Abstract
4.3 Results and Discussion
4.3.3 Silk-based Nanocomposite Bioinks Exhibit Good Flow Behaviour with Faster Thixotropic Recovery and Demonstrates Decent Shape Fidelity for Bioprinting
[323]. Moreover, the permeability of the hydrogels was ascertained through uptake of DilC18
dye by encapsulated porcine endothelial cells within these hydrogels (Figure 4.2F i-iii) advocating for its good diffusion properties. Endothelial cells were used in the study because they are very susceptible to stress (nutritional diffusion constraints) and also angiogenesis is very much essential for regulating osteogenic cell fate. The bioink’s (reported here) hydrogel network provided optimal diffusion properties supporting the endothelial cell survival which is supported by the uptake of DilC18 dye by live endothelial cells within hydrogel (Figure 4.2F i-iii), seen in reference with endothelial cells grown in tissue culture plate (Figure 4.2F iv).
4.3.3 Silk-based Nanocomposite Bioinks Exhibit Good Flow Behaviour with Faster
SR1 bioinks possibly due to a crowding effect which may have retarded the enzyme sites inaccessible for crosslinking. Elastic behaviour of the developed gels was assessed by performing an amplitude sweep at constant angular frequency ω = 10 s-1 and increasing shear strain γ 0.01% to 1000%. At lower shear strains the elastic (G’ > G’’) nature was evident but at higher shear strains the elastic nature was lost and a viscous nature creeped in as G’’ > G’
assuring in the shear thinning effect associated with viscous behaviour of hydrogel ink (Figure 4.3B). Linear viscoelastic range (LVER) were thus calculated for the inks and found to be decent, lying in the range 20-100 %, the workable range before it totally deforms due to applied strain. The calculated rheological parameters of the inks are tabulated in Table A4.2 (Appendix). Similarly, frequency sweep was performed by keeping constant shear strain γ 2%
(set from LVER data) and increasing angular frequency ω = 1 rad.s-1 and to 1000 rad.s-1, where the developed inks resisted the angular distortion beyond 100 rad.s-1 (Figure 4.3 C) substantiating the robustness of the crosslinked hydrogel network. Furthermore, thixotropic nature of the inks was also evaluated (Figure 4.3 D) and the inks were found to regain their nature rapidly post deformation due to shear thinning effect in the LVER region of strain of 100 %, indicating post extrusion the inks would not tend to laterally diffuse.
Compositional features of crosslinked ink were confirmed by fourier-transform infrared (FTIR) spectroscopy. Characteristic peaks for the inks relating to their functional groups were observed at 1650 - 1600 cm-1 indicating presence of amide-I band denoting C=H stretching, 1550- 1500 cm-1 denoting C=N stretching of amide-II band and 1260 - 1210 cm-1 implying amide-III band corresponding to C-N stretching, along with characteristic hydroxyapatite peaks. Bands denoted by a, b, c and d indicated characteristic peaks of hydroxyapatite at 1032 cm-1, 962 cm-1, 574 cm-1 and 561 cm-1 respectively depicting asymmetric and symmetric stretching of P-O and bending of O-P-O as shown in Figure 4.3 E. Moreover, in the crosslinked silk inks a blue-shift in amide-I, amide-II and amide-III peaks was observed in comparison to silk solution, indicating that a β-sheet induction due to enzyme mediated crosslinking (Figure A4.2 (Appendix). We further deconvoluted the amide-I spectra and found that an increase in β- sheet content was evident due to the shift, possibly because of di-tyrosine bridges formed due to horse radish peroxidase (HRP) enzyme mediated crosslinking of tyrosine residues present in the silk fibroins, aided by hydrogen peroxide forming an oxyferryl centre at the HRP active site catalysing the covalent bond formation [312].
Figure 4.3. Characterization of developed nanocomposite silk based bioinks and its bioprinted constructs. Rheological studies on bioink; A) gelation time profile, B) amplitude sweep profile, C) frequency sweep profile and D) thixotropic measurement at alternating low and high oscillating shear and angular frequency for i) SF-PVP, ii) SF-PVP-SR0 and iii) SF- PVP-SR1 bioinks; E) fourier infrared spectrographs of synthesized unmodified (SR0), strontium doped hydroxyapatite (SR1) and different bioinks used in the study; F) Cell viability assessment using calcein-AM (live cell – green clusters) / ethidium homodimer (dead cell – red spots) staining post-printing using, i) SF-PVP, ii) SF-PVP-SR0, iii) SF-PVP-SR1 bioinks and iv) the cell viability percentage calculated using Image-J analysis.
Moreover, this also confirms that the bulking agent, PVP which improved the rheological behaviour of the ink did not hinder the crosslinking but rather formed
interpenetrating network with the crosslinked silk network. The presence of CH2 vibrations at 1460 cm-1, 1416 cm-1 and C-N vibrations at 1290 cm-1 noticed in the infrared spectra also corroborate the same. The cell viability post-printing and the shaped fidelity of the developed inks were tested. For this purpose, porcine adipose derived stem cells (pADSCs) either differentiating towards chondrogenic lineage (in SF-PVP ink) or osteogenic lineage (in SF- PVP-SR0 or SF-PVP-SR1 inks) were bioprinted in different infill patterns as shown in Figure A4.1, (Appendix) i) grid infill or ii) rectilinear infill for constructs of 10 mm x 10 mm x 5 mm in size. Cell densities as low as 6 million cells to 20 million cells per mL bioink were used for printing with viabilities maintained up to ~ 70 % post printing (Figure 4.3F) for the bioprinted constructs after 14 days as seen from the calcein-AM based live cell imaging. Morphological analysis of freeze-dried scaffolds also exhibited good interconnected porosity (Figure A4.3, Appendix) as visualized by electron microscopy. This may be attributed due to the good diffusion properties which was observed earlier, with a decent diffusion coefficient leading to non-Fickian transport. A good permeable hydrogel network is very much essential for nutrient transport, exchange of cytokines or growth factors from one end of the construct to other end facilitating cellular crosstalk and for extracellular matrix remodelling.
4.3.4 Bioprinting of Hierarchically Biomimetic Osteochondral Interfacial Constructs using