Injectable nano-reinforced shape-memory hydrogel system for

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Biological and Medical Applications of Materials and Interfaces

Injectable nano-reinforced shape-memory hydrogel system for regenerating spinal cord tissue from traumatic injury Chong Wang, Haibing Yue, Qian Feng, Bingzhe Xu, Liming Bian, and Peng Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08929 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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ACS Applied Materials & Interfaces

Injectable nano-reinforced shape-memory hydrogel system for regenerating spinal cord tissue from traumatic injury Chong Wang1,2,†, HaibingYue1,†, Qian Feng2, Bingzhe Xu1, Liming Bian2, Peng Shi1,4,* 1

Department of Biomedical Engineering City University of Hong Kong Kowloon, Hong Kong SAR, China

2

College of Mechanical Engineering Dongguan University of Technology Dongguan, Guangdong, China

3

Department of Biomedical Engineering Chinese University of Hong Kong Shatin, NT, Hong Kong SAR, China

4

Shenzhen Research Institute City University of Hong Kong Shenzhen, China

Keywords: Injectable hydrogel; electrospinning; tissue engineering; stem cell; spinal cord injury; composite materials

___________________________________________________________________________ *Corresponding author, Prof. Shi Peng, Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Ave, Kowloon, Hong Kong SAR, China, Email: [email protected] †These authors contributed equally.

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ABSTRACT Traumatic injury in the central nervous system can lead to loss of functional neurons. Transplantation of neural progenitors is a promising therapeutic strategy. However, infusion of dissociated cells often suffers from low viability, uneven cell distribution, and poor in vivo engraftment that could be reinforced by a better cell delivery system. Here, we develop an injectable composite-hydrogel-system for use as a minimally invasive treatment of spinal cord injury (SCI) using motor neurons derived from embryonic stem cells (ESCs). The composite-hydrogel is based on a modified gelatin matrix integrated with shape-memory polymer fibers. The gelatin-matrix creates a local microenvironment for cell assembly and also acts as a lubricant during injection through a fine catheter. Notably, shape-memory fiber scaffolds are able to recover to maintain the microstructures even after dramatic deformation from injection operation, providing the necessary support and guidance for motor neuron differentiation. We find that the composite-hydrogel with an aligned fiber scaffold greatly improves the viability of ESCs and their differentiation towards motor neurons both in vitro and in vivo. When transplanted to SCI animals by injection, the ESC loaded composite hydrogels are identified to significantly enhance tissue regeneration and motor function recovery in mice. With this proof-of-concept study, we believe that the injectable composite-hydrogel system provides a promising solution for in vivo cell delivery with minimum invasiveness and can be readily extended to other stem-cell-based regenerative treatments.


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INTRODUCTION Traumatic injuries in spinal cords often cause neuronal death and axonal degeneration, leading to sensory and motor malfunction

1-3

. Stem cell therapy can provide new neurons to

promote the regeneration of injured spinal cord tissues 4-7. Specifically, motor neurons (MNs) can be derived from neural stem cells (NSCs) 8, induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs)

11

9, 10

. However, transplantation of relevant cells to treat a

spinal cord injury (SCI) still suffers from low cell viability, uneven distribution and poor in vivo engraftment, which can potentially be improved by advanced cellular delivery systems 3. Tissue engineering has been increasingly used to package functional components, including cells, bio-signals, and three-dimensional (3D) scaffolds, for regenerative treatment of spinal cord injuries 12. Particularly for cellular scaffolding, hydrogels are commonly used because of their compositional similarities to extracellular matrix (ECM) and tunable structures for cellular survival and proliferation 13. Electrospun meshes have also been widely used as nanofibrous scaffolds; these are advantageous for controlling the cellular response and biomolecule release because of their interconnected structure, high porosity and ultrahigh surface-area-to-volume ratio 14, 15. The alignment of electrospun fibers can also modulate cell behavior, such as the cytoskeleton orientation, cell differentiation, and so on

16

. For easy in

vivo delivery, increasing efforts have recently been made to develop tissue engineering products that can be implanted with non-/minimum-invasive methods, thus reducing the surgical burden in therapeutic practices. Therefore, injectability is highly preferred and is not permissive for many existing hydrogel or fibrous scaffold systems 17, 18. Here, we developed an injectable cell delivery system with shape recovery capability after dramatic deformation. The system was based on a composite design that combines an injectable hydrogel with electrospun shape-memory meshes. In the composite product, a



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gelatin-matrix was used to create a local environment for cell assembly and was also used as a lubricant during injection via a regular syringe; aligned shape-memory fiber meshes were incorporated to provide structural support and guidance for cell growth and differentiation. We utilized a composite-hydrogel to treat SCI in mice by injecting an ESC-laden composite-hydrogel that was formed by packaging progenitor cells in the composite ex vivo. We found that the composite-hydrogel with an aligned fiber mesh greatly enhanced MN differentiation and neurite extension both in vitro and in vivo and also significantly improved the regeneration of spinal cord tissue and the recovery of motor function after a traumatic injury.

RESULTS Composite system design As shown in Fig. 1, the injectable composite hydrogel was fabricated by embedding an electrospun

nanomesh

in

gelatin-acrylated

β-cyclodextrin

(β-CD)

polyethylene

glycol-hydrogel (GCP-hydrogel) that was formed by a photo-crosslinking process 19. Because of the host-guest complexation between β-CDs and the aromatic groups on gelatin, the hydrogel had excellent stretchability, allowing it to survive extreme deformation that resulted from regular injection. The embedded electrospun nanomesh with a tuned alignment was designed to provide topological cues for ESC maturation and differentiation. To render the final composite injectability, a shape memory material, poly(D,L-lactic acid-co-trimethyl carbonate (P(DLLA-co-TMC)), was used to fabricate the fiber mesh by electrospinning. Accordingly, the resulting fibrous nanomesh also had excellent deformability. The integration of the GCP-hydrogel and nanomesh provides a promising solution for minimally invasive cell delivery to living animals, such that ESCs can be packaged in the composite-hydrogel and


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transplanted to the target tissues (e.g., injury site in mouse spinal cord in this study) via injection. After injection, the shape-memory nanomesh was able to recover to maintain its microstructure in the composite-hydrogel and provide long-term guidance for cell maturation and differentiation in vivo. Characterization of the Injectable Composite Hydrogels To characterize their structural details, different components of the composite-hydrogels were examined by scanning electron microscopy (SEM, Fig. 2). Before embedding in the GCP-hydrogel, the electrospun nanomesh (aligned- or random-) had an average fiber diameter of 800 ± 200 nm (Fig. S1, mean ± s.d., n = 10, 100 fibers were quantified for each sample). After composition, the nanomesh maintained its fibrous structure and alignment within the composite-hydrogels (Fig. 2a, b). The weight to area ratio was dramatically increased when changing from dry to hydrated status (Table S1). We then performed a tensile test to evaluate the mechanical property of the composite-hydrogels at 37 oC, and found that the nanomesh slightly reinforced the GCP-hydrogel (Fig. 2d). But the composite-hydrogel was still relatively soft, showing a modulus around 1kPa (Table S2), which well matched with the stiffness of normal neural tissues of rodent animals

20

.

Immediately after injection, the composite-hydrogels were wrinkled and showed an irregular macroscopic appearance. Further incubating the wrinkled hydrogels at 37 °C allowed the composite-hydrogel to recover to the original morphology with an unaltered fiber alignment (Fig. 2a, b). The shape-memory property of the nanomesh was critical for rendering the GCP-hydrogel with sufficient stretchability to survive an injection procedure 21. Under 100% strain, both the aligned- and random-composite could recover to the original morphology by more than 95% within about 15 seconds, whereas not much shape memory property was observed in Gel-only samples (Table S3). Meanwhile, the GCP-hydrogel worked as a lubricant to facilitate injections because the mesh itself cannot be easily injected through a 5 ACS Paragon Plus Environment


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fine syringe needle (Fig. 2c). We next characterized the degradation of the composite hydrogels. After two week incubation in an environment mimicking the in vivo condition, all the hydrogel samples had some weight loss, possibly due to the degradation of the GCP-hydrogel. But the composite hydrogels showed a slower degrading dynamics, as a result of the scaffolding effects from the fibrous nanomesh. The aligned- and random nanomesh samples lost only 3.5-8% weight and maintained structural morphology and alignment over the same period (Fig. 2e, also see Fig. S2). In vitro MN Differentiation in a Composite Hydrogel The biocompatibility of the composite hydrogel was firstly assessed in vitro by culturing HB9::GFP mouse embryonic stem cells (mESCs). Both types of composite hydrogels (aligned- and random-) were found to be non-toxic to mESC culture, and to promote MN differentiation, which was achieved through a synergy between nanomesh scaffolding and GCP-hydrogel packaging. At the early differentiation stage (2 days after induction), we could observe over 40% HB9+ cells in the composite hydrogels (Fig. S3), which was represented by a significantly less population in cultures within the single-component systems, including nanomesh alone (aligned-composite, 35.0±4.0%; random-composite, 36.0±2.5%) or gel-alone (22.2±2.0%). At later stages of differentiation (6 days after induction), the expression of GFP was reliably observed in the cells, suggesting the successful assumption of the MN phenotype by expressing HB9 (Fig. 3a). Cells in GCP-hydrogel-only samples exhibited as large aggregates with limited neurite outgrowth. By contrast, cells in aligned- or random-composites appeared to be smaller aggregates with a more dispersed distribution and the neurite filaments were also much denser with extended outgrowth (Fig. 3a). Particularly for the aligned-composite, significantly longer neurite filaments were extended (p

Injectable nano-reinforced shape-memory hydrogel system for

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