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Sustained Release of Two Bioactive Factors from Supramolecular Hydrogel Promotes Periodontal Bone Regeneration Jiali Tan,*,† Mei Zhang,† Zijuan Hai,⊥ Chengfan Wu,‡ Jiong Lin,† Wen Kuang,‡ Hang Tang,† Yulei Huang,† Xiaodan Chen,† and Gaolin Liang*,‡ Downloaded via UNIV OF SOUTHERN INDIANA on July 17, 2019 at 08:35:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Department of Orthodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, 56 Lingyuan West Road, Guangzhou, Guangdong 510055, China ‡ Hefei National Laboratory of Physical Sciences at Microscale, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ⊥ Institutes of Physical Science and Information Technology, Anhui University, 110 Jiulong Road, Hefei, Anhui 230601, China S Supporting Information *

ABSTRACT: Intact and stable bone reconstruction is ideal for the treatment of periodontal bone destruction but remains challenging. In research, biomaterials are used to encapsulate stem cells or bioactive factors for periodontal bone regeneration, but, to the best of our knowledge, using a supramolecular hydrogel to encapsulate bioactive factors for their sustained release in bone defect areas to promote periodontal bone regeneration has not been reported. Herein, we used a well-studied hydrogelator, NapFFY, to coassemble with SDF-1 and BMP-2 to prepare a supramolecular hydrogel, SDF-1/BMP-2/NapFFY. In vitro and in vivo results indicated that these two bioactive factors were ideally, synchronously, and continuously released from the hydrogel to effectively promote the regeneration and reconstruction of periodontal bone tissues. Specifically, after the bone defect areas were treated with our SDF-1/BMP-2/NapFFY hydrogel for 8 weeks using maxillary critical-sized periodontal bone defect model rats, a superior bone regeneration rate of 56.7% bone volume fraction was achieved in these rats. We anticipate that our SDF-1/BMP-2/NapFFY hydrogel could replace bone transplantation in the clinic for the repair of periodontal bone defects and periodontally accelerated osteogenic orthodontics in the near future. KEYWORDS: sustained release, supramolecular hydrogel, stromal-derived factor-1, bone morphogenetic protein-2, periodontal bone regeneration

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which can recruit both residing and circulating endogenous stem cells to defect areas and in situ promote the proliferation and differentiation of stem cells, has been a promising bone tissue engineering strategy for periodontal bone regeneration.6−8 Stromal cell derived factor-1 (SDF-1) is one of the most important chemokines. It has complex biological functions, such as augmentation of cell migration9 and regulation of cell growth.10 Importantly, SDF-1 can stimulate the homing of endogenous bone marrow mesenchymal stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs) to the defect areas,11,12 as well as improve their proliferation ability.13,14 Thus, SDF-1 was frequently used to recruit and proliferate

eriodontal bone destruction, which is caused by periodontitis, surgery, or trauma, will advance to the loss of periodontal ligament attachment and subsequent teeth loosening.1 For dentists, intact and stable reconstruction of periodontal bone tissues is an ideal therapeutic strategy for periodontal bone destruction but remains challenging.2 In the clinic, autogenic and allogeneic bone transplantations are two major methods to reconstruct the periodontal bone defects. But they are still facing some problems such as limited bone resources, inevitable trauma by surgery, and immunological rejections to allogenic grafts.3 In research, bone tissue engineering, which uses stem cell- or bioactive factor (or both)-encapsulated biomaterials to fill the destroyed areas, has shown great potential for periodontal bone regeneration.4 Nevertheless, besides the tedious work of their obtainment and culture, the directly delivered extraneous stem cells have low survival rates on site and might cause new infections.5 Therefore, using biomaterials to deliver bioactive factors, © 2019 American Chemical Society

Received: January 28, 2019 Accepted: May 6, 2019 Published: May 6, 2019 5616

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ACS Nano BMSCs and PDLSCs for periodontal tissue reconstruction. After recruitment and proliferation of BMSCs and PDLSCs, their differentiation needs to be initiated. As members of the transforming growth factor beta superfamily, bone morphogenetic proteins (BMPs) can induce BMSCs to differentiate into bone, blood vessel, ligament, and cartilage.15,16 Among them, BMP-2 can significantly induce the differentiation of BMSCs into osteoblasts and improve new bone formation.17 Shen et al. demonstrated that combined utilization of SDF-1 and BMP-2 had synergistic enhanced effects on the angiogenesis and bone regeneration at the calvarial bone defect sites, compared with those of individually used SDF-1 or BMP-2.18 To achieve desired bone regeneration, the above bioactive factors are required to be locally delivered at the defect area at proper levels and durations.19,20 But as we know, bioactive factors are usually unstable and easily degraded.21 Thus, biomaterials are always used to encapsulate the bioactive factors for not only retaining their activity but also sustaining their release at the defect sites.22−25 Among the biomaterials reported, polymer hydrogels are most commonly used as the delivery scaffolds for bone regeneration. The extracellular matrix (ECM)-like, three-dimensional (3D) networks of polymer hydrogels provide a suitable environment for not only the sustained release of the bioactive factors but also the adhesion and growth of the BMSCs and PDLSCs.15,26 Nevertheless, the biocompatibility and biodegradability issues of polymer hydrogels, which are introduced by the toxic reactants and the polymeric covalent bonds during syntheses, limit their biomedical applications.27,28 Supramolecular hydrogels, formed by the self-assembly of small molecules through noncovalent interactions, are much more biocompatible and degradable than polymer hydrogels.29 Recently, we successfully employed supramolecular hydrogels to coassemble and slowly release drugs to enhance the antihepatic fibrosis effect of dexamethasone, enhance the anticancer effect of etoposide, or overcome organ transplant rejection of tacrolimus.30−32 However, to the best of our knowledge, using a supramolecular hydrogel to encapsulate and slowly release bioactive factors to promote periodontal bone regeneration has not been reported. Inspired by previous studies, in this work, we intended to use a biocompatible supramolecular hydrogel to encapsulate SDF-1 and BMP-2 and then slowly release them at the bone defect area to activate the regeneration and reconstruction of periodontal bone tissues in situ. To achieve this, as shown in Figure 1A, a well-studied, biocompatible hydrogelator, NapPhe-Phe-Tyr-OH (NapFFY), was chosen to facilely prepare the supramolecular hydrogel. The rationale for us to choose the supramolecular hydrogel NapFFY is as follows. First, the NapFFY hydrogel is a soft biomaterial, which allows it to well fit the irregular periodontal bone defects. Second, the ECMlike structure of the supramolecular hydrogel could introduce multiple cell signals and then promote the adhesion and proliferation of cells.33 By simply dissolving SDF-1 and BMP-2 in NapFFY phosphate-buffered saline (PBS) solution at pH 8.0 and 37 °C and cooling, a transparent SDF-1/BMP-2/ NapFFY supramolecular hydrogel was obtained. After the hydrogel filled in the bone defect area, as illustrated in Figure 1B, SDF-1 and BMP-2 were slowly released from the hydrogel. Then BMSCs were recruited to the defect sites by SDF-1, and their differentiation was promoted by BMP-2, followed by the initiation of the periodontal bone regeneration process. Specifically, after treating the bone defect areas with our SDF-1/BMP-2/NapFFY hydrogel for 8 weeks using maxillary

Figure 1. (A) Schematic illustration of the formation process of the SDF-1/BMP-2/NapFFY hydrogel. (B) Schematic illustration of promoted periodontal bone regeneration by the SDF-1/BMP-2/ NapFFY hydrogel in the bone defect area. Slowly released SDF-1 from the hydrogel will recruit BMSCs to the defect area; then BMP-2 will promote BMSCs to differentiate into osteoblasts, resulting in the initiation of the periodontal bone regeneration process.

critical-sized periodontal bone defect model rats, we found that a superior bone regeneration rate (i.e., BV fraction, bone volume (BV)/total volume (TV) × 100% = 56.7%) was achieved in these rats, compared with 34.9% for the SDF-1/ NapFFY hydrogel or 36.6% for the BMP-2/NapFFY hydrogel, respectively.

RESULTS AND DISCUSSION Synthesis, Characterization, and Hydrogelation of NapFFY. First, we directly synthesized the hydrogelator NapFFY using solid phase peptide synthesis (SPPS) (Scheme S1) and purified it with high-performance liquid chromatography (HPLC). After NapFFY was fully characterized with 1H NMR, 13C NMR, and electrospray ionization-mass spectrometry (ESI-MS) spectra (Figures S1−S3), we tested its gelation property upon physical adjustment (heated to 55 °C and cooled to 25 °C, pH 8.0). Briefly, 2 mg of NapFFY powder was dispersed in 200 μL of PBS (25 °C, 0.01 M), the pH value of the dispersion was adjusted to 8.0, and then the mixture was heated to 55 °C until the solution became clear. Then the solution was cooled to room temperature (25 °C), and a transparent hydrogel at a concentration of 1.0 wt % was formed 30 min later. Plots of the fluorescence intensity of the emission peak at 315 nm versus the concentration of serial NapFFY dilutions indicated that the critical aggregation concentration (CAC) of NapFFY was 13.62 μM (Figure S4). These results suggested that NapFFY has excellent selfassembly ability. Optimizing the Concentrations of SDF-1 and BMP-2. Before applying NapFFY to coassemble with SDF-1 and BMP2, we optimized the concentrations of two bioactive factors in vitro. First, we isolated rat BMSCs and obtained the thirdgeneration BMSCs for the following experiments. The growth curve (Figure S5) and microscopic image (Figure S6) of the third-generation BMSCs indicated they were in healthy condition. Their osteogenic and adipogenic differentiation abilities were evaluated by Alizarin Red and Oil Red O staining (Figures S7). Then we evaluated the chemotactic effect of SDF-1 on BMSCs using a Transwell migration assay. The BMSCs were allowed to migrate across at different concentrations of SDF-1 for 12 h. The migration number of BMSCs increased with the increase in SDF-1 concentration in 5617

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ACS Nano a dose-dependent manner in the range of 0 to 500 μg/L, but it did not show a difference among SDF-1 concentrations of 500, 1000, and 2000 μg/L (Figure S8). Therefore, we chose 500 μg/L as the optimal concentration of SDF-1 to induce the maximum chemotaxis of BMSCs in vitro. As for BMP-2, its concentration ranging from 300 μg/L to 3000 μg/L was widely investigated in bone tissue engineering.6,34 Overdosed BMP-2 could induce excessive inflammation and adipogenesis rather than osteogenesis and heterotopic bone formation.20,35 Therefore, we chose a relatively low dose of 500 μg/L as the optimal concentration of BMP-2, which is the same as that of SDF-1. Hydrogelations and Characterizations of Four Hydrogels. After confirming the concentrations of SDF-1 and BMP2, we prepared four hydrogels. We first prepared four solutions: NapFFY at 1.0 wt % in PBS; SDF-1/NapFFY solution was 500 μg/L SDF-1 dissolved in 1.0 wt % NapFFY in PBS at 37 °C; BMP-2/NapFFY solution was 500 μg/L BMP-2 dissolved in 1.0 wt % NapFFY in PBS at 37 °C; SDF1/BMP-2/NapFFY solution was 500 μg/L SDF-1 and 500 μg/L BMP-2 dissolved in 1.0 wt % NapFFY in PBS at 37 °C. After cooling to room temperature, all four solutions formed transparent hydrogels 30 min later (insets of Figure 2),

microscopy (TEM) was used to observe the morphology of the four hydrogels. As shown in Figure 2, the TEM image of the NapFFY hydrogel showed dense nanofiber networks with an average diameter of 21.0 ± 9.3 nm. TEM images of SDF-1/ NapFFY, BMP-2/NapFFY, and SDF-1/BMP-2/NapFFY hydrogels showed sparse nanofibers with black granules, which had average diameters of 39.5 ± 28.6, 108.3 ± 33.0, and 44.6 ± 14.3 nm, respectively. The much wider nanofibers with black granules in SDF-1/BMP-2/NapFFY hydrogels compared with the nanofibers in the NapFFY hydrogel additionally suggested the coassembly of NapFFY with SDF1 and BMP-2. Co-assembly of NapFFY with SDF-1 and BMP2 through noncovalent interactions could enable the sustained release of the bioactive factors from the hydrogel. Cumulative Release of SDF-1 and BMP-2 from the SDF-1/BMP-2/NapFFY Hydrogel in Vitro. After the characterizations of hydrogels, we studied the release profile of SDF-1 and BMP-2 from the SDF-1/BMP-2/NapFFY hydrogel in vitro. A 1 mL amount of PBS (0.01 M, pH = 7.4) was added to 200 μL of the SDF-1/BMP-2/NapFFY hydrogel (1.0 wt % for NapFFY, 500 μg/L for the two bioactive factors, respectively) and then incubated at 37 °C. At different times (1, 2, 4, 8, 14, 21, 28, or 35 d), 1 mL of supernatant was collected and the incubation mixture was immediately supplemented with 1 mL of fresh PBS. The concentrations of SDF-1 and BMP-2 in the collected PBS were quantified via their corresponding enzyme-linked immunosorbent assay (ELISA) kits. As shown in Figure 3, constant and sustained

Figure 2. TEM images of four hydrogels (insets: optical images of their corresponding hydrogels). Figure 3. In vitro cumulative release of SDF-1 and BMP-2 from the SDF-1/BMP-2/NapFFY hydrogel in PBS at 37 °C.

indicating successful coassembly of NapFFY with SDF-1 or BMP-2, or both. Then viscoelastic properties of the four hydrogels were characterized. Dynamic strain sweeps of the hydrogels were first determined. Within the strain range of 0.5% to 10%, the storage modulus (G′) of all the hydrogels was higher than their loss modulus (G″), suggesting that these samples are hydrogels (Figure S9). Then the strain amplitude was set at 1.0%, and the dynamic frequency sweep of four hydrogels was tested. As shown in Figure S10, at a frequency from 0.1 to 10 Hz, G′ and G″ values of the four hydrogels slightly increased and the G′ values were larger than their corresponding G′′ values, indicating that these hydrogels are tolerant to external shearing forces. In addition, the G′ and G′′ values between these four groups showed no obvious difference, suggesting that SDF-1 and BMP-2 did not interfere with the mechanical properties of hydrogel NapFFY. The small G′ and G″ values of four hydrogels (around 1 to 30 Pa) indicated these soft hydrogels are injectable and could fill up the irregular periodontal bone defects. Transmission electron

release of both SDF-1 and BMP-2 was observed up to 35 d. It was reported that the suitable release periods of bioactive factors for bone regeneration were longer than 2 weeks but shorter than 4 weeks.36 Therefore, the sustainable release profile of SDF-1 and BMP-2 from our supramolecular hydrogel was applicable for periodontal bone regeneration. In detail, as shown in Figure 3, in the first 2 days, cumulative release of SDF-1 and BMP-2 quickly reached 32.4 ± 2.4% and 38.9 ± 2.2%, respectively. This initial burst release of two factors could afford sufficient stimuli to the defect areas (i.e., instantly concentrated SDF-1 could quickly recruit stem cells, while BMP-2 could quickly promote their differentiation). As we know, the release rate of proteins from the peptide-based hydrogel is associated with their molecular mass.37 Thus, the faster initial release of BMP-2 than SDF-1 from the hydrogel was probably due to the larger molecular mass of BMP-2 (15− 16 kDa) than that of SDF-1 (7 kDa). Then the release rates of 5618

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to the above CCK-8 assay result. Since alkaline phosphatase (ALP) is one of the important biomarkers during the early osteogenic differentiation stage of BMSCs, we used quantitative polymerase chain reaction (qPCR) to quantitate the ALP mRNA expression levels in these five groups at 8 and 14 d. A 24-well Transwell plate was used in this experiment, with BMSCs being seeded in the upper chambers; hydrogels and osteogenic induction media were placed in the lower chambers. At day 8 or day 14, qPCR was carried out to quantitate the ALP mRNA expression levels of BMSCs in the lower chambers. As shown in Figure S16, at both day 4 and day 8, there was no significant difference of ALP expression between the NapFFY group and the Ctrl group, suggesting the NapFFY hydrogel itself could not stimulate the differentiation of BMSCs. Interestingly, although SDF-1 cannot directly stimulate the differentiation of BMSCs, the ALP mRNA expression level of BMSCs in the SDF-1/NapFFY group was close to that in the BMP-2/NapFFY group (p > 0.05), significantly higher than that in the Ctrl and NapFFY groups (p < 0.05). This could explained why the chemotaxis of the BMSCs in the SDF-1/NapFFY group was enhanced by the released SDF-1 from the hydrogel, and more BMSCs migrated to the lower chamber, stimulated to differentiate by the osteogenic induction medium in the lower chamber. As expected, the highest ALP mRNA expression level was observed in the SDF-1/BMP-2/NapFFY group. The above qPCR results of ALP mRNA expression indicated that the chemotaxis and differentiation of BMSCs were effectively enhanced by SDF-1 and BMP-2 released from the NapFFY hydrogels, respectively. Periodontal Bone Regeneration in Vivo. After cytotoxicity assessment of the NapFFY hydrogels, we then explored their bone regenerative capacity using the critical-sized periodontal bone defect models of maxillae in rats (Figure S17). Eight-week-old female Sprague Dawley rats (weighting 180 ± 20 g) with bone defects were randomly divided into five groups (Ctrl, NapFFY, SDF-1/NapFFY, BMP-2/NapFFY, and SDF-1/BMP-2/NapFFY) (n = 3 for each group). After their defects were filled with hydrogels and their wounds were closed, the rats were allowed to recover for 8 weeks with usual feeding. Then their maxillae were analyzed with microcomputed tomography (micro-CT) imaging, hematoxylin and eosin (H&E) staining, and Masson’s trichrome staining. Micro-CT images and quantitative analyses of the BV fractions (BV/TV × 100%) of the defect areas are shown in Figure 4. Generally, after 8 weeks, new bones were found forming from edges to centers of the defect areas in all groups. Micro-CT images of Ctrl group rats at 8 weeks showed that their bone volumes slightly increased by self-repairing. Compared with the low BV fraction (11.2 ± 1.0%) in the Ctrl group, BV fractions of maxillae in other four groups were obviously increased (21.4 ± 1.5% for the NapFFY group, 34.9 ± 1.7% for the SDF-1/NapFFY group, 36.6 ± 2.0% for the BMP-2/ NapFFY group, and 56.7 ± 2.2% for the SDF-1/BMP-2/ NapFFY group), indicating successful accelerated processes of bone bridging and defect reunion by our NapFFY hydrogels. The largely increased BV fraction (1.91-fold) of the NapFFY group indicated that the hydrogel itself had a certain effect on bone defect repairing, which was consistent with the above cytotoxicity results that the NapFFY hydrogel itself could promote the proliferation of BMSCs. The probable mechanisms are as follows: (1) the biomimetic 3D nanofiber network structures of the NapFFY hydrogel could promote the

SDF-1 and BMP-2 were synchronous with time, according to the slopes of the cumulative release rate curves of these two factors in Figure 3. The cumulative release rates approached their plateaus at 21 d. At 35 d, cumulative release rates of SDF1 and BMP-2 were measured to be 74.8 ± 3.5% and 82.1 ± 3.5%, respectively. Therefore, we think that the ideally synchronous sustained release of SDF-1 and BMP-2 from the SDF-1/BMP-2/NapFFY hydrogel could have a coordinated effect on bone regeneration. To directly observe the protein release from the SDF-1/BMP-2/NapFFY hydrogel, we took the TEM images of the hydrogel at day 2, 4, 8, and 14 during the protein release process. As shown in Figure S11, with the increase of incubation time, nanofibers in the hydrogel became sparse and shorter, and the black granules (i.e., SDF-1 or BMP2 proteins) in the nanofibers became less, suggesting slow disassembly of the hydrogel and thereby slow release of the two bioactive factors. Cytotoxicity Assessment of the NapFFY Hydrogels. In order to assess the cytotoxicity of NapFFY hydrogels, we used the cell-counting kit-8 (CCK-8) assay to evaluate the survival and proliferation of BMSCs. First, we suspended BMSCs in culture medium as a control (Ctrl) group and in four solutions of NapFFY, SDF-1/NapFFY, BMP-2/NapFFY, and SDF-1/ BMP-2/NapFFY at 37 °C as experimental groups (1.0 wt % for NapFFY, 500 μg/L for the two bioactive factors). Then the cell dispersions were dropped into a cell culture plate, allowed to stand for 30 min to form a hydrogel at 25 °C, and then incubated at 37 °C for 1, 2, 4, 8, 10, or 14 d. As shown in Figure S12, the cell viability of BMSCs in all four hydrogels increased over time, suggesting that our 1.0 wt % NapFFY hydrogels did not impose toxicity on the cells but enhanced the cell proliferation activity. This might be due to the low stiffness of our supramolecular nanofibers, which allowed the active cellular forces to recruit nearby nanofibers. This type of nanofiber recruitment dynamically increased ligand density on the cell surface and thus promoted focal adhesion formation and signaling.38 In addition, no significant difference was observed between the NapFFY hydrogel and other three hydrogels (SDF-1/NapFFY, BMP-2/NapFFY, and SDF-1/ BMP-2/NapFFY), indicating that neither SDF-1 nor BMP-2 at 500 μg/L exerted side effects on BMSCs. To directly observe the cell viability, we used a calcein-AM/PI (live/dead) kit to stain the BMSCs in the above five groups on days 1, 4, and 8. As shown in Figure S13, consistent with the above cytotoxicity results, the number of live BMSCs increased over time in all groups, and more live BMSCs were observed in experimental groups than the Ctrl group at both day 4 and day 8. Moreover, very rare dead cells were observed in all groups during 8 d of observation. These results indicated that the NapFFY hydrogels did not impose cytotoxicity on BMSCs but stimulated the proliferation of the cells. Stimulated Proliferation of BMSCs by NapFFY Hydrogels. The above results indicated that our hydrogels did not exert cytotoxicity on the BMSCs but stimulated their proliferation. To verify this, we first used a 5-ethynyl-2′deoxyuridine (EdU) assay to label the proliferating BMSCs in the above five groups. At 1, 4, or 8 d, the proliferating BMSCs were stained by EdU and their green fluorescence was imaged under a fluorescence microscope. As shown in Figures S14 and S15, the proliferating BMSCs in four NapFFY hydrogel groups were significantly more than those in the Ctrl group (p < 0.05). Interestingly, there was no significant difference of proliferating BMSCs among the four hydrogel groups, similar 5619

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Figure 5. Histological images of bone defect areas in decalcified maxilla sections by H&E staining (left two columns) and Masson’s trichrome staining (right two columns). Connective tissues (CT) and new bones (NB) with blood-vessel-like structures (indicated by green arrows) in the selected regions (yellow rectangles) are shown at higher magnification in detail. Scale bars = 500 μm for 40× and 100 μm for 200×.

Figure 4. (A) Micro-CT images of the periodontal bone defect areas in the maxillae of rats of different groups (size of each bone defect is about 20 mm3). (B) Bone volume fractions of defect areas in different groups at 8 weeks. Data are expressed as mean ± SD, n = 3. ***p < 0.001, compared with the Ctrl group. Not significant (ns): p > 0.05. Scale bar = 2 mm.

attachment of BMSCs; (2) the low stiffness of the NapFFY hydrogel could allow its network pore size to respond to the mechanical force exerted by the cells when they migrate through the matrix,33 which facilitates the remodeling of the ECM by BMSCs in the bone defect sites. Although certain amounts of bones were found to form in the defects of the SDF-1/NapFFY and BMP-2/NapFFY groups, the central parts of their defect sites were still undergoing bone remodeling. However, in the SDF-1/BMP-2/NapFFY group, the regenerated bones were observed in the whole defect areas. Quantitatively, the BV fraction in the SDF-1/BMP-2/NapFFY group was 1.62-fold and 1.55-fold of that of the SDF-1/ NapFFY and BMP-2/NapFFY groups, respectively. H&E and Masson’s trichrome stainings provided more complementary details of the regenerated tissues, as shown in Figure 5. In accordance with the micro-CT results, staining of the maxilla tissues indicated that maxillae in all five groups were undergoing bone remodeling to some extent. New bones (NB) with blood-vessel-like structures (indicated by green arrows in Figure 5), which suggested a rich vascular supply of the new bone, could be observed in all five groups. In detail, in the Ctrl group, the defects were almost filled with fibrous connective tissue, and a small amount of new bone tissue was formed at the edges of host bones. Compared with the Ctrl group, more new bone tissue and less but denser connective tissue at the defect areas were detected in all four hydrogeltreated groups. The most continuous, compact, and thickest bone tissues were formed at the bone defect areas of the SDF1/BMP-2/NapFFY group. These results suggested that synergistic, sustained release of SDF-1 and BMP-2 from the supramolecular hydrogel could significantly promote the regeneration and reconstruction of bone tissues in the defect areas.

CONCLUSIONS In summary, to effectively promote the regeneration and reconstruction of periodontal bone tissues, we used a wellstudied hydrogelator, NapFFY, to coassemble with SDF-1 and BMP-2 to prepare supramolecular hydrogel SDF-1/BMP-2/ NapFFY for sustained release of these two bioactive factors. Rheology and TEM characterizations of the hydrogels indicated that SDF-1 and BMP-2 coassembled with the hydrogelator NapFFY but did not affect the mechanical strength of the hydrogels, which ensured the sustained release of the bioactive factors from the hydrogels. Cumulative release profiles of SDF-1 and BMP-2 from hydrogel SDF-1/BMP-2/ NapFFY in vitro indicated that ideally synchronous sustained release of the bioactive factors from the hydrogel could afford sufficient and continuous stimuli to bone defect areas. Cytotoxicity and proliferation studies indicated that the hydrogel did not impose toxicity on the cells but enhanced the cell proliferation of BMSCs. In vivo treatment of a periodontal bone defect model in rats with the SDF-1/BMP2/NapFFY hydrogel for 8 weeks indicated that significantly promoted regeneration of bone tissues (BV fraction = 56.7%) was achieved in the defect area. We anticipate that our SDF-1/ BMP-2/NapFFY hydrogel could replace bone transplantation in the clinic for the repair of periodontal bone defects and periodontally accelerated osteogenic orthodontics in the near future. Furthermore, by coassembling with other bioactive factors (e.g., vascular endothelial growth factor or plateletderived growth factor), our hydrogel system may also have potential applications in dental pulp regeneration. This type of work is underway. 5620

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EXPERIMENTAL SECTION

ASSOCIATED CONTENT S Supporting Information *

Chemotactic Effect of SDF-1 on BMSCs. An 8.0 μm pore (Corning 6.5 mm) Transwell system was used to evaluate the chemotactic effect of SDF-1 on BMSCs. The BMSCs were starved for 3 h, trypsinized, resuspended, and loaded into the top chamber of the Transwell system at a density of (1.0−1.5) × 105 cells/insert. Cell culture media containing SDF-1 at different concentrations (0, 1, 10, 100, 200, 500, 1000, or 2000 μg/L) were placed in the lower chamber. After 12 h, migrated BMSCs were fixed with 4% paraformaldehyde for 0.5 h and then stained with 0.2% crystal violet. Migrated cells (on the bottom surface of the insert) were visualized by microscopy. Six random visual fields were selected per well for viewing (100× magnification), and cells were quantified using ImageJ. Animal Model. All animals received care according to the guidelines in the Guide for the Care and Use of Laboratory Animals of Sun Yat-sen University (SYSU, China). The procedures were approved by the Animal Ethics of SYSU Animal Care and Use Committee (Approval No. SYSU-IACUC-2018−000103). Eightweek-old female (weighting 180 ± 20 g) Sprague Dawley rats were placed into five groups randomly (Ctrl, NapFFY, SDF-1/NapFFY, BMP-2/NapFFY, and SDF-1/BMP-2/NapFFY), and each group included three rats. Before the surgery, rats were treated with general anesthesia (intraperitoneal injection of 40 mg/kg pentobarbital sodium). Under an aseptic environment, using the method previously described with minor modification,39 a surgically created periodontal bone defect was made on the mesial area of the randomly selected unilateral maxillary first molar. First, we made a 1 cm relieving incision on the mesial area and the gingival sulcus of the maxillary first molar and evaluated the mucoperiosteal flap. Then, bone tissue was removed by an electric drill with a size-4 round bur at a slow speed under saline irrigation. The defect width was standardized to the width of the round bur (4 mm in diameter) and extended longitudinally (4 mm) to the mesial side of the exposed root (Figure S17). After the critical-sized defects were made, hydrogels were applied to fill these defects in the treatment groups, and in the Ctrl group the defect area was not filled in. Finally, the flaps were replaced and the wound was closed using 5-0 absorbable sutures. Micro-CT Analysis. At 8 weeks, all animals were sacrificed by CO2 inhalation, and the maxillae were obtained for micro-CT and histological examinations. Methods of micro-CT assessments for periodontal defect regeneration were done according to the protocol described by Chang et al.39 Briefly, the obtained specimens were fixed in 10% paraformaldehyde for over 12 h. Then, they were scanned using a micro-CT system (SCANCO Medical AG, Fabrikweg 2, CH8306 Bruettisellen, Switzerland) at a pixel size of 15 μm. The microCT system packaged software was used to 3D reconstruct and quantitatively analyze the specimens. A region of interest (ROI) of the entire bone defect site was selected semiautomatically, and the BV fractions (BV/TV, means ± SD of 3 rats) of the ROI were analyzed by this software. Histology Analysis. After fixation in 10% paraformaldehyde overnight, the maxillae from five groups were decalcified in 14% ethylenediaminetetraacetic acid for 4 weeks, followed by dehydration in gradients of ethanol and embedding in paraffin. Later, the samples were sectioned in a sagittal plane throughout the entire mesial area of the first molars at a section thickness of 5 μm to present the whole surgical area. All the sections were stained with H&E and Masson’s trichrome. The slides were examined under a light microscope (IX71400X, Olympus, Japan) to visualize the formation of newly formed tissues. The formations of new bone within defects were observed at 40× and 200× magnifications. Statistical Analysis. Statistical analyses were measured by oneway ANOVA, and p < 0.05 is considered as statistically significant. All statistical analyses were carried out using the IBM SPSS 20 Statistics software (SAS, Cary, NC, USA). All the data were expressed as means ± SD. All experiments were replicated at least three times.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b00788. Methods; synthesis and characterizations of NapFFY; synthetic route for NapFFY; spectra of NapFFY; CAC for NapFFY; characterizations of BMSCs in vitro; SDF-1 concentration-dependent recruitment of BMSCs; rheological data of the hydrogels; TEM images of the SDF1/BMP-2/NapFFY hydrogel; cytotoxicity and live/dead staining of BMSCs in the hydrogels; fluorescence images and quantifications of proliferating BMSCs in the hydrogels; quantification of the ALP mRNA expression levels of BMSCs in the hydrogels; surgery processes; primer sequences for qPCR (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected] (J. Tan). *E-mail: [email protected] (G. Liang). ORCID

Gaolin Liang: 0000-0002-6159-9999 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81873710, 21725505, 81821001, 81570803), Guangzhou Foundation for Science and Technology Planning Project (201704030083), Guangdong Financial Fund for High-Caliber Hospital Construction, Science and Technology Planning Project of Guangdong Province (2017A050501013), and the Oral Medicine Research Foundation of Guangdong Provincial Key Laboratory (KF2018120102, KF2017120104, KF2016120104). REFERENCES (1) de Coo, A.; Quintela, I.; Blanco, J.; Diz, P.; Carracedo, A. Assessment of Genotyping Tools Applied in Genetic Susceptibility Studies of Periodontal Disease: A Systematic Review. Arch. Oral Biol. 2018, 92, 38−50. (2) Trombelli, L. Which Reconstructive Procedures Are Effective for Treating the Periodontal Intraosseous Defect? Periodontol. 2000 2005, 37, 88−105. (3) Subramaniam, S.; Fang, Y. H.; Sivasubramanian, S.; Lin, F. H.; Lin, C. P. Hydroxyapatite-Calcium Sulfate-Hyaluronic Acid Composite Encapsulated with Collagenase as Bone Substitute for Alveolar Bone Regeneration. Biomaterials 2016, 74, 99−108. (4) Kinane, D. F.; Stathopoulou, P. G.; Papapanou, P. N. Periodontal Diseases. Nat. Rev. Dis. Primers 2017, 3, 17038. (5) Langer, R. Perspectives and Challenges in Tissue Engineering and Regenerative Medicine. Adv. Mater. 2009, 21, 3235−3236. (6) Priddy, L. B.; Chaudhuri, O.; Stevens, H. Y.; Krishnan, L.; Uhrig, B. A.; Willett, N. J.; Guldberg, R. E. Oxidized Alginate Hydrogels for Bone Morphogenetic Protein-2 Delivery in Long Bone Defects. Acta Biomater. 2014, 10, 4390−4399. (7) Martino, M. M.; Briquez, P. S.; Maruyama, K.; Hubbell, J. A. Extracellular Matrix-Inspired Growth Factor Delivery Systems for Bone Regeneration. Adv. Drug Delivery Rev. 2015, 94, 41−52. (8) Chong, L. Y.; Chien, L. Y.; Chung, M. C.; Liang, K.; Lim, J. C.; Fu, J. H.; Wang, C. H.; Chang, P. C. Controlling the Proliferation and Differentiation Stages to Initiate Periodontal Regeneration. Connect. Tissue Res. 2013, 54, 101−107. 5621

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DOI: 10.1021/acsnano.9b00788 ACS Nano 2019, 13, 5616−5622