3D bioprinting is a relatively new but a rapidly evolving biofabrication technology that has created a hope in the tissue engineering world for faster and precise development of 3D tissues and organs. These tissue/organs can be utlized in organ transplant, injury or animal testing models. dECM is a promising biomaterial that can efficiently mimic the complexity of natural ECM of a specific tissue. This property gives advantage in printing a 3D construct that can recreate the microenvironment typical to a living tissue.
The aim of this thesis was to develop a robust biocompatible dLM bioink suitable for bioprinting. In this study, for the first time native liver tissue was decellularized, using both physical and chemical decellularization processes, wherein the structural integrity of the native tissue was lost but, the ECM components like GAGs and collagen were retained and utilized for providing tissue specific microenvironment for cellular activity and viability. DNA quantification confirmed the absence of DNA, while DMMB assay and Hydroxyproline assay confirmed high percentage of GAGs and collagen respectively. These results are in line with the previous work conducted by Pati et al. (2014) with adipose, cartilage and heart tissue. The decellularized liver tissue was transformed into a soluble dLM by digesting the tissue in pepsin that cleaved the telopeptide parts of the collagen allowing its solublization in acetic acid (Lee et al., 2010). This digested dLM solution was transformed to dLM pre-gel by adjusting the pH to 7, which behaved as temperature sensitive material forming a solid gel at a temperature around 37 ºC. The printability and flow characteristics of this ph adjusted dLM was improved by crosslinking using SVA-PEG-SVA with addition of gelatin to form dLM bioink. The rheology characteristics and degree of crosslinking of the dLM bioink was compared and analysed with dLM-gelatin, dLM-PEG and ph adjused dLM. The results illustrated bioink as most suitable for printing with suitable viscosity (flow characteristics), good crosslinking (soft gel) and highest storage modulus than loss modulus among all the gels, which was central to bioprinting. dLM-PEG formed hard gel with low storage modulus, whereas dLM-gelatin formed semi-soft gel with low degree of crosslinking. However, all the gels exhibited shear thinning behaviour.
The developed dLM bioink was cytocompatible and supported cell viability for a period of 7 days. The number of live cells and formazan formation increased from day 1 till day 7 indicating increase in the number of cells. Further, the dLM bioink was used for printing through 3DDiscovery, an extrusion based bioprinter in a 2 layer grid structure.
The dLM bioink was able to retain the 3D structure without any flow characteristic post printing.
Throughout the experiments, repeatability of the dLM bioink concentration was the main concern. This is a common problem when working with natural materials, but it can be fixed by highly optimising the whole process of development of dLM bioink. In addition to this, cell visibility in one plane in the fluorescence microscope was difficult due to the uneven scaffold layer. In the future work, confocal microscopy will be utilized to observe the cells in the 3D environment. In spite of many problems, these results provided a good insight for biofabrication of a 3D construct using dLM bioink based on gelatin and SVA- PEG-SVA.
Previous studies related to dECM of different types of tissues and bioprinting have been succesfully conducted. Whole liver decellularization has been succesfully conducted elsewhere (Mazza, G. et al, 2015), wherein the human liver was perfused with decellularizing agents through the hepatic veins and inferior vena cava. This resulted in preservation of liver structure and vasculature, which can be recellularized with multiple liver specific cell types, like hepatocytes, which will be used in organ transplantaion in future. In contrast to this work, the liver tissue loses its structural characteristics in this thesis work. This results in preservation of the ECM components which can be further designed and bioprinted providing several applications, like organ transplantaion, in-vivo studies or toxicological studies. This thesis work is similar to the work performed previously by Falguni et al. (2014), wherein adipose, heart and cartilage tissues are decellularized and bioprinted on polycaprolactone as secondary scaffold. The bioink in their study was not robust enough to maintain the 3D structure by itself, hence, a secondary support was required. However, in our study crosslinking of the dECM of liver tissue provided enough mechanical strength and robustness that the need for a secondary support was eliminated.
In future, this bioprinting technique of depositing dLM bioink can satisfy several important needs in the field of animal testing models and drug toxicology testing. Further work can be done to develop a hepatic lobule that can mimic the biochemical gradient of the native tissue, since the dLM bioink is robust with high biocompatibility.
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Appendix 1: Calibration curves of DNA quantification, DMMB
assay and Hydroxyproline assay
For the calculation of DNA, GAGs and collagen content, a calibration curve was plotted using Sigma Aldrich DNA fluorescence kit, between the total concentration (ng/ml) of DNA, collagen and GAGs and relative fluorescence units, as seen in graph. The linear square regression equation of the line generated by samples (n=3) was also plotted in form of y=mx+b, where,
y= Emission as relative fluorescence units m= the slope
x= DNA concentration b= intercept
R= Regression value
The regression value R2 demonstrates a fraction between 0.0 and 1.0. If the value is 0, then there is no relation between X and Y axis, but, if the value is near to 1.0, all points or values lie in one lie with minimum or no scatter. Thus, the regression value for the three curves as shown in grapH is 0,9887, 09199 and 0,954, which satisfies the results obtained during the dLM analysis.
DNA concentration with the relative fluorescence units
y = 0,1205x + 225,54 R² = 0,9684 0 200 400 600 800 1000 1200 1400 1600 0 2000 4000 6000 8000 10000 12000 R el ative fl uor es ce nc e unit s DNA concentration (ng/ml) DNA Quantification
Varying GAGs concentration with absorbance at 492 nm
Varying GAGs concentration with absorbance at 492 nm
From the equations obtained from the graph (a), the concentration of DNA in native tissue and dLM was calculated resulting in 1.57 % of DNA present in the dLM relative to native tissue.
On the other hand, the GAGs and collagen content in dLM relative to the native tissue was 26.26% and 91% respectively.
y = 0,0005x + 0,0976 R² = 0,9199 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0 10 20 30 40 50 60 70 A bs or ba nc e (492 n m)
Sulphated GAGs concentration (mg/ml) GAGs quantification y = 0,0343x + 0,0703 R² = 0,9936 0 0,2 0,4 0,6 0,8 1 1,2 0 5 10 15 20 25 30 35 A bs orba nce ( 56 5 nm ) Hydroxyproline (µg/ml) Hydroxyproline assay