Integrated design and fabrication strategies for biomechanically and biologically functional PLA/β-TCP nanofiber reinforced GelMA scaffold for tissue engineering applications

Highlights

• Biomimetic scaffold with channel-like macroporous architecture is developed.

• First time bioactive fluffy type nanofibous mesh is integrated with GelMA hydrogel.

• Nanofibers were efficiently assembled an orientation of hydrogel network.

• Fibers do not interrupt the macroporous architecture of GelMA.

• The pore architecture, stiffness, modulus, and the bioactivity can tailored.

Abstract

We present an integrated design and fabrication strategy for the development of hierarchically structured biomechanically and biologically functional tissue scaffold. An integration of β-TCP incorporated fluffy type nanofibers and biodegradable interpenetrating gelatin-hydrogel networks (IGN) result in biomimetic tissue engineered constructs with fully tunable properties that can match specific tissue requirements. FESEM images showed that nanofibers were efficiently assembled into an orientation of IGN without disturbing its pore architecture. The pore architecture, compressive stiffness and modulus, swelling, and the biological properties of the composite constructs can be tailored by adjusting the composition of nanofiber content with respect to IGN. Experimental results of cell proliferation assay and confocal microscopy imaging showed that the as-fabricated composite constructs exhibit excellent ability for MC3T3-E1 cell proliferation, infiltration and growth. Furthermore, β-TCP incorporated functionalized nanofiber enhanced the biomimetic mineralization, cell infiltration and cell proliferation. Within two weeks of cell-seeding, the composite construct exhibited enhanced osteogenic performance (Runx2, osterix and ALP gene expression) compared to pristine IGN hydrogel scaffold. Our integrated design and fabrication approach enables the assembly of nanofiber within IGN architecture, laying the foundation for biomimetic scaffold.

Introduction

Development of the 3D vascularized tissues has enabled scientific and technological advances in tissue engineering, organ repair and drug screening [[1], [2], [3]]. Ideal scaffolds need the assembly of extracellular matrix (ECM) and vasculature in precise geometry, in which the former provides the structural support for cell proliferation and growth, while the latter is needed to transport the nutrients and waste product, to regulate the delivery factors, and to provide long range signaling route [4,5]. The stiffness of the scaffolds has been identified as a key modulator, especially in osteoblast behavior. Further, the scaffold should be strong enough for facile handling during surgery [6], and should have a comparable absorption rate to that of new tissue regeneration, which could take a few weeks to a few months. Recently, implantation of engineered 3D construct has attracted intense interest for skeletal tissue regeneration [7]. Bone is a highly organized tissue with good regenerative ability, but the extent of bone injury might prevent complete regeneration, especially in terms of functional recovery [8]. Therefore, a significant need still exists to produce clinically relevant biomimetic scaffolds for hard tissue regeneration, with scalable structural properties that can control cellular organization and alignment within a three-dimensional environment.

To date, hydrogels-based scaffold materials have been widely studied for tissue engineering, because of their excellent biological performance and similarities with ECM [9,10]. Among them, collagen-based hydrogels are considered attractive candidates for bone regeneration, due to their unique combination of biological and physical properties, including biocompatibility, and adjustable swelling and degradation properties [11]. Collagen and its denatured form gelatin have abundant cell binding sequences that support cellular activities, such as cell adhesion and migration [12]. Gelatin preserves the matrix degradation sites, and retains the natural RGD-motifs of collagen that arbitrate cell attachment and migration [13]. Additionally, gelatin can be modified with photocrosslinkable methacrylamide (MA) groups to form photopolymerizable gelatin methacrylate (GelMA) that retains the properties of gelatin [14]. Furthermore, when exposed to light irradiation, GelMA solution crosslinks (in the presence of proper photoinitiators, e.g., Irgacure 2959), and endows the material to be solidified from liquid to solid via chemical reaction of the methacrylamide groups [14]. The ideal hydrogel scaffolds should have mechanical properties that are comparable to the target tissues. Usually, those properties are controlled by varying a number of parameters, such as the density and chemistry of the crosslinks, and the concentration and molecular weight of the precursors [15,16]. However, while the stiffness of the 3D hydrogel increases with increasing crosslinking time, it generally compromises the biological performance of the scaffolds, such as limited cell proliferation, migration, and morphogenesis, or requires a long or costly period of pre-culture [17]. Therefore, a method that enhances the mechanical properties as well are bioactivity are of prime concern.

GelMA hydrogel has been reinforced with different materials, such as hydroxyapatite [18,19], carbon nanotubes [20,21], graphene oxide [22], nanofibers [[23], [24], [25]], microfibers [17] and wovens and nonwovens [26]. Considering the structure of native extracellular matrix (ECM), nanofiber reinforcement of a hydrogel is a more attractive approach. Nanofibers mimic collagen fibers that exist in the ECM of the native tissue, and can provide topographical and biochemical clues for shaping cell morphology, guiding cell migration and affecting cell differentiation, when compared to other types of scaffold [[27], [28], [29], [30]]. Owing to its versatility and flexibility, the electrospinning technique is widely adopted for fabricating nanofibers of various polymers with varying diameter in a continuous process, and at a long length scale [[31], [32], [33], [34]]. Integration of such electrospun nanofibers with hydrogel can impart synergistic properties to the scaffolds. Although a few studies have reported hydrogel coating on the electrospun membrane, and then layered to form a multilayered construct, such scaffolds suffer from a 2D distribution of compactly packed nanofibers in a layered manner that hinders cell infiltration. Recently, nanofibers were cut into the micron size and then incorporated into the hydrogel scaffolds where the nanofibers were randomly distributed throughout the scaffold thereby disturbing the macropurous architecture. More recently, macroporous scaffold with channel-like pores are engineered to guide cell migration and to control ECM patterning from porcine collagen [35]. Therefore, nanofiber reinforced hydrogel scaffolds that closely resembles with pore architecture of hydrogel scaffolds is still demanded.

Herein, we fabricated the fluffy-type β-TCP-incorporated PLA nanofibers via electrospinning and post processing. Taking advantage of the fluffy properties, nanofibers were dispersed in GelMA solution, and cross-linked to produce hierarchically structured nanofiber-assembled hydrogel scaffolds. We investigated the effects of the mass composition of the nanofiber component on the pore architecture, mechanical properties, and bioactivities of the scaffolds, using in vitro models to illustrate the potential for bone tissue engineering applications. We believe that this work will provide an integrated design platform to fabricate nanofiber assembled hierarchical nanostructured hydrogel scaffolds for a wide range of biomedical applications.