Thermoresponsive Alginate-Graft-pNIPAM/Methyl Cellulose 3D-Printed Scaffolds Promote Osteogenesis In Vitro.

In this work, a sodium alginate-based copolymer grafted by thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) chains was used as gelator (Alg-g-PNIPAM) in combination with methylcellulose (MC). It was found that the mechanical properties of the resulting gel could be enhanced by the addition of MC and calcium ions (Ca2+). The proposed network is formed via a dual crosslinking mechanism including ionic interactions among Ca2+ and carboxyl groups and secondary hydrophobic associations of PNIPAM chains. MC was found to further reinforce the dynamic moduli of the resulting gels (i.e., a storage modulus of ca. 1500 Pa at physiological body and post-printing temperature), rendering them suitable for 3D printing in biomedical applications. The polymer networks were stable and retained their printed fidelity with minimum erosion as low as 6% for up to seven days. Furthermore, adhered pre-osteoblastic cells on Alg-g-PNIPAM/MC printed scaffolds presented 80% viability compared to tissue culture polystyrene control, and more importantly, they promoted the osteogenic potential, as indicated by the increased alkaline phosphatase activity, calcium, and collagen production relative to the Alg-g-PNIPAM control scaffolds. Specifically, ALP activity and collagen secreted by cells were significantly enhanced in Alg-g-PNIPAM/MC scaffolds compared to the Alg-g-PNIPAM counterparts, demonstrating their potential in bone tissue engineering.

Harnessing the Interplay of Triple Cross-Linked Hydrogels toward Multiresponsive Alginate-Based Injectable Gels for 3D Printing Bioapplications.

We report on a single chain polymer gelator comprising an alginate backbone double grafted with thermoresponsive P(NIPAM86-co-NtBAM14)-NH2 polymer grafts and 3-aminophenylboronic acid moieties. The resulting polymer forms robust polymer networks resulting from three cooperative cross-linking mechanisms: (i) the hydrophobic association of the T-responsive polymer grafts above 24 °C, (ii) the formation of boronate esters between the boronic acid and the diols of the alginate backbone at physiological pH, and (iii) the ionic interactions of the residual carboxylate moieties with Ca2+ ions. The resulting material showed excellent tunability of the mechanical properties driven by stimuli combinations such as temperature, pH, or the addition of glucose as a network disruptor covering a storage modulus range from ∼260 Pa up to ∼1390 Pa by selective stimuli combinations. Also, the material was found to be nontoxic and could form arbitrary structures via 3D printing that can undergo multi-stimuli-responsive erosion profiles.

Dually crosslinked injectable alginate-based graft copolymer thermoresponsive hydrogels as 3D printing bioinks for cell spheroid growth and release.

In this work a dual crosslinked network based on sodium alginate graft copolymer, bearing poly(N-isopropylacrylamide-co-N-tert-butylacrylamide) P(NIPAM-co-NtBAM) side chains was developed and examined as a shear thinning soft gelating bioink. The copolymer was found to undergo a two-step gelation mechanism; in the first step a three-dimensional (3D) network is formed through ionic interactions between the negatively ionized carboxylic groups of the alginate backbone and the positive charges of Ca2+ divalent cations, according to the "egg-box" mechanism. The second gelation step occurs upon heating which triggers the hydrophobic association of the thermoresponsive P(NIPAM-co-NtBAM) side chains, increasing the network crosslinking density in a highly cooperative manner. Interestingly, the dual crosslinking mechanism resulted in a five-to-eight-fold improvement of the storage modulus implying reinforced hydrophobic crosslinking above the critical thermo-gelation temperature which is further boosted by the ionic crosslinking of the alginate backbone. The proposed bioink could form arbitrary geometries under mild 3D printing conditions. Last, it is demonstrated that the proposed developed bioink can be further utilized as bioprinting ink and showcased its ability to promote human periosteum derived cells (hPDCs) growth in 3D and their capacity to form 3D spheroids. In conclusion, the bioink, owing its ability to reverse thermally the crosslinking of its polymer network, can be further utilized for the facile recovery of the cell spheroids, implying its promising potential use as cell spheroid-forming template bionk for applications in 3D biofabrication.

Thermo-Responsive Injectable Hydrogels Formed by Self-Assembly of Alginate-Based Heterograft Copolymers.

Polysaccharide-based graft copolymers bearing thermo-responsive grafting chains, exhibiting LCST, have been designed to afford thermo-responsive injectable hydrogels. The good performance of the hydrogel requires control of the critical gelation temperature, Tgel. In the present article, we wish to show an alternative method to tune Tgel using an alginate-based thermo-responsive gelator bearing two kinds of grafting chains (heterograft copolymer topology) of P(NIPAM86-co-NtBAM14) random copolymers and pure PNIPAM, differing in their lower critical solution temperature (LCST) about 10 °C. Interestingly, the Tgel of the heterograft copolymer is controlled from the overall hydrophobic content, NtBAM, of both grafts, implying the formation of blended side chains in the crosslinked nanodomains of the formed network. Rheological investigation of the hydrogel showed excellent responsiveness to temperature and shear. Thus, a combination of shear-thinning and thermo-thickening effects provides the hydrogel with injectability and self-healing properties, making it a good candidate for biomedical applications.

Alginate-g-PNIPAM-Based Thermo/Shear-Responsive Injectable Hydrogels: Tailoring the Rheological Properties by Adjusting the LCST of the Grafting Chains.

Graft copolymers of alginate backbone and N-isopropylacrylamide/N-tert-butylacrylamide random copolymer, P(NIPAMx-co-NtBAMy), side chains (stickers) with various NtBAM content were designed and explored in aqueous media. Self-assembling thermoresponsive hydrogels are formed upon heating, in all cases, through the hydrophobic association of the P(NIPAMx-co-NtBAMy) sticky pendant chains. The rheological properties of the formulations depend remarkably on the NtBAM hydrophobic content, which regulates the lower critical solution temperature (LCST) and, in turn, the stickers' thermo-responsiveness. The gelation point, Tgel, was shifted to lower temperatures from 38 to 20 °C by enriching the PNIPAM chains with 20 mol % NtBAM, shifting accordingly to the gelation temperature window. The consequences of the Tgel shift to the hydrogels' rheological properties are significant at room and body temperature. For instance, at 37 °C, the storage modulus increases about two orders of magnitude and the terminal relaxation time increase about 10 orders of magnitude by enriching the stickers with 20 mol % hydrophobic moieties. Two main thermo-induced behaviors were revealed, characterized by a sol-gel and a weak gel-stiff gel transition for the copolymer with stickers of low (0.6 mol %) and high (14, 20 mol %) NtBAM content, respectively. The first type of hydrogels is easily injectable, while for the second one, the injectability is provided by shear-thinning effects. The influence of the type of media (phosphate buffer (PB), phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM)) on the hydrogel properties was also explored and discussed. The 4 wt % NaALG-g-P(NIPAM80-co-NtBAM20)/DMEM formulation showed excellent shear-induced injectability at room temperature and instantaneous thermo-induced gel stiffening at body temperature, rendering it a good candidate for cell transplantation potential applications.

pH/Thermo-Responsive Grafted Alginate-Based SiO2 Hybrid Nanocarrier/Hydrogel Drug Delivery Systems.

We report the preparation of mesoporous silica nanoparticles covered by layer by layer (LbL) oppositely charged weak polyelectrolytes, comprising poly(allylamine hydrochloride) (PAH) and a sodium alginate, highly grafted by N-isopropylacrylamide/N-tert-butylacrylamide random copolymers, NaALG-g-P(NIPAM90-co-NtBAM10) (NaALG-g). Thanks to the pH dependence of the degree of ionization of the polyelectrolytes and the LCST-type thermosensitivity of the grafting chains of the NaALG-g, the as-prepared hybrid nanoparticles (hNP) exhibit pH/thermo-responsive drug delivery capabilities. The release kinetics of rhodamine B (RB, model drug) can be controlled by the number of PAH/NaALG-g bilayers and more importantly by the environmental conditions, namely, pH and temperature. As observed, the increase of pH and/or temperature accelerates the RB release under sink conditions. The same NaALG-g was used as gelator to fabricate a hNP@NaALG-g hydrogel composite. This formulation forms a viscous solution at room temperature, and it is transformed to a self-assembling hydrogel (sol-gel transition) upon heating at physiological temperature provided that its Tgel was regulated at 30.7 °C, by the NtBAM hydrophobic monomer incorporation in the side chains. It exhibits excellent injectability thanks to its combined thermo- and shear-responsiveness. The hNP@NaALG-g hydrogel composite, encapsulating hNP covered with one bilayer, exhibited pH-responsive sustainable drug delivery. The presented highly tunable drug delivery system (DDS) (hNP and/or composite hydrogel) might be useful for biomedical potential applications.

Injectable self-assembling hydrogel from alginate grafted by P(N-isopropylacrylamide-co-N-tert-butylacrylamide) random copolymers.

Sodium alginate grafted by a thermo-responsive copolymer of N-isopropylacrylamide, enriched with the hydrophobic N-tert-butylacrylamide monomer, (P(NIPAM-co-NtBAM)-NH2) was synthesized and its thermo- and shear-induced responsive capabilities were studied through rheology. The graft copolymer formed a 3D network through thermo-induced hydrophobic association of the thermo-responsive P(NIPAM-co-NtBAM) side chains in water. By applying the frequency-temperature superposition principle, the terminal relaxation time, τ and the shear viscosity, as a function of temperature were evaluated. Both parameters increased exponentially upon heating orders of magnitude, 15 °C above the onset of gelation (35 °C). It is shown that the thermo-induced thickening effect was mainly due to the slowdown of the P(NIPAM90-co-NtBAM10) associative side chains exchange dynamics. Moreover, combination of shear- and thermo-responsiveness provided excellent hydrogel injectability with instantaneous gelation at physiological temperature. The better insight of the thermo-thickening mechanism through oscillatory rheology allows precise tuning of the carbohydrate-based hydrogel properties towards potential bioapplications.