Abstract
4.3 Results and Discussion
4.3.1 Hierarchically Biomimetic Nature of Synthesized Nano-apatites and Strontium Substituted Nano-apatites
Naturally in the bone the mineral homeostasis is maintained due to substitutions in its three sub lattices of apatites (M10(ZO4)6X2), i.e. (M = Ca, Sr, Pb, Na, K etc.; Z = P, CO3, V, As, Cr, B, etc.; and X = OH, CO3, Br, Cl, BO2), thus achieving the regulation for mineral homeostasis [319]. Utilising this notion we introduced Sr substitution in hydroxyapatite ((Sr0.5Ca0.5)5(PO4)3.OH) via wet chemical ammoniacal precipitation method, where Sr2+
underwent substitutions with Ca2+ cations in the apatite hexagonal crystal lattice structure. The synthesized nano-apatites Sr doped (SR1) and unmodified (SR0) along with control hydroxyapatite (HA) (Sigma-Aldrich, USA) were validated with wide-angle X-ray diffraction studies where characteristic peaks (2θ) corresponding to the signature miller indices were observed (Figure 4.1A). The peaks 2θ = 31.86°, 32.20° and 32.90° corresponding to miller indices 211, 112, 300 in SR1 was slightly broader in comparison to SR0 and HA indicating that SR1 was less crystalline. We further calculated the crystal parameters such as crystallinity (Xc), d spacing and crystallite size D (Table 4.3) and found that Sr substitution reduced the crystallinity and increased the d spacing along the 002 c-axis and 300 a-axis planes. Moreover, the crystallite size (D) was determined along ‘c’ and ‘a’ axis and observed to be around 25-50 nm which is the same range as noticed in the native bone’s apatite, thereby bearing greater
likelihood to it. Morphological analysis carried out using FETEM also corroborated this hierarchical biomimicry wherein rod-shaped crystals of individual crystallite size ~ 50 nm were noticed (Figure 4.1 Bi-ii, 1 Ci-ii).
Figure 4.1. Hierarchically biomimetic nature of synthesized nano-apatites – physicochemical characterizations. A) Wide angle X-ray diffraction analysis with corresponding 2θ (degrees) for hydroxyapatite (HA) peaks represented, followed with their miller indices in brackets: a – 25.90° (002), b – 31.86° (211), c – 32.20° (112), d – 32.90°
(300), e – 46.69° (222), f – 49.51° (213); Field emission transmission electron microscopy analysis of synthesized hydroxyapatites B) i-ii SR0 apatite, iii) Selected area energy diffraction (SAED) pattern, iv) elemental analysis to confirm stoichiometry of apatite; C) i-ii SR0 apatite, iii) SAED pattern, iv) elemental analysis to confirm stoichiometry of apatite.
Table 4.3. Crystal parameters of synthesized hydroxyapatites
Sample
Crystallinity Crystal Plane (002) Crystal Plane (300) (XC)
(%)
d Spacing
(nm)
Crystallite Size (D) Along c-axis
d Spacing
(nm)
Crystallite Size (D) Along a-axis
HA 65.79402 0.20369 46.75786 0.154343 30.60398
SR0 63.05164 0.224185 52.13054 0.154022 37.88596
SR1 42.23993 0.785786 25.42422 0.176133 24.93892
The selected area energy diffraction (SAED) patterns also confirmed the presence of 211, 102 and 002 planes of apatite crystal with 211 plane appearing slightly diffused in SR1 than SR0 (Figure 4.1 Biii, Ciii) confirming its relative amorphous nature. Elemental analysis also confirmed the molar stoichiometry Ca/P ratio of ~1.6 in SR0 and SR1 whereas the experimental Sr doping values also matched with the theoretical values Ca:Sr:P (0.3:0.3:0.4) in atomic weight ratios approximately (Figure 4.1 Biv, Civ). Furthermore, we looked into the dispersity and Zeta potential (ζ) of aqueous dispersions of apatites (Table A4.1, Appendix) by dynamic light scattering experiments. The hydrodynamic radius of nano-apatite agglomerates in dispersion were found to be ~800 nm, a range consistent enough to be printed and also appropriate for not invoking immune response. The incorporation of Sr however improved the polydispersity index (PDI), Zeta potential (ζ) and conductivity of the apatite whereby demonstrating that SR1 could form much stable suspensions ideal for printing applications, where sedimentation of additives always pose a great hindrance for bioinks. The synthesized apatites were hierarchically similar to the native bone’s apatite in size and morphology. The hydrodynamic radius (~800 nm) of the dispersion of the apatite agglomerates were found to be appropriate for bioprinting without harming the encapsulated cells. It has also been reported that particle size and shape of apatites are few of the important factors that influence inflammatory responses, where smaller particle < 500 nm enhanced the NLPR3 inflammasome while larger particle ~1 μm did not evoke any response [320]. The introduction of Sr also decreased the crystallinity of the apatites. Crystalline apatites have very poor dissolution rates (Kd) of the order 10-40 to 10-59 [31, 321] and addressing it becomes essential, our method here shows SR1 has crystallinity % ~ 40 which desirably would have better resorption rates. It was noticed the workable range of ceramic additives in the bone bioink was found to be in range 0.1 to 0.75 w%, anything above clogged the 21 G needle used in the study.
4.3.2 Silk-based Bioinks Exhibit Shear Thinning Behaviour and Non-Fickian Transport The bioink composition listed in Table 4.1 consists of 4 (w/v) % SF, 8 (w/v) % PVP, culminating in total mass percentage of 12 % (w/v) which was found to be working optimum for bioprinting applications reported here. A range of concentrations for SF-PVP inks (Figure 4.2A) was assessed for printability at print speeds 10 – 20 mm/s and at extrusion pressures of 13-16 psi using 21 G blunt ended stainless-steel needles to identify ink concentrations with suitable viscosities for printing. However, it was found the ink was printable only in the ranges of mass percentages of 8 – 12 % (w/v) exhibiting shear thinning nature (Figure 4.2B) which is ideal for any good bioink. Concentrations of inks, anything lower resulted in lateral dispersion of the ink owing to lack of adequate viscosity of ink or anything above resulted in rigid ink with poor flow properties. Workable range of ceramic additives was found to be in range 0.1 to 0.75 wt%, anything above clogged the 21 G needle used in the study.
Factors such as permeability and associated diffusion parameters are poorly understood in hydrogel-based microenvironments, especially in bioprinted constructs which mostly rely on printing cell encapsulated crosslinked hydrogels. Better survival and extracellular matrix (ECM) turnover in chondrocytes were facilitated in hydrogel matrices with better permeability [322]. The permeability of gels was analysed in ranges 6 – 12 % (wt/v), close to our printable bioink gel’s range to have an idea whether increase in concentration affected the permeability.
In order to evaluate the diffusion properties, three different molecular weight, fluorescein isothiocyanate conjugated (FITC) conjugated biomolecules: FITC-glutathione (FITC-GSH ~1 kDa, FITC-Inulin ~ 5 kDa, FITC-Dextran ~ 70 kDa) was encapsulated in different weight percentages (6 w %, 9 w % and 12 w%) of ink hydrogels. The release profiles (Figure 4.2 C,D,E i) were correlated with their diffusion mechanism by fitting the data in Korsmeyer- Peppas model (Mt/Mα = ktn), where Mt/Mα represents the cumulative fraction of biomolecule released at time “t”, with “k” denoting release rate constant and “n” denoting release exponent (Figure 4.2 C,D,E ii). All the gels exhibited a non-Fickian diffusion behaviour and the diffusion coefficient (De) was calculated by plotting the Mt/Mα against square of root of time
“t” and the resulting slope yielded the De [322]. Concentration dependent release of different weight biomolecules were observed, where 70 kDa FITC-dextran’s cumulative release was relatively retarded in comparison to ~1 kDa FITC-GSH (~90 % release vs. ~ 15 % release at 60 h, for 6% gel). Similarly, when comparing the low percentage gels (6%) had a better release kinetics in comparison to higher percentage gels (8 %, 12 %) owing to the micro-nano structures resulting in lesser pore sizes, albeit not significantly different between the gels.
Figure 4.2. Characterization of developed silk-based nanocomposite bioinks. A) Printabilility chart for the bioink; B) Shear thinning effect of the bioinks; complex viscosity vs. increasing angular frequency; Diffusional studies using FITC conjugated biomolecules, C) FITC- Glutathione (GSH) (~1kDa), D) FITC-Inulin (~5 kDa), E) FITC-Dextran (~70 kDa) i) cumulative percentage release and ii) respective Korsmeyer-Peppas model fitting; F) Dil C18
stain uptake by porcine endothelial cells to study diffusional properties on i) 6% ii) 9%, iii) 12% bioink and iv) on tissue culture plate.
The De of all the gels was found to be of the order ~3 to 5 x 10 -11 m2/s which was close to range of few reported hyaluronic acid based hydrogels where good viability of encapsulated chondrocytes was reported earlier [322]. Similar diffusion kinetics were also noticed in polyethylene glycol based hydrogels which reported better survival of encapsulated pancreatic islet cells and exchange of secreted of insulin into outer environment from the hydrogel matrix
[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