Black Phosphorus Hydrogel Scaffolds Enhance Bone Regeneration

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Biological and Medical Applications of Materials and Interfaces

Black Phosphorus Hydrogel Scaffolds Enhance Bone Regeneration via a Sustained Supply of Calcium-Free Phosphorus Keqing Huang, Jun Wu, and Zhipeng Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21179 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Applied Materials & Interfaces

Black Phosphorus Hydrogel Scaffolds Enhance Bone Regeneration via a Sustained Supply of Calcium-Free Phosphorus

Keqing Huang†, Jun Wu† *, Zhipeng Gu†*

†

Key Laboratory of Sensing Technology and Biomedical Instrument of Guangdong

Province, School of Biomedical Engineering; State Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou, 510006, PR. China

ABSTRACT Effective bone regeneration remains a challenge for bone tissue engineering. In this study, we propose a new strategy to accelerate bone regeneration via a sustained supply of phosphorus without providing foreign calcium. Herein, a black phosphorus nanosheet (BPN)-based hydrogel platform was developed, and the BPNs were used to stably and mildly provide phosphorus. The hydrogel was fabricated by photocrosslinking of gelatin methacrylamide (GelMA), BPNs, and cationic argininebased unsaturated poly(ester amide)s [U-Arg-PEAs]. This platform combines the following advantages: the hydrogel scaffold would keep BPNs inside, and the encapsulated BPNs can degrade into phosphorus ions and capture calcium ions to accelerate biomineralization in a bone defect. The introduction of BPNs helped to enhance the mechanical performance of hydrogels, photoresponsively release phosphate, and accelerate mineralization in vitro. Moreover, BPN-containing hydrogels improved osteogenic differentiation of human dental pulp stem cells (hDPSCs) via the BMP–RUNX2 pathway. In vivo results from a rabbit model of bone defects revealed that the BPNs helped to accelerate bone regeneration. All these results strongly suggest that the strategy of a sustained supply of calcium-free phosphorus and this BPNcontaining hydrogel platform hold promise for effective bone regeneration. KEYWORDS: black phosphorus hydrogel, bone regeneration, calcium-free phosphorus, cationic arginine-based unsaturated poly(ester amide)s, gelatin methacrylamide

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INTRODUCTION The effective bone regeneration in tissue engineering so far remains a challenge owing to the complexity of a bone microenvironment in vivo. In the past decades, with the aim to accelerate bone regeneration, many strategies have been proposed and investigated1. Among them, mimicking the composition and function of natural bone, inorganic– organic or organic-composite–based scaffolds have shown promising results and a good potential 2. To the best of our knowledge, the strategies reported so far for inorganic– organic composites or related bone-repairing scaffolds have been mainly based on the supply of calcium, especially a certain Ca/P ratio, as the major inorganic component, such as calcium carbonate, calcium phosphate3, tricalcium phosphate4, calcium polyphosphate5, and hydroxyapatite6. Combined with three-dimensional (3D) printing or nanotechnology, the calcium strategies have aroused great interest and achieved obvious and encouraging progress in bone regeneration. Nonetheless, the performance of scaffolds supplying calcium or calcium with phosphorus is still not so satisfactory according to clinical feedback. Thus, a new strategy that could better meet the requirements of bone regeneration is urgently needed. According to the above description, as another major constituent of bone minerals, phosphate plays an important role in bone regeneration7. Nevertheless, there are no systematic studies focusing on a calcium-free phosphorus strategy for bone regeneration. Furthermore, problematic disadvantages cannot be overlooked, namely, phosphorus-containing materials either show an uncontrollable phosphorus release (such as phosphate-based biomaterials) or manifest toxicity (e.g., red phosphorus, white phosphorus). Fortunately, the recently developed black phosphorus (BP), another main allotrope

of

phosphorus,

possesses

stable

chemical

properties

and

good

biocompatibility. Lately, several-layer black phosphorus nanosheets (BPNs) found some promising biomedical applications, especially for drug delivery, photothermal therapy, and imaging8. Herein, a calcium-free phosphorus strategy for enhancing bone regeneration via a BP hydrogel scaffold was investigated for the first time (Scheme 1). Offering some


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advantages (the hydrogel scaffold will encapsulate and keep BPNs inside and serve as an extracellular matrix with good biocompatibility), a BPN-based hydrogel is hypothesized here to provide extra phosphorus for enhanced bone regeneration. In this report, the hydrogel was fabricated via photopolymerization of gelatin methacrylamide (GelMA) and cationic arginine-based unsaturated poly(ester amide)s [U-Arg-PEAs]. GelMA has been widely used in bone tissue engineering and has shown excellent biocompatibility9. U-Arg-PEAs have been incorporated into hydrogels not only for crosslinking to form a hydrogel with improved cellular interactions but also for the resulting positive charge that facilitates a strong electrostatic attachment of the hydrogel to BPNs10, promoting a phosphate release in a sustained manner. The fabricated platform may supply phosphorus, and encapsulated BPNs can capture calcium ions to accelerate biomineralization in a bone defect. Our in vitro and in vivo experiments evaluated photoresponsive release behavior of phosphate of BPN-containing hydrogels and its effect on mineralization. An in vivo rabbit model of bone defects was utilized to evaluate the effect of BPNs on bone regeneration performance.



Scheme 1. A 3-D hydrogel platform follows a new strategy by supplying phosphorus without extra calcium and offers combined advantages: the hydrogels with encapsulated BPNs can capture calcium ions to accelerate biomineralization in a bone defect and enhance bone regeneration.

RESULTS AND DISCUSSION Fabrication and characterization of BP/PEA/GelMA hydrogels GelMA with a predesigned high degree of methacrylation and U-Arg-PEAs were synthesized according to previous reports11. BPNs were prepared from bulk BP by a liquid exfoliation method using N-methylpyrrolidone12. Transmission electron microscopy (TEM) was carried out to characterize the morphology of BPNs. TEM images (Fig. 1a) illustrated that the BPNs were successfully exfoliated with lateral diameters from 20 to 100 nm. More details about the preparation of GelMA, U-ArgPEAs, and BPNs can be found in Supporting Information (Figure S1, S2 and S3). The BPN-containing hydrogels were prepared by the photopolymerization of PEA and


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GelMA in an aqueous solution with the Irgacure 2959 photoinitiator and were named as BP/PEA/GelMA hydrogels. Before the ultraviolet (UV) irradiation, BPNs were added into the solution, which was then well mixed. As presented in Fig. 1b, the BPNcontaining hydrogels showed an obvious color difference when compared to the PEA/GelMA hydrogels, indicating successful encapsulation of BPNs. The silvery gray shimmering appearance was dependent on the amount of BPNs, and the greater the BPN content, the deeper was the color; this phenomenon helped us to understand the dynamic change of the concentration of BPNs in the hydrogels. The surface morphology of the hydrogels was studied by scanning electron microscopy (SEM). A typical macroporous sponge-like structure was seen in PEA/GelMA hydrogels with 50 μm mean pore diameter (Fig. 1c), whereas the BP/PEA/GelMA hydrogel showed a thicker wall with holes and encapsulation of BPNs, indicating a possible improvement on the network structure of hydrogels. The porous structures also indicated good water absorption capacity, which benefits a nutrient supply for cell growth in tissue engineering13. Capacity for water absorption was next studied by evaluating the swelling behavior of the hydrogels. The swelling kinetics of the hydrogels immersed in ultrapure water was analyzed over a period of 2 days at room temperature. As illustrated in Fig. 1d, BP/PEA/GelMA hydrogels had higher swelling rates compared with the PEA/GelMA hydrogels (without BPNs). The swelling ratios of the BPN-containing hydrogels showed no obvious differences when immersed in ultrapure water, phosphate buffer, the DMEM medium, or citrate buffer (Fig. 1e). For better bone regeneration, the scaffold degradation rate should match the rate of new bone tissue formation with suitable structural integrity. In vitro biodegradation of BP/PEA/GelMA hydrogels was also investigated in the four above-mentioned solutions. Figure 1f indicates that the remaining mass of the hydrogels was ~40% after 21-day immersion in the solutions. For bone tissue engineering applications, adequate mechanical properties of scaffolds are required. To analyze the mechanical properties of the hydrogels, an Instron 5944 testing system was used. Figure 1g depicts a comparison of the mechanical properties of the swollen hydrogels with or without BPNs. Compared with PEA/GelMA hydrogels,



the hydrogels with BPNs showed a slight increase in compression modulus (Fig. 1h); this finding was in good agreement with the SEM results, implying possible better mechanical support in the period of bone regeneration.

Photoresponsive in vitro mineralization As to the scaffolds for bone regeneration, the abilities to generate a mineralized matrix and to form a strong mechanical bond to bone tissue are important and could be simply evaluated in vitro by mineralization14. Some reports have shown that with light exposure, BPNs in an environment containing oxygen and water can be photo-oxidized and degraded into phosphate and phosphate ions15. Furthermore, BPNs have been reported to possess the capture capacity toward calcium ions16. Hence, the spontaneous binding of BPNs and calcium ions may benefit biomineralization in a bone defect, thereby leading to enhanced bone regeneration. To determine these effects, photoresponsive phosphate release testing and an in vitro mineralization experiment were conducted. Three groups of BP/PEA/GelMA hydrogels were immersed in pure water without phosphorus and were incubated in the dark, under natural light, or in the dark but with 5 min 808 nm near-infrared (NIR) laser irradiation every 12 h. The molybdenum blue phosphorus method was used to determine the phosphorus concentration in an aqueous solution17. The results showed that the concentration of phosphate in the group treated with light was significantly higher than that in group “in the dark” (Fig. 1i). The group treated with 808 nm NIR laser irradiation released the largest amount of phosphate, whereas the group treated with natural light showed a more sustained phosphate release. A small amount of phosphate was still present in group “in the dark” possibly because the light was not completely shielded during the experiment. All these results indicated that BPN-containing hydrogels provided extra phosphorus in a photoenergy-related manner. To confirm the effect of degraded BPNs on the mechanical performance, the mechanical properties of the BP/PEA/GelMA hydrogels were tested after three sessions of 808 nm NIR laser irradiation within 48 h. Both BP/PEA/GelMA hydrogels and PEA/GelMA hydrogels showed slightly lower compression modulus after 48 h owing


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to biodegradation (Fig. 1h). Compared with the PEA/GelMA hydrogels subjected to the same treatment, BP/PEA/GelMA hydrogels still showed higher compression modulus, indicating good mechanical performance with little influence on the phosphate release. According to some reports, BPNs can efficiently convert NIR light into thermal energy18. In particular, the temperature of aqueous solutions containing BPNs at the concentration of 50 ppm increased by 31.4°C after 5 min 808 nm NIR Laser irradiation, while the temperature of water only increased by 2.9°C. Thus, in terms of thermal energy, which may worsen a bone defect, the subsequent operations were carried out under natural light. To test the capacity for mineralization in vitro, the hydrogels were soaked in simulated body fluid (SBF) under natural light for 15 days. Notably, the BP/PEA/GelMA hydrogel with the supply of calcium-free phosphorus showed obvious mineralization characteristics with a white appearance (Fig. 1j). SEM images revealed significantly enhanced mineral formation on the surface of BP/PEA/GelMA hydrogels compared with PEA/GelMA hydrogels, suggesting that the extra phosphorus may benefit in vitro mineralization. Both BP/PEA/GelMA hydrogels and PEA/GelMA hydrogels showed lower compression modulus after immersion in SBF for 15 days, while mineralization and biodegradation coexisted (Fig. 1h). Identical conclusion was obtained in the X-ray photoelectron spectroscopy (XPS) study where calcium element was observed after mineralization (Figure S4). Remarkably, even after 15 days, BP/PEA/GelMA hydrogels still showed high compression modulus and better mechanical performance as compared with PEA/GelMA hydrogels. All the results indicated that BPNcontaining hydrogels possess the capacity for a photoresponsive phosphate release and for accelerated in vitro mineralization with the supply of extra phosphorus.



Figure 1. Characterization of BP/PEA/GelMA hydrogels: (a) A TEM image of BPNs. (b) An image of PEA/GelMA hydrogels and BP/PEA/GelMA hydrogels. (c) SEM images of freeze-dried PEA/GelMA hydrogels and BP/PEA/GelMA hydrogels. (d) Swelling kinetics of the PEA/GelMA hydrogels and BP/PEA/GelMA hydrogels in ultrapure water at room temperature. (e) Swelling ratios and (f) in vitro biodegradation of BP/PEA/GelMA hydrogels in ultrapure water, phosphate buffer, DMEM medium, or citrate buffer. (g) Stress–strain curves of BP/PEA/GelMA hydrogels and PEA/GelMA hydrogels. (h) Statistics on the compression modulus of BP/PEA/GelMA


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hydrogels and PEA/GelMA hydrogels (*P

Black Phosphorus Hydrogel Scaffolds Enhance Bone Regeneration

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