Sustained Release of Two Bioactive Factors from Supramolecular

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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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b00788 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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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‡,* †

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 ABSTRACT Intact and stable bone reconstruction is ideal for the treatment of periodontal bone destructions but remains challenging. In research, biomaterials are used to encapsulate stem cell or bioactive factor for periodontal bone regeneration but, to the best of our knowledge, using supramolecular hydrogel to encapsulate bioactive factors for their sustained release in bone defect area 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, sustained released from the hydrogel to effectively

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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 (BV) fraction was achieved in these rats. We anticipate that our SDF-1/BMP-2/NapFFY hydrogel could replace bone transplantation in clinic for the repair of periodontal bone defect and periodontally accelerated osteogenic orthodontics (PAOO) in near future.

KEYWORDS: sustained release, supramolecular hydrogel, stromal-derived factor-1, bone morphogenetic protein-2, periodontal bone regeneration

Periodontal bone destructions, which are 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 destructions but remains challenging.2 In 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 destructive areas, has shown great potential for periodontal bone regeneration.4 Nevertheless, besides the tedious works of their obtainment and culture, those directly delivered extraneous stem cells have low survival rates on site and might bring new


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infections.5 Therefore, using biomaterials to deliver bioactive factors, 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 BMSCs and PDLSCs for periodontal tissue reconstruction. After recruitment and proliferation of BMSCs and PDLSCs, their differentiation is needed 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, 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 easy to be degraded.21 Thus, biomaterials are


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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-dimension (3D) networks of polymer hydrogels provide 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 non-covalent 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, or enhance the anticancer effect of etoposide, or overcome organ transplant rejection of tacrolimus.3032

However, to the best of our knowledge, using 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 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 Nap-Phe-Phe-Tyr-OH (NapFFY) was chosen to facilely


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prepare the supramolecular hydrogel. The rationales for us to choose supramolecular hydrogel NapFFY are as following. First, the NapFFY hydrogel is a soft biomaterial which allows it to well fit the irregular periodontal bone defects. Second, the ECM-like structure of 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, transparent SDF-1/BMP-2/NapFFY supramolecular hydrogel was obtained. After the hydrogel was filled in the bone defect area, as illustrated in Figure 1B, SDF1 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 critical-sized periodontal bone defect model rats, we found that superior bone regeneration rate (i.e., BV fraction, bone volume (BV)/total volume (TV) × 100% = 56.7%) was achieved in these rats, compared with that 34.9% of SDF-1/NapFFY hydrogel or 36.6% of BMP-2/NapFFY hydrogel, respectively.


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Figure 1. (A) Schematic illustration of the formation process of SDF-1/BMP2/NapFFY hydrogel. (B) Schematic illustration of promoted periodontal bone regeneration by SDF-1/BMP-2/NapFFY hydrogel in bone defect area. Slowly released SDF-1 form the hydrogel will recruit BMSCs to the defect area, then BMP-2 will promote BMSCs to differentiate into osteoblasts, resulting in the initiation of periodontal bone regeneration process.

RESULTS AND DISCUSSION Synthesis, Characterization, and Hydrogelation of NapFFY. Firstly, 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,

13

C NMR, and electrospray

ionization-mass spectrometry (ESI-MS) spectra (Figures S1-S3), we tested its gelation property upon physical adjustment (heat up to 55 °C and cool down to 25 °C, pH 8.0). Briefly, 2 mg NapFFY powder was dispersed in 200 μL PBS (25 °C, 0.01 M) and pH value of the dispersion was adjusted to 8.0, then heated up to 55 °C until the solution became clear. Then the solution was cooled down to room temperature (25 °C) and a transparent hydrogel at the 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 the critical aggregation concentration (CAC) of NapFFY was 13.62 μM (Figure S4). These results suggested that NapFFY has excellent self-assembly ability.


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Optimize the Concentrations of SDF-1 and BMP-2. Before applying NapFFY to co-assemble with SDF-1 and BMP-2, we optimized the concentrations of two bioactive factors in vitro. At first, we isolated rat BMSCs and obtained the third generation BMSCs for 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 of SDF-1 concentration at a dose-dependent manner in the range of 0 to 500 μg/L but it did not show 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 BMP2 could induce excessive inflammation and adipogenesis rather than osteogenesis and heterotopic bone formation.20,35 Therefore, we chose a relative low dose 500 μg/L as the optimal concentration of BMP-2, which is same to that of SDF-1. Hydrogelations and Characterizations of Four Hydrogels. After confirming the concentrations of SDF-1 and BMP-2, we prepared four hydrogels. We firstly 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 ℃; BMP-2/NapFFY


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solution was 500 μg/L BMP-2 dissolved in 1.0 wt% NapFFY in PBS at 37 ℃; 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 ℃. After cooling to room temperature, all above four solutions formed transparent hydrogels 30 min later (insets of Figure 2), indicating successful co-assembly 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 firstly determined. Within the strain range of 0.5% to 10%, the storage modulus (G′) of all the hydrogels were 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 were tested. As shown in Figure S10, at the frequency from 0.1 to 10 Hz, G′ and G′′ values of four hydrogels slightly increased and the G' values were larger than its 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 SDF1 and BMP-2 did not interfere 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 microscopy (TEM) was used to observe the morphology of the four hydrogels. As shown in Figure 2, TEM image of NapFFY hydrogel showed dense nanofiber networks with an average diameter of 21.0 ± 9.3 nm. TEM images of SDF1/NapFFY, BMP-2/NapFFY, and SDF-1/BMP-2/NapFFY hydrogels showed the sparse nanofibers with black granules, which had average diameters of 39.5 ±28.6 nm,


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108.3 ± 33.0 nm, and 44.6 ± 14.3 nm, respectively. The much wider nanofibers with black granules in SDF-1/BMP-2/NapFFY hydrogels compared with those nanofibers in NapFFY hydrogel additionally suggested the co-assembly of NapFFY with SDF-1 and BMP-2. Co-assembly of NapFFY with SDF-1 and BMP-2 through non-covalent interactions could enable the sustained release of the bioactive factors from the hydrogel.

Figure 2. TEM images of four hydrogels (insets: optical images of their corresponding hydrogels). Cumulative Release of SDF-1 and BMP-2 from 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. 1 mL PBS (0.01 M, pH = 7.4) was added to 200 μL SDF-1/BMP-2/NapFFY hydrogel (1.0 wt% for NapFFY, 500 μg/L for 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 supernatant was


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collected and the incubation mixture was immediately supplemented with 1 mL 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 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 to 32.4 ± 2.4% and 38.9 ± 2.2%, respectively. This initial burst releases 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 differentiations). As we know, the release rate of proteins from 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 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 SDF-1 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 SDF-1/BMP-2/NapFFY hydrogel could have a coordinated effect on bone regeneration. To directly observe the protein release


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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 BMP-2 proteins) in the nanofibers became less, suggesting slow disassembly of the hydrogel and thereby slow release of the two bioactive factors.

Figure 3. In vitro cumulative release of SDF-1 and BMP-2 from SDF-1/BMP2/NapFFY hydrogel in PBS at 37 ℃.

Cytotoxicity Assessment of the NapFFY Hydrogels. In order to assess the cytotoxicity of NapFFY hydrogels, we used cell-counting kit-8 (CCK-8) assay to evaluate the survival and proliferation of BMSCs. Firstly, we suspended BMSCs in culture medium as control (Ctrl) group and in four solutions of NapFFY, SDF1/NapFFY, BMP-2/NapFFY, and SDF-1/BMP-2/NapFFY at 37 ℃ as experimental groups (1.0 wt% for NapFFY, 500 μg/L for the two bioactive factors). Then the cell dispersions were dropped into cell culture plate, waited for 30 min to form hydrogel at 25 ℃, and then incubated at 37 ℃ for 1, 2, 4, 8, 10, or 14 d. As shown in Figure S12,


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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 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 NapFFY hydrogel and other three hydrogels (SDF1/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 above five groups at day 1, 4, and 8. As shown in Figure S13, consistent with 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 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. Above results indicated that our hydrogels did not exert cytotoxicity on the BMSCs but stimulated their proliferation. To verify this, we first used 5-Ethynyl-2'-deoxyuridine (EdU) assay to label the proliferating BMSCs in 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-S15, the proliferating BMSCs in


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four NapFFY hydrogel groups were significantly more than those in Ctrl group (p < 0.05). Interestingly, there was no significant difference of proliferating BMSCs among the four hydrogel groups, echoing with 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 d and 14 d. 24transwell 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 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 NapFFY group and the Ctrl Group, suggesting NapFFY hydrogel itself could not stimulate the differentiation of BMSCs. Interestingly, although SDF-1 cannot directly stimulate the differentiation of BMSCs, ALP mRNA expression level of BMSCs in SDF-1/NapFFY group was close to that in BMP-2/NapFFY group (p > 0.05), significantly higher than that in Ctrl and NapFFY groups (p < 0.05). This could be explained that the chemotaxis of the BMSCs in 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. Above qPCR results of ALP mRNA expression


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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). 8-weekold male sprague dawley (SD) 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 micro-computed 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 (bone volume (BV)/total volume (TV) × 100%) of the defect areas were 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 w showed that their bone volumes slight increased by self-repairing. Compared with the low BV fraction (11.2  1.0%) in Ctrl group, BV fractions of maxillae in other four groups were obviously increased (21.4  1.5% for NapFFY group, 34.9  1.7% for SDF-1/NapFFY group, 36.6  2.0% for BMP-2/NapFFY group, and 56.7  2.2 % for 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 NapFFY group indicated that the hydrogel itself had a certain


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effect on bone defect repairing, which was consistent with above cytotoxicity results that NapFFY hydrogel itself could promote the proliferation of BMSCs. And the probable mechanisms are as follows: (1) the biomimetic 3D nanofiber network structures of NapFFY hydrogel could promote the attachment of BMSCs; (2) the low stiffness of NapFFY hydrogel could allow its network pore size responding to the mechanical force exerted by the cells when they migrate through the matrix,33 which facilitates the remodeling of ECM by BMSCs in the bone defect sites. Although certain amounts of bones were found formed in the defects of SDF-1/NapFFY and BMP2/NapFFY groups, central parts of their defect sites were still undergoing bone remodeling. But in SDF-1/BMP-2/NapFFY group, the regenerated bones were observed in the whole defect areas. Quantitatively, the BV fraction in SDF-1/BMP2/NapFFY group was 1.62-fold and 1.55-fold of that of SDF-1/NapFFY and BMP2/NapFFY groups, respectively. H&E and Masson's trichrome stainings provided more complementary details of the regenerative 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 at 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. Detailedly, in Ctrl group, the defects were almost filled with fibrous connective tissue (CT) and small new bone tissues were formed at the edges of host bones. Compared with Ctrl group, more new bone tissues, less but denser connective tissues at the defect areas were detected in all four hydrogel-treated groups. The most continuous, compact, and


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thickest bone tissues were formed at the bone defect areas of SDF-1/BMP-2/NapFFY group. These results suggested that synergistic, sustained release of SDF-1 and BMP-2 from supramolecular hydrogel could significantly promote the regeneration and reconstruction of bone tissues in the defect areas.

Figure 4. (A) Micro-CT images of the periodontal bone defect areas at maxillae in 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 were expressed as mean ± SD, n = 3. ***p < 0.001, compared with the Ctrl group. Not significant (ns): p > 0.05. Scale bar = 2 mm.


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Figure 5. Histological images of bone defect areas in decalcified maxillae 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) were shown at higher magnification in detail. Scale bars, 500 µm for 40 × and 100 µm for 200 ×.

CONCLUSIONS In summary, to effectively promote the regeneration and reconstruction of periodontal bone tissues, we used a well studied hydrogelator NapFFY to coassemble


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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 co-assembled 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/BMP2/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 area. 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 periodontal bone defect model in rats with SDF-1/BMP-2/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/BMP2/NapFFY hydrogel could replace bone transplantation in clinic for the repair of periodontal bone defect and periodontally accelerated osteogenic orthodontics (PAOO) in near future. Furthermore, by co-assembling with other bioactive factors (e. g., vascular endothelial growth factor or platelet derived growth factor), our hydrogel system may also have potential application in dental pulp regeneration. This type of work is underway.

EXPERIMENTAL SECTION


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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). 8-week-old male (weighting 180  ±  20  g) sprague dawley (SD) rats were selected 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 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 1st molar. Firstly, we made a 1 cm relieving incision on the mesial area and the gingival sulcus of the maxillary first molar, and evaluated


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mucoperiosteal flap. Then, removed bone tissue 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 mesial side of the exposed root (Figure S17). After the critical-sized defects were made, hydrogels were applied to fill these defects in treatment groups, and nothing was filled in the defect area in Ctrl group. Finally, replaced flaps and closed wound using 5-0 absorbable sutures. Micro-CT Analysis. At 8 week, 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 defects 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, scanned them using a micro-CT System (SCANCO Medical AG, Fabrikweg 2, CH-8306 Bruettisellen, Switzerland) at a pixel size of 15 μm. The micro-CT 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 semi-automatically and the BV fractions (bone volume (BV)/total volume (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 dehydrated in gradients of ethanol and embedded in paraffin. Later, the samples were sectioned in a sagittal plane throughout the entire mesial area


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of the 1st molars at a thickness of 5 μm sections to present the whole surgically areas. All the sections were stained with H&E staining and Masson’s trichrome staining. The slides were examined under a light microscope (IX71-400X, 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 one-way ANOVA, and p

Sustained Release of Two Bioactive Factors from Supramolecular

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