Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs

Abstract

Bioprinting has exciting prospects for printing three-dimensional (3-D) tissue constructs by delivering living cells with appropriate matrix materials. However, progress in this field is currently extremely slow due to limited choices of bioink for cell encapsulation and cytocompatible gelation mechanisms. Here we report the development of clinically relevant sized tissue analogs by 3-D bioprinting, delivering human nasal inferior turbinate tissue-derived mesenchymal progenitor cells encapsulated in silk fibroin–gelatin (SF–G) bioink. Gelation in this bioink was induced via in situ cytocompatible gelation mechanisms, namely enzymatic crosslinking by mushroom tyrosinase and physical crosslinking via sonication. Mechanistically, tyrosinases oxidize the accessible tyrosine residues of silk and/or gelatin into reactive o-quinone moieties that can either condense with each other or undergo nonenzymatic reactions with available amines of both silk and gelatin. Sonication alters the hydrophobic interaction and accelerates self-assembly of silk fibroin macromolecules to form β-sheet crystals, which physically crosslink the hydrogel. However, sonication has no effect on the conformation of gelatin. The effect of optimized rheology, secondary conformations of silk–gelatin bioink, temporally controllable gelation strategies and printing parameters were assessed to achieve maximum cell viability and multilineage differentiation of the encapsulated human nasal inferior turbinate tissue-derived mesenchymal progenitor cells. This strategy offers a unique path forward in the direction of direct printing of spatially customized anatomical architecture in a patient-specific manner.

Introduction

Despite fascinating advances in the field of tissue engineering, the fabrication of anatomically relevant three-dimensional (3-D) engineered tissues still presents a major hurdle, due to our inability to replicate complex tissue architecture, composition and biomechanical functionality by using pre-formed polymeric scaffolds. Bioprinting has exciting prospects for fabricating complex 3-D multicellular tissue analog architecture by delivering a precisely characterized progenitor cell population, along with an appropriate biomaterial in a defined and organized manner, at the targeted location, in adequate numbers and within the right environment, by dispensing cell suspension or cell-laden hydrogels in a layer-by-layer process [1], [2], [3], [4], [5], [6]. But after the initial enthusiasm, progress in effective bioprinting was severely slowed down due to limited choices of bioink for cell encapsulation and cytocompatible gelation mechanisms.

During extrusion-based bioprinting, sol-gel transition is induced for gelatin [7], gelatin/chitosan [8], [9], gelatin/alginate [9], gelatin/fibrinogen [10], [11], Lutrol F127/alginate [12] and alginate [13], [14] by either thermal processes or post-print crosslinking. However, there is tremendous scope for improvement in the design of bioink. Alginate-based bioink has major limitations, such as quick loss of mechanical properties during in vitro culture (∼40% within 9 days) [15], different responses of human and animal cells [14], lack of bioactive binding sites [13] and resistance to protein adsorption [14]. Collagen ink suffers from issues of qualitative batch-to-batch variations, loss of shape and consistency due to shrinkage and poor mechanical property [16]. Pluronic F-127 (Poloxamer 407) dissolves in culture medium, resulting in the collapse of the construct architecture. Furthermore, encapsulated human BMP-2 gene-transduced bone marrow stromal cells could not grow well within the pluronic gel in vitro and failed to induce bone formation in vivo [17]. Fibrin hydrogels possess poor mechanical property and undergo fast disintegration (and hence proteinase inhibitor is needed for stabilizing up to 4 weeks in vitro [10], [11]), and fail to induce bone formation in vivo [11]. Thus, there is an urgent need to develop a novel bioink for printing 3-D tissue analog with tailored mechanical properties, instantaneous cytocompatible gelation, tailorable degradation rate, tissue specificity and adaptability to clinical set-up.

In our previous study, an optimized blend ratio of silk fibroin and gelatin was used for the fabrication of microperiodic scaffolds via the 3-D printing (direct-write) technique, to induce redifferentiation of expanded chondrocytes [18]. The primary structure of Bombyx mori silk fibroin predominantly consists of [GAGAGS]_n (where G is glycine, A is alanine and S is serine) repeat sequences, which form physical crosslinks though β-sheet crystallization. The cell adhesion mechanism on B. mori silk fibroin is still not properly understood, as it is devoid of known cell adhesion peptides, but it offers robust mechanical properties and tailorable degradability. On the other hand, the primary sequence of gelatin commonly contains GXY (where G is glycine, and X and Y are usually proline and hydroxyproline) and RGD (arginine–glycine–aspertate) amino acid sequences, but it suffers from a faster degradation rate. Though their blend overcomes the limitation of the individual material, certain points need to be taken into account to develop silk–gelatin bioink. During bioprinting, the bioink should come out of the micronozzle smoothly with minimal pressure. After extrusion, bioink should undergo rapid gelation and minimum deformation with deliverance of excellent cell viability. The gelation process of the hydrogel used as bioink should be mild and cell friendly. The composition and rheological features of the hydrogel bioink must support cell survival and targeted differentiation [19]. Hence, by considering these criteria, we have developed a directly printable bioink of silk fibroin–gelatin (SF–G) blend, which can be crosslinked in situ without any cytotoxic effect. The two different types of in situ crosslinking strategies evaluated in this study were physical crosslinking using probe-based sonication and enzymatic crosslinking using mushroom tyrosinase.

Tyrosinases belong to the group of oxidative enzymes and oxidize the accessible tyrosine residues of proteins into reactive o-quinone moieties without breaking the peptide bond. It was reported that tyrosinase can oxidize ∼10–11% and 20% of the tyrosine residues of silk and gelatin, respectively [20], [21]. Oxidized tyrosyl residues (i.e., quinone residues) can either condense with each other or undergo nonenzymatic reactions with available nucleophiles, such as amines of both gelatin and silk [20], [21]. Thus, tyrosinase can be applied for in situ crosslinking of cell encapsulated silk–gelatin hydrogel by sol-to-gel conversion of both silk and gelatin. On the other hand, sonication is a mechanical vibration that increases localized dynamics of polymer chains, causing a localized heating effect, the formation and collapse of bubbles, modulation in pressure and strain rates. Further, sonication induces crystalline β-sheets by the alteration of hydrophobic hydration and self-assembly of silk macromolecules [22].

To evaluate the cellular responses, we used human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs), a promising cell source due to their intrinsic capacity to proliferate, thereby providing large cell numbers, and high multilineage differentiation potential [23], [24]. These cells can be isolated from the inferior turbinate tissue that was discarded after a frequently performed surgery to relieve nasal obstruction resulting from turbinate hypertrophy [24]. Most interestingly, passage numbers and donor age could not affect differentiation characteristics of hTMSCs significantly, unlike bone-marrow-derived or adipose-derived mesenchymal stromal cells, [25]. The hTMSCs exhibited approximately five times higher proliferation compared to that of bone-marrow-derived MSCs [26]. Although adipose-derived mesenchymal stromal cells (ASCs) could easily be isolated by a minimally invasive procedure from aspirated fat tissue, hTMSCs displayed ∼30 times higher yield than adipose-derived MSCs at early passage [27].

In the present study, we explored the strategy of 3-D printing hTMSCs encapsulated within optimized SF–G solution. Furthermore, we focused on the effect of secondary conformations and supramolecular structures developed due to either tyrosinase- or sonication-induced gelation on the printing process and cell survival within the cell-laden 3-D constructs. Targeted multi-lineage differentiation of the encapsulated hTMSCs within silk–gelatin constructs was evaluated by gene expression, immunofluorescence and histological studies. To evaluate the potential of silk–gelatin bioink compared to the present standard procedure, we made a comparison with alginate because it is a widely used bioink for cell encapsulation and printing due to its easy processability [4], [28], [29], [30]. The outcomes provided a valuable insight into bioprinting of hTMSCs to engineer custom-made 3-D tissue constructs.