactone) (PCL), (Li et. al, 2005), have been intensively investigated for bone tissue engineering because of their good mechanical property and controllable degradation (Xiaoran li et. al, 2008). However, most of these polymers are unable to specifically interact with the cells due to their high hydrophobicity as compared to the ECM. Poly(Îµ-caprolactone), which is a regulatory approved biodegradable polymer, has been proven to be tissue compatible (Pitt C., 1990). Thus, it has been taken as the model polymer in our experiment because it is a resilient material and shows sufficient mechanical properties. It is also, less hydrophobic in nature and hence, mimics the ECM to a good extent. PCL has low toxicity, low cost and it degrades slowly in vivo. (Shea LD et. al, 2000)
For our experiment, we are using the MG-63 cell line. These cells are derived from the human Osteosarcoma. They are very resilient and strong cells, and divide rapidly. MG-63 cells are characterised as osteoblast like cells in morphology and they secrete the osteogenic ECM in culture media (Tomoyuki K. Et al, 2005).
Several studies have incorporated electrospinning for bone tissue engineering. For example, the study by Yoshimoto H. Et al in 2003 showed the potential of electrospun PCL nanofibres for bone tissue engineering using Mesenchymal stem cells (MSCs) derived from rat neonatal. Osteogenic supplements were added and after 4 weeks the results showed mineralization and presence of collagen type I (H. Yoshimoto et. al, 2003). Another study by McCullen S.D. et al in 2009, used electrospun composite scaffolds consisting of Î²-tricalcium phosphate (TCP) crystals and poly(L-lactic acid) (PLA), and assessed their ability to induce proliferation and osteogenic differentiation in human Adipose derived Stem cells (hASCs). Their results suggested an increase in the osteogenic differentiation of hASCs due to the biochemical nature of the scaffold (S.D. McCullen et al, 2009). Thus, many such studies have been done to assess the influence of electrospinning on bone tissue engineering and have shown better results with electrospun fibres.
The aim of our experiment is to investigate the cell morphology of MG-63 osteoblast like cells and to calculate the number of cells that adhere to random and aligned fibres. Electrospinning process has been used to fabricate the scaffolds to produce nanometer diameter fibres of 12.5% PCL. Picogreen assay has been used for quantitative analysis of the amount of DNA in each scaffold type and thus, investigating the adhesion activity of MG-63 osteoblast like cells.
MATERIALS AND METHODS
Fabrication of electro-spun fibre scaffolds
The electro-spinning set up was used to produce randomly oriented fibres and aligned fibres to form porous scaffolds. Poly-ï¥-caprolactone (PCL) polymer solution at a concentration of 12.5% was obtained by dissolving it in a solvent mixture of Chloroform and Dimethylformamide (DMF) with a volume ratio of 7:3. Dissolving in two mediums gives the polymer charge. The polymer solution was loaded into a syringe with a 22 gauge needle attached and connected to a high voltage power supply. The solution was delivered at a constant flow rate of 0.01 ml/min. The collector was plastic cover slips of size 24mmx24mm.
When voltage was applied (4KV for random and 4.5KV for aligned), a fluid jet was ejected from the syringe. As the jet travelled towards the ground collector, the solvent evaporated and a charged polymer fibre was deposited on the collector. For random fibres, the cover slips were directly stuck to the ground collector and the working distance was about 15cm. However, in order to get aligned fibres, the cover slips were mounted onto a rotating mandrill and the working distance was increased to 20cm. A volume of 0.006 ml was used for random fibre orientation, whereas, for aligned fibres, 0.016 ml fluid volume was utilised. Once the scaffolds were prepared, silicon rubber attachments were stuck on every corner of each cover slip in order to avoid detachment of fibre.
Scaffold sterilization and cell Trypsinization
For sterilization, scaffolds are placed in a 6 well plate and immersed in 70% Industrial Methylated Spirit (IMS) for about an hour. The forceps used for handling the scaffolds also need to be sterilized in 70% IMS before use. After sterilization, remove all the IMS and let the scaffolds dry completely, as even a single drop could kill the cells.
The MG-63 cells were cultured in Dulbeccco's Modified Eagles Medium - high glucose (DMEM-HG), supplemented with 10% Fetal Calf Serum (FCS), 1% L - Glutamine and 1% Antibiotics and Antimyotics. The medium was changed twice every week and the culture was incubated at 37oC in a humidified atmosphere containing 5% CO2. The MG-63 cells used in our study are in 96 to 97 passage and are 100% confluent. To lift the cells from the T-75 flask we need three things: PBS, Trypsin EDTA and DMEM - HG medium.
Firstly, aspirate the medium from the flask and wash the cells thoroughly with about 5ml of Phosphate Buffer Saline (PBS), in order to ensure that no medium still remains on the cells. Then the cells are trypsinized using 3 ml of Trypsin EDTA and incubated again for about 5 minutes. Deactivate trypsin by adding about 5ml of quenching media. Remove the cell suspension to a universal tube and centrifuge it, at about 1200 revolutions per minute (rpm) for 5 minutes. After centrifugation, the cells settle at the bottom of the tube and can be separated by removing the supernatant on top. The remaining cell pellet is resuspended in 5 ml of basic medium. Mix the solution homogeneously several times, such that all the cells mix properly with the medium. Pipette out about 10 ïl of this cell suspension into a haemocytometer for counting the number of cells.
We got too many cells in the suspension; this made the counting of cells difficult. Thus, in order to count the number of cells, the solution was further diluted with 5ml of quenching medium and the cells were counted from a haemocytometer.
Cells are counted from counting chamber known as haemocytometer under an optical microscope. The total numbers of cells in the flask are estimated by finding the average number of cells from five grids, multiplying it with the dilution factor and 10,000. Thus, from our experiment we got the average value as approximately 187 cells. Thus, the total numbers of cells in the flask were about:
187 x 10,000 x 10ml = 1.87 million cells
Number of samples: we have 3 random samples and 3 aligned samples. This gives us a total of 6 scaffolds (take 2 extra for error margin). Therefore, the total number of scaffolds is 8. We need to seed 100,000 cells per scaffold. Thus, the total number of cells needed is 800,000. For these many cells, the volume of cell suspension required is 0.428 ml (428 ïl). Therefore, about 450 ïl is taken from the cell suspension.
Cell seeding protocol
We have a total of 8 scaffolds, and for each scaffold 100,000 cells are required, which are to be suspended in 500 ïl of solution. Thus, the total cell seeding solution required is 4 ml. So, we re-suspend 450 ïl of cell suspension in 4 ml of media. Mix thoroughly, such that the cells are well suspended. Now, using a Gilson pipette, add 500 ïl of this cell solution onto each scaffold. Care must be taken to ensure that the solution does not spread out into the plate. After adding the cell solution to each scaffold, cover the plate and incubate at 37oC and 5% CO2 for about an hour and a half.
Analysing the number of adhered cells
After about 1.5 hours, remove plate from the incubator and picture it. This gives us images showing how well the cells have adhered to random and aligned fibres. These images also show the effect of fibre orientation on the morphology of the cells.
After imaging the scaffolds, remove the media and wash thoroughly with PBS. Then, prepare the Lysis buffer solution with 1:20 diluted TE buffer and 0.1% Triton X. Add 400 ïl of this lysis buffer solution to each scaffold. Leave for about 5 minutes for the cells to lyse. TE buffer helps to lift the cells up from the scaffold and also damages the cell membrane, thereby, causing the release of DNA. Fluorescence is initiated by Triton X solution. After about 5 minutes, using a cell scrapper, scrap off all the cells and fibre from the coverslip and transfer this solution to 1.5 ml, labelled ependorphs for freezing till stained. Vortex these tubes before freezing, this ruptures the cell membrane and cause more release of DNA. Freeze the tubes at -20oC for a few days.
The samples must be allowed to gradually thaw down before doing Picogreen assay. Prepare the 1x TE buffer and Picogreen working solution, needed for analysis. The working DNA solution of 2 ïg/ml is prepared by diluting stock DNA with 1x TE buffer. This working solution is diluted with 1x TE buffer to get the 8 different concentrations of DNA standards needed, as specified in Table 1.
Picogreen analysis is done in duplicates. Thus, we add 100 ïl of each standard to the wells of a 96 well plate (in duplicates). Use columns 11 and 12 of the plate for the DNA standards. Then, add 100 ïl of each sample (in duplicates) to the wells of the 96 well plate. Label the plate before in order to avoid confusion. Take blank controls, that is, 100 ïl of lysis buffer in row A (in duplicate). Put the aligned samples in row B and the random samples in row D. Then, add 100 ïl of the Picogreen working solution to every sample, blank and every standard. This should be done fast in order to prevent photo-bleaching of the picogreen reagent. When finished, wrap the plate in foil and read on plate reader at 485/435 nm (emission/excitation) wavelength.
MG-63 osteoblast like cells were seeded on random and aligned fibres in a 6 well plate for about 1.5 hours and then imaged using optical microscope. The microscopic images (Figure 1) of these scaffolds show that the cells adhered to aligned fibres faster than random fibres. Also, a change in cell morphology was seen in the cells that were seeded on aligned fibres. The cells appeared to be more aligned and showed elongation in the direction of the fibre in aligned scaffolds. Whereas, the cells seeded on random fibres did not show any change in cell morphology and appeared to be rounded.
The Picrogreen assay provides a quantitative analysis of the amount of DNA for each scaffold type. 100 ïl of picogreen was added to every sample and every standard, and the amount of DNA absorbed was read out using the BioTek Synergy 2 plate reader at 485/435 nm (emission/excitation) wavelength. Table 2 shows the DNA readings.
We are using commercial DNA at different concentrations as standard and quantifying the amount of DNA that has been absorbed. From the standard values (column 11 & 12), we plot a standard curve of concentration against absorbance. This curve gives us the equation of the best fit line and also the R squared value. (Figure 2)
It can be seen from the graph that the R2 value is around 0.996. We know that, the closer this value is to 1, more significant are the readings. Since, 0.996 is very close to 1, it shows that we have got a fairly linear relationship between the concentration and absorbance of DNA. From the equation of this line we can calculate the concentration of DNA for aligned and random samples.
Average values for each sample is calculated and blank absorbance is subtracted from all the values. Then, each value is multiplied by 4, this is because initially we had 400 ïl solution, but for the test only 100 ïl is taken from that solution. Therefore, it is 4 times diluted and thus, multiplying by 4 gives us the actual fluorescence. Then, the DNA concentration is calculated using the equation of the line from the standard curve. The above calculations are summarized in table 3.
Once we know the DNA concentration, we can statistically analyse the data by finding the average, the standard deviation and by performing t-test. From statistics point of view, we notice a higher DNA concentration in random fibres as compared to aligned fibres (Figure 3 and 4).
The standard deviation, statistically, shows us the variation of the data values from the average. We see a higher standard deviation for aligned fibres than for random fibres. For our data set, we perform a 2 tailed, paired t-test. The t-test value is 0.368906, which shows it is not statistically significant, that is, the amount of DNA (number of cells) for aligned and random fibres is almost the same. There is no significant difference between the two. Table 4 gives us the following values.
This study focuses on the use of electrospinning process to produce random and aligned nanofibre scaffolds of Poly-(ï¥-Caprolactone), (PCL) and investigate the adhesion activity of MG-63 osteoblast like cells on these scaffolds. Electrospinning process is driven by electrostatic forces and fibres of nanometer diameter range are deposited on the target due to the stretching and bending on the polymer solution jet, while the solvent gets evaporated. The fibre diameter attained on the target in the electrospinning process, depends on the type of polymer and the process condition (H. Yoshimoto et al, 2003). In our study, fibre diameter obtained was between 600 - 800 nm.
Aligned nanofibres were produced using a rotating mandrill, whereas, random fibres were obtained directly on the ground collector. MG-63 osteoblast cells, which are derived from human osteosarcoma, were seeded onto the scaffolds and left to attach for about 1.5 hours. Microscopic images were then taken to analyse the effect of fibre orientation on cell morphology. From the microscopic images (Figure 1), it can be seen that there is a change in cell morphology of the cells seeded on aligned fibres, but not much difference on random fibres. The cells appear to be elongated and tend to align themselves along the direction of the fibre in the aligned scaffolds, whereas, in random scaffolds, the cells appeared to be more spherical and rounded, and did not show any alignment. A significant number of cells adhered to both aligned and random scaffolds after just an hour and a half in DMEM - HG medium. However, the results showed that cells adhere faster to aligned fibres as compared to the random ones.
Picogreen assay was used for the quantitative analysis of cell adhesion on aligned and random nanofibres. Picogreen is a highly sensitive fluorescent nucleic acid stain and it selectively binds to the double stranded DNA. It has an emission maximum at 520nm wavelength and an excitation peak at 480nm wavelength (Susan J. Ahn et al, 1995). But, in our study, we are reading the data at 485/435 nm (emission/excitation). Very little background is produced when picogreen binds to the dsDNA, this is because the unbound dye has virtually no fluorescence. The picogreen fluorescence enhancement for dsDNA is exceptionally high (Susan J. Ahn et al, 1995). In our study, we have used picogreen assay for the quantitation of the amount of DNA in multiple samples and comparing DNA measurements between fibre orientations.
The results show that the average DNA concentration for random samples is 6.01726 whereas, for aligned samples it is 5.5097. This indicates more DNA concentration for random fibres and thus, shows more number of cells adhered to randomly oriented fibres as compared to aligned fibres. One reason for this could be the spreading of the medium while seeding the cells. Statistical analysis using 2-tailed and paired t-test was carried out to assess the significance of our result. The t-test value indicates that the result is not statistically significant, that is, there is not much difference in the adhesion activity of MG-63 cells between random and aligned fibres. This could be attributable to the time given for the cells to adhere. In our study, the cells were allowed to adhere for just 1.5 hours. Future studies could be carried out by increasing the time and investigating the change in cell adhesion.
Ploy - (ï¥-Caprolactone), (PCL) was chosen as the model polymer for our study because it is a biodegradable polymer (approved by the FDA), non toxic and degrades slowly in vivo (H. Yoshimoto et al, 2003). It is currently being considered as the candidate polymer for bone tissue engineering because it has sufficient mechanical properties to serve as a scaffold where more resilient material is required (G. Ciapetti et al, 2003). One issue with most of the polyesters is that they are unable to interact with cells due to their high hydrophobicity as compared to the natural ECM. PCL is less hydrophobic and hence, has an advantage over the other polymers (Xiaoran Li et al, 2008). Xiaoran et al. (Xiaoran Li et al, 2008) showed that coating the PCL scaffolds with gelatine and calcium phosphate makes them completely hydrophilic and their results showed better cell adhesion and higher proliferation rate. Ashammakhi et al (N. Ashammakhi et al, 2007) formed 3D scaffolds of PCL by a combination of nanofibres and microfibers, obtained by electrospinning and fibre bonding. They showed that these scaffolds had higher ability to enhance cell adhesion and organisation and also showed better AP activity.
Li et al. (Li et al, 2006) studied the multifunctional silk fibroin fibre-based scaffolds and combined it with either BMP-2 or nanoparticles of hydroxyapatite (nHAp). They used human bone marrow derived MSCs and seeded the cells for 31 days. Their results showed that scaffolds containing BMP-2 displayed higher calcium deposits and enhanced levels of bone-specific markers, whereas, the scaffolds containing nHAp were associated with improved bone formation. However, they concluded that Silk fibroin combined with both BMP-2 and nHAp were associated with highest calcium deposits and an upregulation of BMP-2, making them potential scaffolds for bone tissue engineering.
McCullen et al. (S.D. McCullen et al, 2009) investigated electrospun composite scaffolds of Î²-Tricalcium Phosphate (TCP) crystals and Ploy (L-Lactic acid) (PLA), at varying levels of TCP and assessed the ability of these scaffolds to induce proliferation and osteogenic differentiation in human Adipose Derived Stem cells (hASCs) in the presence of osteogenic media. Their findings showed that electrospun PLA w/10% TCP increased the proliferation of hASCs, whereas, electrospun PLA w/20% TCP accelerated osteogenic differentiation. Thus, their work suggested that heterogeneous scaffolds provide a superior biomaterial for electrospun bone-tissue enginnering scaffolds.
In our study, we just investigated the cell adhesion activity and the change in cell morphology on electrospun scaffolds. Future work can be done to investigate other osteogenic markers like calcium deposition, presence of collagen type I, alkaline phosphatase, to name a few. Also, the study can be further improved by investigating different cell types like primary cells or osteoblast cells, and comparing how does the proliferation, cell attachment and cell differentiation change.
In the present study, electro-spinning process was used to produce electrospun fibre mats of diameter 600-800nm of poly (ï¥-caprolactone), (PCL) from a solution of 12.5% w/v PCL with 7:3 ratio of chloroform and dimethylformamide. Random and aligned elctrospun fibre mats were obtained and were used as scaffolds to seed human osteosarcoma derived MG-63 osteoblast like cells. The study aimed at investigating the cell adhesion activity on these scaffolds and to assess the change in cell morphology. The cells appeared to be more elongated and showed alignment in the direction of the fibre in the aligned scaffolds, whereas, the cells appeared more rounded and spherical, and showed no alignment in the random scaffolds. Thus, fibre orientation showed to have an effect on the cell morphology. Picogreen assay was used for quantitative analysis of the data. Picogreen is a fluorescent nucleic acid dye, only specific to DNA double strands. The amount of DNA was measured for each scaffold type and compared using statistical analysis. The measure of DNA gives an estimate of the number of cells that adhered to the scaffold. The results were not statistically significant and the amount of cells that adhered was almost the same for both scaffold type. This could be due to the time given to the cells to adhere was not sufficient to get a significant difference. Thus, we can conclude that fibre orientation influences the cell morphology and also, nanometer diameter fibres enhance the cell attachment. But, for significant results, the cells should be allowed to adhere for a longer duration.