Functionalization of Novel Theranostic Hydrogels with

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Biological and Medical Applications of Materials and Interfaces

Functionalization of Novel Theranostic Hydrogels with Kartogenin Grafted USPIO Nanoparticles to Enhance Cartilage Regeneration Wei Yang, Ping Zhu, Huanlei Huang, Yuanyuan Zheng, Jian Liu, Longbao Feng, Huiming Guo, Shuo Tang, and Rui Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b12288 • Publication Date (Web): 02 Sep 2019 Downloaded from pubs.acs.org on September 2, 2019

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ACS Applied Materials & Interfaces

Functionalization of Novel Theranostic Hydrogels with Kartogenin Grafted USPIO Nanoparticles to Enhance Cartilage Regeneration

Wei Yang

a, 1,

Ping Zhu

b, 1,

Huanlei Huang b, Yuanyuan Zheng a, Jian Liu b, Longbao Feng c,

Huiming Guo b *, Shuo Tang d *, Rui Guo a *

a

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of

Biomedical Engineering, Jinan University, Guangzhou 510632, China b

Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong

Academy of Medical Sciences, Guangzhou 510100, China c

Beogene Biotech (Guangzhou) CO., LTD, Guangzhou 510663, China

d

Department of Orthopaedics, the Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen

517000, China

1

These two authors contributed equally to this work.

* Corresponding author Email: [email protected], [email protected], [email protected] Tel/Fax: +86-20-85222942

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ABSTRACT Here, kartogenin (KGN), an emerging stable nonprotein compound with the ability to promote bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into chondrocytes, was grafted onto the surface of modified ultrasmall superparamagnetic iron-oxide (USPIO), and then integrated into cellulose nanocrystal/dextran (CNC/Dex) hydrogels. The hydrogels served as a carrier for the USPIO-KGN and a matrix for cartilage repair. We carried out in vitro and in vivo studies, the results of which demonstrated that KGN undergoes long-term stable sustained release, recruits endogenous host cells, and induces BMSCs to differentiate into chondrocytes, thus enabling in situ cartilage regeneration. Meanwhile, the USPIO-incorporated theranostics hydrogels exhibited distinct magnetic resonance (MR) contrast enhancement, and maintained a stable relaxation rate, with almost no loss, both in vivo and in vitro. According to noninvasive in vivo observation results and immunohistochemistry analyses, the regenerated cartilage tissue was very similar to natural hyaline cartilage. This innovative diagnosis and treatment system increase the convenience and effectiveness of chondrogenesis. KEYWORDS: hydrogels, dextran, magnetic resonance imaging (MRI), kartogenin, cartilage regeneration


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INTRODUCTION Articular cartilage is an elastic tissue that covers the surfaces of bone joints and protects subchondral bone tissue.1 Cartilage defects are often caused by aging, obesity, or mechanical damage. Cartilage tissue is very poor at self-repair and regeneration due to the natural characteristics of avascular tissue, nerves, lymphoid tissues and its low cell density.2-4 Despite traditional therapies, including marrow stimulation, autografts, and matrix-induced autologous chondrocyte implantation, it is difficult to obtain regenerative cartilage that is similar to natural hyaline cartilage.5-6 Autografts cause new trauma and autologous chondrocyte implantation causes dysimmunity and fibrocartilage formation. Hence, many researchers have begun to focus on the preparation of scaffolds that stimulate cartilage regeneration by tuning their physicochemical properties and improving their biocompatibility.2, 7-8 However, sufficient regeneration is limited by the innate simple cellular characteristics of cartilage.9 Subsequently, ideas based on integrating chondrocytes with a three-dimensional scaffold in vitro and then implanting them into cartilage defects has been suggested.10-11 However, the regulation of the number of transplanted cells remains a problem although there will be no immunogenicity.12-13 Recently, stem cell therapy has been proposed as an emerging strategy for cartilage regeneration.14-15 Compared to chondrocytes, bone marrow-derived mesenchymal stem cells (BMSCs) have many advantages including facile extraction,16 differentiation into end-stage lineage cells,17 and immunoregulation capacity.18 However, the differentiation of BMSCs into chondrocytes requires the intervention of an important cytokine. Transforming growth factor-β3 (TGF-β3) is a promising factor that can induce the differentiation of BMSCs into chondrocytes.11 However, its short half-life means that it can only repair cartilage defects for 3 months.19 Due to



exogenous protein components, TGF-β is easily denatured and inactivated, leading to immunogenicity.20 Bone morphogenetic protein (BMP-9), which is considered the best potential chondrogenic bone morphogenetic protein, has been used for chondrogenesis in BMSCs. However, BMP-9 treatment mainly stimulates an increase in sulfated glycosaminoglycans, and does not increase mineralization.21 Kartogenin (KGN), a small, non-protein compound, is stable for storage and transportation at room temperature and can induce BMSCs differentiation into chondrocytes by adjusting the CBF-RUNX1 signaling pathway.22-23 Furthermore, it can recruit endogenous host MSCs for homing, thus achieving cartilage regeneration without cell transplantation.9 Despite the fact that KGN can be delivered into articular cavities via intra-articular injection, most of the KGN will be absorbed by the circulatory system.24 Hence, researchers have considered loading KGN into a three-dimensional scaffold to facilitate effective cartilage regeneration. For example, Chen et al. prepared KGN by incorporating it into a thermogel to obtain better-integrated and more smoothly regenerated cartilage, with almost no degeneration of the adjacent normal cartilage.11 Sun et al. coated KGN with poly (L-lactide-co-glycolide) PLGA microspheres, which they loaded into collagen-based porous scaffolds to enable effective KGN release for in vivo cartilage repair.25 Therefore, a sustained release system is necessary to reduce the KGN dose and loss, prolong the duration of the KGN activity, and match with the repair cycle required for damaged cartilage.26 To attenuate secondary surgical trauma, we could use noninvasive imaging modalities that provide useful feedback information about the regenerated tissue or implanted materials to monitor cartilage regeneration therapies.27-28 Meanwhile, noninvasive monitoring of in vivo dynamic distributions will help to reduce the number of unnecessary animal sacrifices at different


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points in time and provide enough data so that researchers can make the treatment more accurate and effective. Magnetic resonance imaging (MRI), which has deep penetration capabilities, has served as a safe and noninvasive tool for monitoring molecular changes, the absorption states of implanted materials, and the remodeling of regenerated tissue. It is widely and effectively applied in clinical investigations. In the past, MRI has been used to visualize and assess tissue-engineered constructs due to its superior distinguishing capabilities for soft tissue.27 Recently, the enhancement of imaging contrast by incorporating various magnetic materials, such as gadolinium or iron oxide, into chondrocytes and MSCs has enabled researchers to detect scaffolds and regenerated cartilage and visually localize cells and scaffolds based on particle-labeling techniques.29-31 In our previous research, USPIO labeled collagen scaffolds with KGN were prepared and characterized in vitro.32 We found that the labeled composite scaffolds obtained stable imaging ability and favorable cytocompatibility. The sustained release of KGN can promote the proliferation and migration of BMSC, and moreover up-regulate the expression level of cartilage-specific genes. But SOX9 and COL1A1 genes showed higher expression, which could cause a tendency that BMSC was induced differentiation for the formation of fibrotic cartilage instead of hyaline cartilage. Furthermore, CNC enhanced collagen scaffold with higher mechanical properties could cause mechanical stress damage in vivo. Dextran is a natural polysaccharide with structure similar to chondroitin sulfate, which can obtanin high water absorption, good biocompatibility, and provide a suitable ECM environment after crosslinking to form hydrogels.33 The hydrogels can serve as candidate material better for further study of cartilage regeneration mechanism in vivo.



Based on the background presented above, we propose a novel strategy of grafting KGN onto the surface of USPIO, then incorporating it into cellulose nanocrystal/dextran (CNC/Dex) hydrogels. The hydrogels serve as a microenvironmental matrix to support the subsequent sustained release of the hydrophobic drug. The prepared functional system can initiate BMSCs homing in the host, and thus cell differentiation without cell transplantation. Moreover, USPIO-labeled hydrogels provide a stable imaging contrast for in vitro MRI; indeed, in R2 relaxometry analyses, changes in the R2 value were reflected by the consistent degradation of the corresponding hydrogels. Furthermore, we used a rabbit defect model to successfully demonstrate noninvasive monitoring of USPIO-labeled hydrogels. The monitoring results were consistent with the observed neo-cartilage morphology and degradation of hydrogels, which we evaluated using histological and pathological analyses (Figure 1A).

Figure 1. Characterization of physicochemical properties of CNC/Dex/USPIO-KGN hydrogels in vitro. (A) Schematic illustration of preparation and utilization of USPIO-labeled Dex/CNC/USPIO-KGN hydrogels for engineering artificial cartilage repair. (B) TEM images and (C) DLS of CNC. (D) FTIR characterization of the USPIO-KGN and modified dextran (M-Dex) hydrogels. (E) Scanning electron microscopy observations of dextran hydrogels with the various CNC. (a, 0%), (b, 1%), (c, 3%), (d, 5%) and (e, 7%). The scale bar indicates 20 μm.


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RESULTS AND DISCUSSION USPIO, with good biocompatibility, has been widely applied to stem cell tracking and cancer treatment.34-35 Owing to the abundance of hydroxyl groups on the surface of USPIO, it is widely used as a drug carrier for tissue engineering after surface modification. In this study, USPIO was prepared using a precipitation method.36 Successful synthesis was demonstrated by the absorption bands at 457 cm-1 and 637 cm-1, which were attributed to Fe-O stretching vibrations, 798 cm-1 and 1217 cm-1 due to Si-O stretching vibrations, and 1535 cm-1 from NH2 stretching.37 Fe3O4 nanoparticles (NPs) were coated with (3-aminopropyl) trimethoxysilane (APTES). The KGN absorption peak at 1710 cm-1 reflected that the carboxyl group appeared in the Fe3O4@SiO2-KGN, and the absorption bands at 1535 cm-1 was reduced, demonstrating that the KGN was conjugated with Fe3O4@SiO2-NH2 (Figure 1D). According to the EDX results of Fe3O4 (Figure S2C), we observed significant Fe-K and O-K peaks corresponding to an atomic ratio of approximately 3/4 (Fe/O) (Table S1). While coated with SiO2, Fe3O4@SiO2-NH2 exhibited a distinct Si-K peak (Figure S2D). According to transmission electron microscopy (TEM), the SiO2-coated Fe3O4 had a diameter of 14  2 nm (Figure S2A). Interestingly, Fe3O4@SiO2-NH2 NPs could uniformly disperse for a period after ultrasonication in the aqueous phase, without intervention using a magnetic field, but the dispersed magnetic NPs completely aggregated after the strong magnetic field was applied (Figure S2B). The results vibrating sample magnetometry (VSM) showed that Fe3O4 and Fe3O4@SiO2-NH2 NPs had coincident magnetization curves and almost no hysteresis, coercive force, or remanence, all of which were approximately zero. We observed a symmetric “S” type magnetization curve, indicating that the prepared Fe3O4; Fe3O4@SiO2-NH2 NPs were super-paramagnetic.38 Compared to that of Fe3O4, the magnetic saturation strength of



Fe3O4@SiO2-NH2 decreased slightly, which could have been caused by the encapsulation of SiO2. CNC, which has good biocompatibility and biodegradability and is inexpensive, is one of the most widely available natural resources.39 Due to its excellent mechanical properties, it is widely used as nanofiller. In a previous study, we enhanced different scaffolds with CNC, then used them for biomedical and tissue engineering applications.40-41 The rod-shaped CNC NPs with lengths of 87 ± 14 nm and diameters of 7.3 ± 1.8 nm were prepared using a previously reported acid hydrolysis method (Figure 1B-C).40 Dex, which has excellent biocompatibility and nontoxicity and no immunogenic properties, has been investigated as a potential hydrogel for repairing cartilage.4, 8, 42 Based on the results of those studies, we designed CNC-enhanced Dex hydrogels. Water-soluble linear Dex was cross-linked with sodium trimetaphosphate (STMP) to obtain a hydrogel structure, as previously reported.43-44 According to the reaction mechanism of STMP, the final product were crosslinking dextran and nontoxic, well-known sodium phosphates (Figure S1). Compared to pure Dex without treatment, some absorption bands at 1100-1300 cm-1, which we ascribed to P=O stretching vibrations, appeared in the cross-linked Dex.45-46 This demonstrated that the Dex was successfully cross-linked (Figure 1D). Incorporated CNC was used to enhance the hydrogel structure. According to SEM results (Figure 1E), cross-sections of CNC/Dex hydrogels with 1%, 3%, and 5% CNC contents exhibited mesh pore interconnectivity with similar pore sizes and porosities. For example, the pore size and porosity were 45  11 μm and 92.8  8.7%, respectively, in the case of 1% CNC/Dex hydrogels, 45  13 μm and 90.5  6.9% for 3% CNC/Dex hydrogels, and 47  9 μm and 92.6  6.3% for 1% CNC/Dex hydrogels (Table S2) with a smooth pore wall, but the 7% CNC/Dex hydrogels had rough pores and agglomerated pore walls, which reduced the


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porosity to 87.3  5.8%. This may have been due to the excessive dispersion of CNC, which was difficult to disperse uniformly in Dex solution due to its high viscosity, thus causing accumulation inside pores. According to the swelling results (Figure S3A), CNC/Dex hydrogels with various CNC contents were immersed in aqueous solution and had almost reached equilibrium after 1 h. The swelling rate increases significantly with the CNC content. Representative stress–strain curves indicated that pure Dex hydrogels exhibited lower fracture stress, which was improved by incorporating the CNCs (Figure S3B). For instance, the stress at maximal strain was 264 KPa for M-Dex hydrogels, 414 KPa for 1% CNC/Dex hydrogels, 481 KPa for 3% CNC/Dex hydrogels, 537 KPa for 5% CNC/Dex hydrogels, and 648 KPa for 7% CNC/Dex hydrogels. These results demonstrate that the incorporation of CNC enhanced the mechanical performance of the hydrogels. The effects of CNC on CNC/Dex were evaluated by thermogravimetric analyses (TGAs) (Figure S3C). All the samples were thermally degraded through three steps. First, pure CNC was thermally degraded at high temperatures and modified Dex (M-Dex) was degraded at low temperatures. In the case of temperatures ranging from 0°C to 100°C, the initial mass loss was attributed to water loss. We observed a sharper decrease in the weight for temperatures between 250 °C and 375 °C, corresponding to the thermal decomposition of the hydrogels. The last thermal process involved the elimination of carbon material. We also evaluated the degradation performance of CNC/Dex hydrogels (CNC, 5% [w/w]) in a phosphate-buffered saline (PBS, pH 7.4) and trypsin environment (Figure S3D). Cartilage regeneration is a prolonged process due to its lack of healing ability.6 It is imperative to match the rate of degradation of the filling nutrition matrix with the rate of cartilage regeneration. Furthermore, the controlled degradation of hydrogels is vital for drug release, as the degradation



rate can disrupt the release of the encapsulated drug from the hydrogel structures. Over time, the CNC/Dex hydrogels degraded in PBS, which may have been due to the favorable hydrophilicity of the linear Dex itself. However, the degradation rate was significantly accelerated in a trypsin environment, which may have been due to the enzyme causing the long-chain molecules to be broken into small molecules, thus accelerating the degradation process. The degradation rate decreased on the 10th day and the mass residue remained at 42.32% at the 28th day, indicating that the prepared CNC/Dex hydrogels were degradable. We also investigated the rheological performance of the CNC/Dex hydrogels with different CNC contents after incubation in a PBS and trypsin environment for 28 days (Figure 2A-D). We found that the saturation values of G’ and G’’ were positively correlated with the CNC content, but saturation was lower in the trypsin environment than in the PBS environment. These results indicate that the CNC/Dex hydrogels degraded more and faster in trypsin. Based on physicochemical results, 5% CNC enhanced CNC/Dex hydrogels with the compressive modulus near to the natural cartilage, 6 served as the optimal content for further research.

Figure 2. In vitro properties characterization of CNC/Dex/USPIO-KGN hydrogels with different content of USPIO. (A) Storage modulus (G’) against frequency of enzyme-degraded CNC/Dex hydrogels. (B) Storage modulus (G’) against frequency of PBS-degraded CNC/Dex hydrogels. (C)


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Loss modulus (G’’) against frequency of enzyme-degraded CNC/Dex hydrogels. (D) Loss modulus (G’’) against frequency of PBS-degraded CNC/Dex hydrogels. (E) Scanning electron microscopy (SEM) observations demonstrating the interconnected structures of the Dex/CNC/USPIO-KGN hydrogels. The scale bar indicates 50 μm. Prussian blue staining shows the distribution of USPIO within the hydrogels. The scale bar indicates 50 μm. T2WI indicated increasing signal contrast with USPIO content. USPIO has been applied as an emerging MRI contrast agent due to its penetration depth and high spatial resolution, without ionizing radiation, thus providing both biocompatibility and effectiveness.47 USPIO-KGN conjugation ability was assessed using ultraviolet-visible (UV-vis) spectrophotometry (Figure S4A). We observed the maximum absorption peak at a scanning wavelength of 287 nm. Prior to adding USPIO, the maximum absorption peak was at 0.8986, whereas it was 0.7528 after adding USPIO. We calculated that the conjugated KGN contributed approximately 16% to the absorption. The USPIO optimization of CNC/Dex/USPIO-KGN hydrogels was determined by structural analyses using Prussian blue for MRI. According to gross observations (Figure 2E), the dry hydrogels were uniformly faint yellow, indicating that the USPIO-KGN was uniformly dispersed in the hydrogels. The presence and distribution of USPIO was also assessed using Prussian blue staining. The SEM results showed that pore size (51  9.2 μm) and porosity were similar across every group, regardless of the amount of USPIO, as previously reported.47 Efficient MRI visualization was investigated by passively incorporating between 0.06% and 0.3% USPIO into hydrogels. The composite hydrogels were visually assessed using T2-weighted imaging (T2WI). T2* and T2 mapping sequences, corresponding to R2* and R2, respectively,



were calculated based on the relaxation time (Figure 3A). USPIO concentrations ranging from 0.06–0.3% did not cause sensitive artifacts, but 0.06% USPIO exhibited a hyperintense signal, whereas the hyperintense signal gradually became hypointense as the USPIO content increased. The hydrogels with 0.3% USPIO exhibited an obvious dark border. The corresponding R2 and R2* values were also positively correlated with the increase in the amount of USPIO. Previous reports have indicated that a USPIO content of more than 25 μg Fe/mL inhibits chondrogenesis.48 To ensure clear MR imaging, 0.1% USPIO was considered the optimal concentration for further research. We evaluated the KGN release behavior of loading KGN-USPIO into CNC/Dex hydrogels (Figure S4B). Compared to CNC/Dex/KGN, CNC/Dex/USPIO-KGN released less KGN after the same length of time, with no obvious burst release. These observations can be explained by the USPIO breaking from the amide bond under the action of water molecules, then breaking through the hydrogel barrier to enter the environmental liquid. The long-term presence of residual hydrophobic KGN in the hydrogels caused a low burst release. The KGN release from the CNC/Dex/USPIO-KGN hydrogels reached 32% after 144 h. Gu et al. coated KGN with poly(lactic-co-glycolic acid), which was loaded onto photo-cross-linkable acrylate hyaluronic acid hydrogels. They obtained a KGN release of about 20% on the 15th day, thus promoting effective cartilage regeneration.9 Chen et al. prepared KGN-incorporated thermogels with a KGN release of approximately 32% after 144 h, supporting BMSCs for remarkable cartilage regeneration.11


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B

100

C 2.0 1.8

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1.4 1.2

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R2*/S

-1

-1

8 R2/S

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6 4

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CNC/Dex CNC/Dex/KGN KGN

E

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Figure 3. Characterization of physicochemical properties and biocompatibility of CNC/Dex/USPIO-KGN hydrogels in vitro. (A) Calculated R2* and R2 relaxometry rates increased linearly with USPIO content. (B) Hemolysis indicates that Dex/CNC/USPIO-KGN hydrogels have regular blood compatibility. (C) Cell proliferation results show that the Dex/CNC/USPIO-KGN supported the growth of BMSCs without toxicity. (D) Cell morphologies seeded on different hydrogels for 2 days, visualized by scanning electron microscopy (SEM). (a) CNC/Dex, (b) CNC/Dex/USPIO, (c) CNC/Dex/KGN, (d) CNC/Dex/USPIO-KGN. Scale bar indicates 50 μm. (E) Live/dead assay was used to evaluate cell survival on day 2. (a) The control group, (b-f) CNC/Dex, CNC/Dex/KGN, CNC/Dex/USPIO, KGN, CNC/Dex/USPIO-KGN, respectively. The scale bar indicates 100 μm. (F) Cell migration with KGN (a-c) and without KGN (a’-c’). (a, 0h), (b, 12h), (a, 24h). The scale bar indicates 100 μm. Hemolysis results from red blood cells (RBCs) rupturing to escape hemoglobin. Mature human RBCs are biconcave disks that lack cell nuclei and organelles and are sensitive to membrane-active materials. For our hemolysis experiment (Figure 3B), the hemolysis rate in all the hydrogels increased slowly over time, but less than 5%. This did not cause significant cytolytic rupture, as previously reported.49 These results can be attributed to the fact that both CNC and Dex



are natural biocompatible polysaccharides, and Dex has a similar structure to chondroitin sulfate,50 which can adapt to blood. On the other hand, the interaction between RBCs and extraneous polymers was mainly driven by electrostatic attraction between positive charges of polycations and negative charges at the surfaces of the RBCs, or the hydrophobic interaction between the lipid bilayer of the RBC membrane and hydrophobic groups of amphiphilic polymers, or the van der Waals’ forces and hydrogen bonds.49 CNC prepared by sulfuric acid hydrolysis was negatively charged due to the sulfonic acid group on the surface,51 which adsorbs few RBCs, thus preventing aggregation. We investigated BMSCs proliferation on hydrogels using a Cell Counting Kit-8 (CCK-8) assay. In general, the results always showed high viability in all samples (Figure 3C). No significant differences appeared on days 1 and 4, but CNC/Dex/USPIO-KGN was more viable than the control group after 7 days. This can be attributed to the fact that CNC/Dex/USPIO-KGN provided a more favorable growth environment for BMSCs. For the BMSCs live/dead staining assay (Figure 3E), we found that BMSCs maintained enough viability after 2 days of culture in all groups, with almost all cells remaining alive (green) and few dead cells (red). These results are consistent with the CCK-8 results. A previous study indicated that KGN is not toxic to BMSCs and chondrocytes at high concentrations (100 mM) and protects existing chondrocytes.52-53 Jiang et al. injected KGN into full-thickness cartilage defects to initiate remarkable defect filling and increase hyaline-like cartilage formation.22 Li et al. investigated the effects of KGN at various concentrations on fibroblasts and observed it had no evident impact on fibroblast viability.54 Yi et al. prepared KGN-conjugated chitosan-hyaluronic acid hydrogels, and subsequent continuous KGN release in hydrogels promoted adipose-derived stem cell proliferation.55


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We assessed the cell adhesion and morphological properties of the hydrogels using SEM (Figure 3D). The BMSCs adhered to all the hydrogel surfaces. Interestingly, it was found that some cells spread on the wall with visual and filamentous ECM like cell pseudopod in the CNC/Dex/USPIO-KGN group after culturing for 2 days. Cells on the CNC/Dex, CNC/Dex/USPIO hydrogels were round and partially attached to the hydrogels. In cell migration experiments, BMSCs migrated faster after KGN stimulation than they did without KGN stimulation in a wound-healing model. This suggests that adding KGN to hydrogels may attract host BMSCs to enhance hydrogel–host tissue integration in vivo. These results are consistent with previously reported results, that the released KGN in photo-cross-linked scaffolds achieves cartilage regeneration without BMSCs transplantation (Figure 3F).9 These results suggest that CNC/Dex/USPIO-KGN exhibits good biocompatibility properties and can promote in vivo migration and homing of BMSCs. The imaging stability of USPIO-labeled CNC/Dex/USPIO-KGN hydrogels after coculture with BMSCs on days 1, 7, 14, and 21 was evaluated with T2W1, T2 mapping, and T2* mapping scanning sequences. There were no remarkable differences in the PBS over time (Figure S5A). However, the shadow shrunk after coculture with BMSCs, indicating that the hydrogels could be degraded due to enzymes secreted by BMSCs. The relaxation rate was directly proportional to the amount of iron and inversely proportional to the calcium concentration, thus degradation of the hydrogels reduces the relaxation rate (Figure S5B). This demonstrates the effectiveness of noninvasive MRI monitoring of hydrogel degradation in vivo. Next, we evaluated the morphologies and secreted BMSCs matrices seeded in hydrogels for 14 days (Figure S6). Hematoxylin and eosin (H&E) staining showed that the amounts of BMSCs



in the CNC/Dex/KGN and CNC/Dex/USPIO-KGN hydrogels appeared in clusters within the mesh interstices, more so than CNC/Dex and CNC/Dex/USPIO hydrogels. This difference was consistent with the difference in cell proliferation on day 7. In the same way, the Toluidine-blue and Safranin O staining observations revealed more secreted matrix in mesh pores in CNC/Dex/USPIO-KGN hydrogels than in CNC/Dex and CNC/Dex/USPIO hydrogels. BMSCs differentiation was observed by Alcian blue staining. CNC/Dex/KGN and CNC/Dex/USPIO-KGN groups exhibited similar degrees of differentiation, which were significantly larger than the others. The amount of secreted COL1A2, which was vital to the synthesis of hyaline cartilage, was positively correlated with the secreted matrix. Periodic acid-schiff (PAS) staining showed that similar amounts of glycosaminoglycan (GAG) were secreted by each group. These results indicate that the prepared CNC/Dex/USPIO-KGN hydrogels were nontoxic and that the KGN promoted aggregation and differentiation of BMSCs. We assessed the gene expression levels of BMSCs cultured within hydrogels (Figure S7). After 14 days of induction, BMSCs in CNC/Dex/USPIO-KGN hydrogels exhibited significantly higher expression of chondrogenic markers than hydrogels without KGN. For example, mRNA expression in CNC/Dex/USPIO-KGN hydrogels was enhanced by about 214.2  39.5%, 308.2  31.5%, and 197  10.3% for aggrecan, COL1A2, and SOX 9 compared to that of the CNC/Dex groups at day 14, respectively. The expression levels of COL1A1 were similar to those of other groups, with a low value. This indicates that CNC/Dex/USPIO-KGN hydrogels promote the hyaline cartilage phenotype instead of fibrocartilage and maintain chondrocytes in a matrix-producing phenotype. Inspired by the in vitro efficiency of promoting hyaline cartilage and stable USPIO-labeled


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hydrogels imaging features, we further examined the diagnosis effect of CNC/Dex/USPIO-KGN hydrogels in vivo. Samples of rabbit articular cartilage were harvested during the 6th and 12th weeks and photographed (Figure 4A). The CNC/Dex/USPIO-KGN group had the best cartilage regeneration, and the defect was mostly repaired after the 6th week. Regenerated cartilage-like tissue with smooth surfaces integrated into the adjacent host cartilage without boundaries was observed in the 12th week, indicating the inhibition of normal cartilage degeneration that results from defects. We attributed this to the protective effects of the released KGN. Untreated cartilage contained obvious defects at week 12, with no new tissue. This was related to fact that the untreated cartilage lacked the innate ability to mount enough healing response. In the CNC/Dex/USPIO group, the defects contained thick and irregular neocartilage by the 6th week and were mostly filled with white neocartilage by the 12th week. These results demonstrate that CNC/Dex/USPIO promotes cartilage repair and that KGN accelerates the process of cartilage regeneration. According to International Cartilage Repair Society (ICRS) grades, which are based on macroscopic observations, the untreated group was severely abnormal (Grade IV), the CNC/Dex/USPIO groups were abnormal (Grade III), and the CNC/Dex/USPIO groups were nearly normal (Grade II) at 6 weeks. After 12 weeks, the untreated group was severely abnormal (Grade IV), and the other two groups were both abnormal (Grade II) (Figure S9).



Figure 4. Characterization of the rabbit cartilage defect model experiment. (A) Observations of cartilage harvested during weeks 6 and 12 (black circle: degenerated cartilage). The scale bar indicates 4 mm. (B) MR 3D_WATSc for neocartilage assessment at 3, 6, 9 and 12 weeks after surgery. (C) MR PDWI was used for the morphological observations. (D) Semiquantitative R2* and R2 relaxometry rate of the different hydrogels. The degradation of hydrogels will be accelerated by the complex body fluid environment and will cause KGN and USPIO release. Noninvasive MRI monitoring of USPIO-labeled hydrogels can be used to assess the degree of degradation residue and the relative amount of KGN released. USPIO with good biocompatibility and no cytotoxicity can effectively enhance MR contrast. USPIO-labeled hydrogels were visualized sensitively using MRI. Proton-density-weighted imaging (PDWI) and T2* and T2 sequence scans were performed using MRI in the 3rd, 6th, 9th, and 12th weeks after transplantation surgery. We found that the MR signals in the


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CNC/Dex/USPIO and CNC/Dex/USPIO-KGN groups decreased gradually over time. However, no significant differences were observed in the untreated group. The contrast signal of CNC/Dex/USPIO-KGN was lower than that of the CNC/Dex/USPIO group in the 9th and 12th weeks (Figure 4B). Both the R2 and R2* values increased over time in the untreated group, but decreased and then increased in the CNC/Dex/USPIO and CNC/Dex/USPIO-KGN groups (Figure 4D). Because of incorporated USPIO on the accumulation of calcium, we inferred that the untreated group accumulated calcium deposits. The USPIO detected in the treated groups was reduced by the degradation of the USPIO-labeled hydrogels. We attributed this to the fact that the absorption of sodium chloride increased the R2 and R2* values prior to surgery, which the body fluids then replaced, leading to the observed decrease in sodium chloride.47 The increase mainly resulted from the later-stage accumulation of calcium. This may have been due to parts of the hydrogels remaining in the defect due to their partial degradation after the 6th week. MR water-selective cartilage scan (3D_WATSc) sequences, which can be used to obtain high signal contrast with a short scanning time,56 were carried out to noninvasively monitor the neo-cartilage morphology after surgery (Figure 4C). In the untreated group, the hyperintense regions were maintained over time, which we attributed to the accumulation of tissue fluid at the defect site, as observed in the gross morphology. In the CNC/Dex/USPIO group, we observed that thin neocartilage had extended into the defect alongside the adjacent normal cartilage by the 12th week. In the CNC/Dex/USPIO-KGN group, hyperintense regions resulting from neocartilage formation were observed at the edge of the defect in the 6th week, and a similar morphology to the adjacent normal cartilage was observed in the hypointense region in the 9th and 12th weeks. This indicates that the hydrogels may have degraded completely and the released USPIO was absorbed



by the tissue.

Figure 5. Post-surgery histological assessment, after 6 and 12 weeks. H&E staining shows that the neocartilage integrated with the adjacent normal cartilage. (N: normal cartilage; R: repair cartilage; the arrows indicate the margins of the normal cartilage and repaired cartilage). Masson staining was used to visualize the deposited collagen. Toluidine blue staining and Safranin O staining shows deposited cartilage-specific matrix. Prussian blue staining shows the distribution of USPIO in tissue. (The red arrows indicate USPIO). The expression of vital cartilage proteins including SOX9 and aggrecan is revealed by immunohistochemical staining. PAS staining images show cartilage-deposited polysaccharides. All scale bars indicate 200 μm. We further examined the therapeutic efficacy of CNC/Dex/USPIO-KGN hydrogels by the typical tissue section staining assay (Figure 5). At the 6th week, H&E staining showed that almost no cartilage regeneration had taken place in the defect of the untreated group. In the


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CNC/Dex/USPIO group, the defect section was partially integrated with discontinuous neocartilage, and there were partially degraded hydrogels. The cartilage matrix is shown by the Toluidine blue and Safranin O staining. A small amount of collagen, which we stained with Masson, glycosaminoglycan, and SOX9, indicated that cartilage matrix production was underway. However, in the CNC/Dex/USPIO/KGN group, the defect contained abundant cartilage matrix, including collagen, SOX9, aggrecan, and polysaccharide, which were almost continuously integrated with the adjacent normal cartilage, which was repaired better than the CNC/Dex/USPIO group. In the 12th week, the H&E staining showed that the untreated group still had a discontinuous section, and the defect was filled with thin, fibrous cartilage tissue. In the CNC/Dex/USPIO group, the Toluidine blue and Safranin O staining showed that more cartilage matrix had grown, and there was more aggrecan and SOX9 polysaccharide, which are vital cartilage components, than in the 6th week. Despite the cartilage being more regenerated than in the

untreated

group,

there

were

still

discontinuities

in

the

neocartilage.

In

the

CNC/Dex/USPIO-KGN group, the continuous neocartilage was similar to and well-integrated with the normal cartilage. The Toluidine blue and Safranin O staining showed that there was an abundant cartilage matrix, and the arrangement of the collagen was consistent with that of normal cartilage. No residual hydrogels were observed, which indicated that the hydrogels were almost completely degraded. According to the Prussian blue staining, USPIO was observed across the entire group in the 6th week, but less residual USPIO remained during the 12th week. We attributed this to the degradation of the hydrogels. The pathology results were consistent with the MRI results. Hence, we can noninvasively monitor the degradation of USPIO-labeled hydrogels and neocartilage regeneration in real time by MRI tool. Meanwhile, we investigated the inflammatory



response of the cartilage defect location after implantation surgery and found no obvious signs of inflammation in any group (Figure 6A), in confirmation of the quantification of inflammatory factors within defect (Figure 6B-C). After initially increasing within the acute period (1 week), inflammatory factors (IL-1, TNF-) were maintained at a very low-level during repair. These results demonstrate that implantation of the functionalized hydrogels did not induce inflammation or a rejection reaction. The CNC/Dex/USPIO-KGN group had a significantly higher reduced modulus and hardness, with a value close to the normal cartilage (Figure 6D-E). The above results indicated that the CNC/Dex/USPIO/KGN supported effective cartilage regeneration.

Figure 6. Inflammation and mechanical property evaluation of repaired cartilage. (A-C) Evaluation of inflammation: (A) Histological appearance of the neo-cartilage at 6 and 12 weeks .


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The entire photographs were corresponding to the implant tissue. (B) Content of IL-1 in joint fluid; (C) Content of TNF- in joint fluid. The biomechanical properties of repaired cartilage in different groups: (D) reduced modulus and (E) hardness. Recently, the limited integration of repaired hyaline cartilage and adjacent normal tissue has presented a challenge to researchers.57 Discontinuous cartilage surfaces cause high peak forces that can lead to cartilage degeneration.58 This could cause serious matrix damage in the treated joint compartment, thus damaging the entire knee and inducing contralateral compartments and lesions.59 The prepared CNC/Dex/USPIO-KGN hydrogels induced a higher degree of cartilage repair, with fewer tissue borders than the control group in the 12th week. Surprisingly, the original defect vacancy always remained present in the untreated group, indicating that the released KGN promoted BMSCs differentiation and even exhibited chondro-protective effects in the CNC/Dex/USPIO-KGN hydrogels. In vitro evidence indicated that the expression levels of the cartilage matrix genes encoding COL1A2, SOX9, and aggrecan were higher in the KGN group than in the other groups, whereas there were no remarkable differences in the COL1A1 expression levels across all groups. The released KGN induced the BMSCs to differentiate into chondrocytes and mainly promoted the hyaline cartilage matrix phenotype. After in vivo implantation, the cartilage matrix gene was clearly expressed at 6 and 12 weeks (according to SOX9, aggrecan, and Masson staining), which potentially improved the quantity and quality of repaired tissue at the chondral interface. These observations were consistent with the in vitro gene expression results. CONCLUSIONS In conclusion, we successfully prepared CNC/Dex/USPIO-KGN hydrogels with good mechanical strength, long-term sustained KGN release, and stable MR imaging capabilities in



vitro and in vivo. We controlled their properties using CNC and USPIO. Furthermore, the hydrogels recruited host BMSCs without cell transplantation, thus promoting continuous hyaline cartilage regeneration. The results of real-time noninvasive MRI monitoring of in vivo hydrogel degradation and neocartilage morphology were consistent with histological results. Our pathological results showed that CNC/Dex/USPIO-KGN produced the highest level of cartilage matrix deposition and the most effective integration between all the groups. The combined diagnostic and treatment design provide a promising method for future cartilage regeneration research. Supporting Information Synthesis of CNC, amino-functionalized USPIO nanoparticles, materials and methods, and additional data ACKNOWLEDGEMENTS This study was supported financially by the Natural Science Foundation of China (No.81570279, No.81974019, No. 81573708 and No. 31271019), the National Key Research and Development Program of China (2018YFA0108700 and 2017YFA0105602), the Science and Technology Program of Guangzhou (No. 201601010270, No. 2017010160489, No. 201704030083, No. 201907010032, No. 201907010037, No. 805212639211), the Pearl River S&T Nova Program of Guangzhou (No. 201710010155, No. 201806010072), and the Science and Technology Project of Guangdong province (No. 2015A010101313, No. 2017A050506011, No. 2017A030312007, No. 2017A050501013, No. 2017B090911012, No. 2018A050506040, No. 2018A050506019, No. 2018A050506021). The Special Project of Dengfeng Program of Guangdong Provincial People's Hospital.


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TOC Graphic


Functionalization of Novel Theranostic Hydrogels with

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