Photo-Cross-Linked Scaffold with Kartogenin-Encapsulated

Jan 12, 2016 - ... by its lack of innate abilities to mount a sufficient healing response. .... Hong Chen , Feng Jia , Chengcheng Zhu , Jumei Xu , Xia...
0 downloads 0 Views 8MB Size
Download PDF
Photo-Cross-Linked Scaffold with KartogeninEncapsulated Nanoparticles for Cartilage Regeneration Dongquan Shi,†,‡ Xingquan Xu,†,‡ Yanqi Ye,#,§ Kai Song,†,‡ Yixiang Cheng,∥ Jin Di,#,§ Quanyin Hu,#,§ Jianxin Li,⊥ Huangxian Ju,⊥ Qing Jiang,*,†,‡ and Zhen Gu*,#,§,¶ †

The Center of Diagnosis and Treatment for Joint Disease, Drum Tower Hospital, Medical School, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Zhongshan Road 321, Nanjing 210008, Jiangsu China ‡ Joint Research Center for Bone and Joint Disease, Model Animal Research Center (MARC), ∥School of Chemistry and Chemical Engineering, and ⊥State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, Jiangsu China # Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States § Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ¶ Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, United States S Supporting Information *

ABSTRACT: The regeneration of cartilage, an aneural and avascular tissue, is often compromised by its lack of innate abilities to mount a sufficient healing response. Kartogenin (KGN), a small molecular compound, can induce bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. The previous in vitro study showed that kartogenin also had a chondrogenesis effect on synovium derived mesenchymal stem cells (SMSCs). Herein, we present the effect of an ultraviolet-reactive, rapidly cross-linkable scaffold integrated with kartogenin-loaded nanoparticles using an innovational one-step technology. In vivo studies showed its potential role for cell homing, especially for recruiting the host’s endogenous cells, including BMSCs and SMSCs, without cell transplantation. Of note, the regenerated tissues were close to the natural hyaline cartilage based on the histological tests, specific markers analysis, and biomechanical tests. This innovative KGN release system makes the chondrogenesis efficient and persistent. KEYWORDS: drug delivery, tissue engineering, hydrogel, regenerative medicine, nanoparticles

C

physicochemical properties of the cellular scaffolds, including tuning chemical composition, nano- or microscaled morphology, and light-reactive properties, in order to improve cell behaviors such as cell attachment, proliferation, differentiation, and extracellular matrix (ECM) secretion.4−7 Recently, a small molecular organic compound, kartogenin (KGN), was found to induce the bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes.8 KGN stimulates runt-related transcription factor (RUNX) family members, mainly RUNX1 expression,8,9 which has been shown to play a critical role in chondrogenesis, chondrocyte proliferation, and survival.9,10

artilage, an aneural and avascular tissue, lacks innate abilities to mount a sufficient healing response and is difficult to regenerate. Traditional therapies include marrow stimulation (microfracture), autografts as autologous chondrocyte implantation (ACI), and matrix-induced autologolus chondrocytes implantation (MACI).1 However, it remains challenging to get natural hyaline cartilage with normal anatomy and function in clinical application.2 Repaired cartilage lacks the appropriate mechanical properties and zonal organization of intact cartilage, which may subsequently lead to further degeneration. Even when the cartilage is well prepared, the autograft is dependent on sufficient integration and additional defects.3 Attempts have been made to repair cartilage through restoration of the hyaline cartilage, but there is still no consensus on an ideal therapy.2 To date, researchers have investigated a dozen of methods to optimize the © XXXX American Chemical Society

Received: October 22, 2015 Accepted: January 8, 2016

A

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

www.acsnano.org

Article

ACS Nano

Figure 1. (A) Schematic of KGN-loaded PLGA nanoparticles, molecule structures of KGN, and acrylated hyaluronic acid (m-HA). (B) Schematic of the surgical procedure for the cartilage defects repair. (C) Schematic of the hyaline cartilage chondrogenesis with photo-crosslinked HA scaffold encapsulated with KGN-loaded nanoparticles.

that the group treated with KGN only could not form a smooth, tightly packed pellets structure (Figure 2B), indicating that KGN alone failed to induce chondrogenesis. Interestingly, in the group pretreated with TGF-β3 for 1 week and then induced by KGN for 2 weeks, an increase in pellet size was observed (Figure 2C), suggesting that KGN could induce human SMSCs into chondrocytes under specific early phase conditions. Human SMSCs exposed to TGF-β3 + BMP-2 showed a slow increase of the pellet size as well as an accumulation of the extracellular matrix (Figure 2C) while the addition of KGN could accelerate the pellet growth. We also validated that the combination of TGF-β3 + BMP-2 + KGN resulted in the largest pellet size (Figure 2C). After sectioning the pellets, we performed the Hematoxylin and Eosin (H&E) staining and immunofluorescence staining. H&E staining showed that there was no cartilage-like tissue in the control (Figure 2D1), or KGN-alone group (Figure 2D2). The lacunalike structures were observed in the center of the pellet sections in these two control groups. Interestingly, a few clusters of cartilage-like tissue were observed in the TGF-β3 + KGN group (Figure 2D3) and the TGF-β3 1 week + KGN 2 weeks group (Figure 2D4), which indicated that KGN supplementation could enhance chondrogenic induction. Groups supplemented with KGN had less lacuna-like structures in the pellet sections. In addition, the large areas of cartilage-like tissue in the TGF-β3 + BMP-2 group (Figure 2D5) and the TGF-β3 + BMP-2 + KGN group (Figure 2D6) were also observed. Moreover, the cartilage showed a larger quantity and a more uniform distribution in the latter group than the former one. Furthermore, we detected a strong expression of collagen type II in the TGF-β3 + BMP-2 + KGN group (Figure 2E) and the TGF-β3 1 week + KGN 2 weeks group (Figure 2F) shown by the immunofluorescence staining. Expression Levels of Chondrocyte Marker Genes and Hypotrophy-Related Genes after 21 Days of Induction. To further evaluate the chondrogenic ability of KGN, we

Herein, we described a novel strategy by utilizing an ultraviolet (UV) light-reactive, rapidly cross-linkable matrix integrated with KGN-loaded nanoparticles to obtain the natural hyaline cartilage with a simple procedure (Figure 1). First, KGN is encapsulated into the biodegradable poly(lactic-coglycolic acid) (PLGA) nanoparticles through an emulsionbased formulation method. To enhance the stability of nanoparticles, alleviate burst release, and provide matrix for cell homing and regeneration, photo-cross-linkable acrylated hyaluronic acid (m-HA) is then utilized to form an in situformed hydrogel scaffold under UV light treatment. The KGNloaded PLGA nanoparticles are encapsulated inside the HA matrix for the sustained release of KGN. Without cell transplantation, this system may recruit the host’s endogenous cells, including BMSCs from marrow clot, synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane, and chondrocytes from surrounding healthy cartilage. Utilizing the skeletally mature female New Zealand White rabbit as an animal model, we demonstrated that after a convenient onestep procedure, this KGN-based release strategy could efficiently and persistently promote chondrogenesis.

RESULTS AND DISCUSSION Chondrogenic Potential of Human MSCs Derived from SMSC Induced by Chondrogenic Medium Supplemented with KGN. To confirm that KGN can induce human MSCs into chondrocytes, we compared the isolated SMSCs from the synovium tissue and the used pellets culture system with or without KGN. Our results showed that most of the cells adhered to the bottom of the culture dishes 24 h after the cell suspensions had been plated and the adherent cells exhibited a flattened and fibroblast-like morphology (Figure 2A) and characteristics of MSCs. Flow cytometric analysis showed that the majority of isolated cells expressed CD90 and CD105, and were negative for CD34, CD45, CD11b (Supporting Information, Figure S1). After 21 days induction, we found B

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 2. Chondrogenic potential of human MSCs derived from the synovium (SMSC) induced by chondrogenic medium supplemented with KGN. Passage-3 (P3) cells were pelleted and induced for 21 days in vitro. (A) P3 SMSCs. (B) MSC pellets induced by 10 μM KGN alone, cultured for 21 days, cannot form a regular pellet. (C) Addition of KGN significantly increased the pellet size. P3 SMSCs pretreated with TGF-β3 for 1 week and induced with KGN for 2 weeks exhibited a similar pellet size to that of the TGF-β3 + KGN group and the TGF-β3 + BMP-2 group. (D1−D6) HE staining of the sections to reveal chondrocyte-like cells induced by different factors. Sections were numbered consistently with the pellets groups (1, control group; 2, KGN group; 3, TGF-β3+KGN group; 4, TGF-β3 1W, KGN 2W group; 5, TGFβ3+BMP-2 group; 6, TGF-β3+BMP-2+KGN group). (E,F) Immunofluorescence staining for collagen type II on pellets of the TGF-β3 + BMP2 + KGN group (E) and the TGF-β3 1 week + KGN 2 weeks group (F) (n = 3). Scale bar: 100 μm in panels A, D, E, and F.

RUNX2 (Figure S3D) nor collagen type X (Figure S3E) were significantly different. Characterization of the KGN-Encapsulated PLGA Nanoparticles and Integration with Hyaluronic Acid Hydrogel. Next, the KGN-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were synthesized by an emulsion method. As shown in the scanning electron microscopy (SEM) image in Figure 3A, the resulting KGN-loaded PLGA nanoparticles (KGN-NPs) displayed spherical shapes and narrow size distribution. The average diameter of the KGNNP was 270 nm, identified by dynamic light scattering (DLS) (Figure 3B), which was consistent with the result of SEM imaging. The KGN loading capacity was identified as 7.5 wt % and encapsulation efficiency was 66.8 wt %. To create a biocompatible and biodegradable matrix that can encapsulate KGN-loaded nanoparticles for sustained release and subsequent regeneration, photo-cross-linkable acrylated m-HA was prepared (Figure 1A).11,12 The HA-based hydrogel scaffold

examined the expression of related marker genes. We sectioned the pellets samples and conducted histological and histochemical staining. We found that the expression of collagen type II could not be detected in the control group, whereas it was barely detectable in the KGN-treated group (Figure S3A). The expression level was significantly higher in the other chondrogenic groups. There was no significant difference observed between the TGF-β3 + BMP-2 and the TGF-β3 1 week + KGN 2 weeks groups. Interestingly, the addition of KGN to the chondrogenic medium significantly up-regulated the expression of collagen type II (Figure S3A). For the expression of chondrogenic regulator SOX9, the combination of TGF-β3 + BMP-2 + KGN induced the highest expression among all other groups (Figure S3B). The collagen type I expression trend was different as chondrogenic medium supplemented with KGN evidently down-regulated its expression (Figure S3C). Neither the expression level of C

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 3. Characterization of the KGN-encapsulated PLGA nanoparticles and integration with hyaluronic acid hydrogel. (A) The TEM image of the KGN-encapsulated PLGA nanoparticles coated with chitosan. Scale bar: 1 μm. (B) The hydrodynamic size of the KGN-encapsulated PLGA nanoparticles measured by DLS. (C) The hyaluronic acid (HA) hydrogel encapsulating PLGA nanoparticles can be formed by UV irradiation for 1 min (left, before irradiation; right, after irradiation). (D) In vitro release of the KGN from PLGA nanoparticles and HA hydrogel encapsulating PLGA nanoparticles at 37 °C (n = 3).

hydrogel scaffold for regeneration, the mixture of m-HA and nanoparticle was injected into the defects site, immediately followed by the UV irradiation applied for 1 min in the experimental group. The defects of the control group were left without treatment. After 4 weeks, the experimental group (Figure 4B1) showed more remarkable filling of cartilage defects in comparison to the control group (Figure 4A1). The majority of the repair tissue was stained positively in the experimental group (Figure 4B2), while negatively toluidine blue staining was only observed in the control group (Figure 4A2). The ECM of the repair tissue in the experimental group (Figure 4B3) was stained homogeneously with Safranin O. Although the demarcation between the repair tissue and native cartilage was still clear, only a slight section of regenerated ECM was observed in the control group (Figure 4A3). The following macroscopic evaluation in the experimental group at 12 weeks was displayed in Figure 4D1. The repaired tissue almost fully recovered the defects flushing with the native tissue. In contrast, in the control group (Figure 4C1), fibrouslike tissue was observed in the cartilage defects, and surface tissues were evident in some specimens. Toluidine blue staining of the repaired tissue in the experimental group (Figure 4D2) was almost the same as that of the native cartilage, but a little thinner. The majority of the repaired tissue in the control group (Figure 4C2) was stained negatively with toluidine blue. For the Safranin O staining, the ECM in the repaired tissue of the experimental group (Figure 4D3) had a homogeneous intensity and obvious integration. The ECM Safranin O staining was not noticeable in most of the repaired tissues of the control group (Figure 4C3). For the cartilage defects repaired by the UVcross-linked scaffold without KGN, no positive staining of toluidine blue and Safranin O were detected under the macroscopic observation (Figure S2).

containing KGN nanoparticles was quickly formed through a free-radical polymerization with exposure to UV light for 1 min, in the presence of cross-linker (N,N-methylenebis(acrylamide) (MBA)) and photoinitiator (Figure 3C). Such enzymatically degradable scaffold can enhance the stability of nanoparticles, alleviate burst release of KGN, and shield from immunogenicity.13,14 The release kinetics of KGN from drug-loaded PLGA nanoparticles (KGN-NPs) and HA matrix containing KGN-NPs scaffold were conducted using HPLC as a function of time. As shown in Figure 3D, the release rate of KGN associated with the HA matrix integrated with KGN-NPs was almost linear without remarkable burst release during the two months experimental period. At the initial 20 days, for example, the cumulative KGN release was found to be 2-fold less in the case of the conjugated scaffold than that of the PLGA nanoparticles without the HA matrix. The more sustained release behavior and alleviated burst release could be attributed to the long-term retention of the hydrophobic KGN within the cross-linked polymeric matrix. Moreover, the release profile can be tuned by varying the weight ratio of m-HA and MBA. With the increase of the cross-linking density, the release rate of KGN further decreased (Figure S4). Collectively, these results substantiated that the KGN release from the HA scaffold loaded with KGN nanoparticles underwent a sustained releasing manner, which offered promise for the long-term administration using KGN in vivo. In vivo Cartilage Defects Repair Using KGN-Loaded Photo-Cross-Linked Scaffold. To test the in vivo efficacy of the HA scaffold integrated with KGN-loaded nanoparticles, the skeletally mature female New Zealand White rabbits were utilized as the animal models. The full-thickness cartilage defects (3.5 mm in diameter, 3.0 mm in depth) were created in the center of the trochlear groove using an osteochondral transplantation instrumentation in both knees. To form the D

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 4. Results of in vivo cartilage defects repair using KGN-loaded photo-cross-linkable scaffold. (A1−D1) Macroscopic appearance of the specimens harvested at 4 (A1, B1) and 12 (C1, D1) weeks after operation. (A2−D2) Toluidine blue staining of the sections in the experimental groups (B2,D2) and the control groups (A2,C2). (A3−D3) Safranin O staining of the sections showed hyaline-like cartilage formation in the experimental groups. (E−H) Immunohistochemistry staining showed no type I collagen and increased type II collagen formation in the experimental groups (F, H) compared with the control groups (E, G). Scale bar: 2 mm in (A2−D2), (A3−D3), and (E−H).

Figure 5. Results of histological scoring system for cartilage defects repair quality evaluation. (A) Total score. (B) Hyaline cartilage content. (C) Structure characteristics. (D) Freedom from cellular changes. (E) Freedom from adjacent cartilage degeneration. (F) Subchondrol bone reconstruction. (G) Bonding at cartilage defects margins. (H) Safranin O staining of the repair tissue.

Quality Evaluation for Cartilage Defects Repair. At 12 weeks after the surgery, the repaired tissue treated with hydrogel showed strong type II collagen staining in the defects (Figure 4H) and slight type I collagen staining (Figure 4F). In the control group, there was minimal type II collagen staining

(Figure 4G), but obvious type I collagen staining compared with that of the experimental group (Figure 4E). At 12 weeks, significant improvements in ICRS scores were assessed in the experimental group compared with the control group (Figure S5). In addition, the histological scoring evaluation showed that E

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

studies demonstrated that treatment of KGN with transforming growth factors could significantly increase the expression of type II collagen and reduce the expression of type I collagen. The mechanism of interaction between KGN and transforming growth factors remains unclear. Several studies have shown that the TGF-β superfamily and RUNX protein have cooperative function in regulating cell growth and differentiation. Shi et al. suggested that RUNX3 and TGF-β superfamily cooperatively played important roles in IgA synthesis in B lymphocytes.19 BMP and RUNX2 synergistically induced the osteoblastic differentiation.20,21 Recently, it was reported that RUNX1 could interact with intracellular TGF-β Smad signaling pathways in the processes of microglia cell activation and proliferation as well as the neural stem cell proliferation after traumatic brain injury.22 Our work indicated that KGN enables interaction with the TGF-β superfamily in regulating chondrocyte differentiation. Furthermore, the safety of constitutive overexpression of growth factors in previous studies was often a concern. To circumvent this, we replaced the growth factors with KGN, which could confer the control of the duration and magnitude of the transgene expression. KGN also protects the existing chondrocytes under the pathological conditions of OA.8 Moreover, the molecular weight of KGN is too small to initiate the immune response, and no toxicity was observed with KGN at 100 mM in hMSCs, chondrocytes, osteoblasts, and synoviocytes.

the experimental group had significantly higher scores than the control group for all the variables except for the degeneration of the repaired tissue (Figure 5). At 3 months after the surgery, biomechanical testing of the repaired cartilage zones was performed via nanoindentation. On the basis of the load− displacement curves, the biomechanical properties of cartilage repaired by KGN had a significantly higher reduced modulus value than the control groups (Figure S6). Similarly to the intact cartilage in the same joint, cartilage repaired by KGNreleasing system was mechanically strong enough to withstand the exterior pressure. Previous tissue repair had been challenging in the clinical and preclinical studies because of the limited integration of the native and the repaired hyaline cartilage.15,16 In this study, we sought to improve the integration of the regenerated cartilage at the chondral surface. From our assessment, the repaired tissue borders with KGN-releasing system were less demarcated compared with those in the control groups. Furthermore, there was a significant improvement in the scoring of the integration at the cartilage interface from the histological assessment. Chondrocyte viability and proliferation were increased in the presence of the released KGN. Additionally, BMSCs cultured with KGN increased their proteoglycan deposition and type-II collagen content, potentially increasing the quality and quantity of repaired tissue at the chondral interface. The bleeding osteochondral defects may also recruit BMSCs to the lesion following curettage at the site of the osteochondral lesion. KGN may increase cartilage repair at the scaffold interface through recruitment of mesenchymal stem cells, as demonstrated by the positive effect on the migration of the mesenchymal stem cells. Furthermore, in vitro evidence has indicated that KGN promotes BMSCs chondrogenic differentiation, and consequent deposition of proteoglycan and type-II collagen. It is likely that the cumulative effects, including the inhibition of intra-articular catabolic activity, led to an improved graft chondral integration and repaired cartilage degeneration. Besides, the subchondral bone formation was more intact compared to the control group. The cooperative function of KGN, TGF-β superfamily, and RUNX protein may promote the advanced subchondral bone formation, while the mechanism remains to be clarified.8 Various tissues such as bone marrow, adipose tissue, synovial membranes, and trabecular bone generate MSCs. BMSCs have the best capacity for chondrogenesis and cartilage self-repair. Johnson et al. found that KGN promoted the differentiation of BMSCs into chondrocytes and also showed chondro-protective effects in two osteoarthritis animal models.8 In our study, BMSCs in situ are involved in cartilage regeneration. Inducing BMSCs differentiation in situ with KGN could significantly enhance the chondrogenesis and tissue regeneration by eliminating the needs for in vitro culture of chondrocytes. Patient-derived cells could be seeded onto scaffolds in a single surgery following the UV irradiation which makes the procedure easier and safer.17 Sakaguchi et al. demonstrated SMSCs had as high a proliferation and chondrogenesis as BMSCs.18 By harvesting SMSCs from knee joints and culturing with KGN and/or growth factors, our results showed that the addition of KGN to TGF-β3 or to TGF-β3 + BMP-2 significantly increased the size of the pellets and up-regulated the expression of cartilagerelated genes. These results indicated that KGN with transforming growth factors had a significant synergistic effect. In addition, KGN may help form hyaline cartilage, as our

CONCLUSIONS In summary, compared with the traditional microfracture and ACI, our hydrogel scaffold with sustained-release property of KGN facilitated the filling of the defects fast and the generation of hyaline cartilage. The convenient and less-invasive procedure holds great promise for clinical translation.23 These findings have important implications for the design of strategies for tissue regeneration in a small molecule-induced manner. Further investigation includes thorough assessment of systemic and long-term biocompatibility of the scaffold as well as the relationship among biodegradation of scaffold, drug release profile, and regeneration process. METHODS Materials. All chemicals unless mentioned were purchased from Sigma-Aldrich. Sodium hyaluronic acid (HA, the molecular weight of 77 kDa) was purchased from Freda Biochem Co., Ltd. (Shandong, China). Preparation and Characterization of the Drug Release Scaffold. 1). Preparation of Drug-Loaded PLGA Nanoparticles. PLGA nanoparticles loaded with KGN were prepared by a single oilin-water (O/W) emulsion/solvent evaporation method. First, 45 mg of PLGA (actide/glycolide (50:50); MW, 40−75 kDa) was dissolved in 4.5 mL of dichloromethane, and then 5 mg of KGN was dissolved in 0.5 mL acetone. The KGN in acetone was added to the PLGA in dichloromethane to form the organic phase. Then 5 mL of the oil phase was added dropwise to a 0.1% chitosan (Mn, 612 kDa; degree of deacetylation, 96.1%) solution through a syringe pump (1 mL/min). The oil-in-water (O/W) emulsion was formed using a high speed homogenizer at 16 000 rpm in an ice bath to prevent overheating. Then the emulsion was added to a 160 mL 0.1% chitosan solution, and the mixture was stirred at 200 rpm overnight to evaporate the organic phase. KGN-loaded nanoparticles were then collected by centrifugation (12 000 rpm, 30 min). The nanoparticles were centrifuged and resuspended using deionized water three times before removing excess surfactant and free drug. A fine powder of drug-loaded nanoparticles was obtained by lyophilization for 2 days and stored at 4 °C. F

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Statistical Analysis. All the results were reported as mean ± standard deviations. The unpaired t-test was used to carry out statistical analysis. P ≤ 0.05 is considered to be a significant difference. All the values were analyzed by using the SPSS software (version 20.0; IBM, America).

2). Preparation of Photopolymerizable Hyaluronic Acid.24 Hyaluronic acid (HA) was modified with a double bond by reacting with the methacrylic anhydride (MA). Two grams of HA was dissolved in 100 mL of distilled (DI) water with stirring in a cold room overnight, followed by the addition of 1.6 mL of MA into the HA solution. The pH of the reaction was maintained between 8 and 9 by adding 5 N NaOH and kept at 4 °C under continuous stirring for 24 h. Subsequently, m-HA was precipitated in acetone, washed with ethanol, and then dissolved in DI water. After dialysis against DI water for 48 h, the purified m-HA with a yield of 87.5% was obtained by lyophilization and characterized by 1H NMR (Varian Gemini 2300). The degree of modification (DM) was determined to be about 15% by comparing the ratio of the areas under the proton peaks at 5.74 and 6.17 ppm (methacrylate protons) to the peak at 1.99 ppm (N-acetyl glucosamine of HA) after performing a standard deconvolution algorithm to separate closely spaced peaks. m-HA: 1H NMR (D2O, 300 MHz, δ ppm): 1.85−1.96 (m, 3H, CH2C(CH3)CO), 1.99 (s, 3H, NHCOCH3), 5.74 (s, 1H, CH1H2C(CH3)CO), 6.17 (s, 1H, CH1H2C(CH3)CO). The hydrogel was formed by photopolymerization of m-HA (2%, w/v) and N,N-methylenebis(acrylamide) (MBA) (MBA/m-HA, 0.5:1, w:w) with photoinitiator (Irgacure 2959; 0.1%, w/v) via UV irradiation for 1 min using a BlueWave 75 UV Curing Spot Lamp (DYMAX). In vivo Study for Cartilage Repair. All the animal procedures were approved by Institutional Animal Care and Use committee of Drum Tower Hospital, Medical School, Nanjing University. The skeletally mature female New Zealand White rabbits (2.0−2.5 kg) were put in the supine position after general anesthetization. A medial para-patellar incision was made to dislocate the patellar and expose the articular surface. Full-thickness cartilage defects (3.5 mm in diameter, 3.0 mm in depth) were created in the center of the trochlear groove using an osteochondral transplantation instrumentation in both knees. The knees were randomly divided into two groups, experimental group and control group. The mixture of m-HA, MBA, and nanoparticle was injected into the defects site, and hydrogel was formed via UV irradiation for 1 min in the experimental group. The defects of the control group were left untreated. The animals were allowed to have free movements in their cages postoperation. The limbs were allowed to bear the whole weight. General health status was monitored by a veterinarian. The rabbits were sacrificed at 4 and 12 weeks, respectively. Macroscopic Evaluation. The regenerated tissue was assessed using the International Cartilage Repair Society (ICRS) macroscopic score which contains three categories: degree of defect repair, integration to board zone, and macroscopic appearance. The scoring was performed by three different investigators. Histology Evaluation. The samples were fixed in 10% formalin for 7 days and then decalcified in 15% EDTA for 14 days. After being embedded in paraffin, the samples were cut into 5 μm sections. Then, the sections were stained with toluidine blue and Safranin O/fast green to examine the morphology and glycosaminoglycan content. Observation was performed under a light microscope (Olympus, Japan). The repaired tissue was graded by three different investigators, using a modified O’Driscoll histology scoring system. Immunohistochemistry. For immunochemical evaluation, primary antibodies, mouse anti col II (Calbiochem, Merckmillipor), mouse anti col I (abcam), mouse anti col II (abcam), and mouse anti col X were used in the present study. Biotinylated secondary antimouse antibody (GE Healthcare) was used. The sections were first incubated with 0.4% pepsin (Roche) at 37 °C for 1h for antigen retrieval. Three % H2O2 in methanol was used to block the endogenous peroxidase, and 1% BSA (Sigma) was used to block nonspecific protein binding. After overnight incubation with primary antibody at 4 °C, the sections were incubated with the secondary antibody for 1 h at room temperature. The DAB substrate system was used to develop the color. Biomechanical Test. The biomechanical analysis of repaired tissues was performed using nanoindentation by the Institute of Sports Medicine, Peking University Third Hospital.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b06663. Experimental details for synthesis route of kartogenin (KGN), chondrogenesis of synovial-derived mesenchymal stem cells, and in vitro release study of the KGNloaded PLGA nanoparticles (KGN-NPs), HA containing KGN-NPs (HA/KGN-NPs) (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (81101338) and National Natural Science Foundation of China (81572129) (to D.S.), the Distinguished Young Investigator Project of Nanjing (to D.S.), and the startup package of UNC-Chapel Hill and NC State University (to Z.G.). We thank Professor Xingxu Huang for kindly providing advice for our experiments. We also thank Yeshuai Shen, Long Xue, Qian Huang, Zhihong Xu, Dongyang Chen, and Huajian Teng for helping our project. REFERENCES (1) Hollander, A. P.; Dickinson, S. C.; Kafienah, W. Stem Cells and Cartilage Development: Complexities of A Simple Tissue. Stem Cells 2010, 28, 1992−1996. (2) Huey, D. J.; Hu, J.; Athanasiou, K. A. Unlike Bone, Cartilage Regeneration Remains Elusive. Science 2012, 338, 917−921. (3) Natoli, R. M.; Skaalure, S.; Bijlani, S.; Chen, K.; Hu, J.; Athanasiou, K. Intracellular Na+ and Ca2+ Modulation Increases the Tensile Properties of Developing Engineered Articular Cartilage. Arthritis Rheum. 2010, 62, 1097−1107. (4) Matta, C.; Zákány, R. Calcium Signalling in Chondrogenesis: Implications for Cartilage Repair. Front. Biosci., Scholar Ed. 2013, 5, 305−324. (5) Mahmood, T. A.; Shastri, V. P.; van Blitterswijk, C. A.; Langer, R.; Riesle, J. Evaluation of Chondrogenesis within PEGT: PBT Scaffolds with High PEG Content. J. Biomed. Mater. Res., Part A 2006, 79, 216− 222. (6) Keselowsky, B. G.; Collard, D. M.; García, A. J. Integrin Binding Specificity Regulates Biomaterial Surface Chemistry Effects on Cell Differentiation. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5953−5957. (7) Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.; Nealey, P. F. Effects of Synthetic Micro- and Nano-Structured Surfaces on Cell Behavior. Biomaterials 1999, 20, 573−588. (8) Johnson, K.; Zhu, S.; Tremblay, M. S.; Payette, J. N.; Wang, J.; Bouchez, L. C.; Meeusen, S.; Althage, A.; Cho, C. Y.; Wu, X.; Schultz, P. G. A Stem Cell−Based Approach to Cartilage Repair. Science 2012, 336, 717−721. (9) Wang, Y.; Belflower, R. M.; Dong, Y.; Schwarz, E. M.; O’Keefe, R. J.; Drissi, H. Runx1/AML1/Cbfa2Mediates Onset of Mesenchymal Cell Differentiation toward Chondrogenesis. J. Bone Miner. Res. 2005, 20, 1624−1636. G

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano (10) Wotton, S.; Terry, A.; Kilbey, A.; Jenkins, A.; Herzyk, P.; Cameron, E.; Neil, J. C. Gene Array Analysis Reveals a Common Runx Transcriptional Programme Controlling Cell Adhesion and Survival. Oncogene 2008, 27, 5856−5866. (11) Yu, J.; Zhang, Y.; Ye, Y.; DiSanto, R.; Sun, W.; Ranson, D.; Ligler, F. S.; Buse, J. B.; Gu, Z. Microneedle-Array Patches Loaded with Hypoxia-Sensitive Vesicles Provide Fast Glucose-Responsive Insulin Delivery. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 8260−8265. (12) Mo, R.; Jiang, T.; DiSanto, R.; Tai, W.; Gu, Z. ATP-Triggered Anticancer Drug Delivery. Nat. Commun. 2014, 5, 3364−3373. (13) Di, J.; Yao, S.; Ye, Y.; Cui, Z.; Yu, J.; Ghosh, T. K.; Zhu, Y.; Gu, Z. Stretch-Triggered Drug Delivery from Wearable Elastomer Films Containing Therapeutic Depots. ACS Nano 2015, 9, 9407−9415. (14) Gu, Z.; Dang, T. T.; Ma, M.; Tang, B. C.; Cheng, H.; Jiang, S.; Dong, Y.; Zhang, Y.; Anderson, D. G. Glucose-Responsive Microgels Integrated with Enzyme Nanocapsules for Closed-Loop Insulin Delivery. ACS Nano 2013, 7, 6758−6766. (15) Hunziker, E. B. Articular Cartilage Repair: Basic Science and Clinical Progress. A Review of the Current Status and Prospects. Osteoarthritis Cartilage 2002, 10, 432−463. (16) Bekkers, J.; Creemers, L.; Tsuchida, A.; van Rijen, M.; Custers, R.; Dhert, W.; Saris, D. One-Stage Focal Cartilage Defect Treatment with Bone Marrow Mononuclear Cells and Chondrocytes Leads to Better Macroscopic Cartilage Regeneration Compared to Microfracture in Goats. Osteoarthritis Cartilage 2013, 21, 950−956. (17) Brunger, J. M.; Huynh, N. P.; Guenther, C. M.; Perez-Pinera, P.; Moutos, F. T.; Sanchez-Adams, J.; Gersbach, C. A.; Guilak, F. ScaffoldMediated Lentiviral Transduction for Functional Tissue Engineering of Cartilage. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, E798−E806. (18) Sakaguchi, Y.; Sekiya, I.; Yagishita, K.; Muneta, T. Comparison of Human Stem Cells Derived from Various Mesenchymal Tissues: Superiority of Synovium as A Cell Source. Arthritis Rheum. 2005, 52, 2521−2529. (19) Shi, M.; Stavnezer, J. CBFα3 (AML2) Is Induced by TGF-β1 to Bind and Activate the Mouse Germline Ig α Promoter. J. Immunol. 1998, 161, 6751−6760. (20) Lee, K.; Kim, H.; Li, Q.; Chi, X.; Ueta, C.; Komori, T.; Wozney, J. M.; Kim, E.; Choi, J.; Ryoo, H.; Bae, S. Runx2 Is A Common Target of Transforming Growth Factor Beta1 and Bone Morphogenetic Protein 2, and Cooperation Between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12. Mol. Cell. Biol. 2000, 20, 8783−8792. (21) Zhang, Y.; Yasui, N.; Ito, K.; Huang, G.; Fujii, M.; Hanai, J.; Nogami, H.; Ochi, T.; Miyazono, K.; Ito, Y. A RUNX2/ PEBP2αA/ CBFA1Mutation Displaying Impaired Transactivation and Smad Interaction in Cleidocranial Dysplasia. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 10549−10554. (22) Logan, T. T.; Villapol, S.; Symes, A. J. TGF-β Superfamily Gene Expression and Induction of the Runx1 Transcription Factor in Adult Neurogenic Regions after Brain Injury. PLoS One 2013, 8, e59250− e59263. (23) Mitragotri, S.; Anderson, D. G.; Chen, X.; Chow, E. K.; Ho, D.; Kabanov, A. V.; Karp, J. M.; Kataoka, K.; Mirkin, C. A.; Petrosko, S. H.; Shi, J.; Stevens, M. M.; Sun, S.; Teoh, S.; Venkatraman, S. S.; Xia, Y.; Wang, S.; Gu, Z.; Xu, C. Accelerating the Translation of Nanomaterials in Biomedicine. ACS Nano 2015, 9, 6644−6654. (24) Jiang, T.; Mo, R.; Bellotti, A.; Zhou, J.; Gu, Z. Gel−LiposomeMediated Co-Delivery of Anticancer Membrane-Associated Proteins and Small-Molecule Drugs for Enhanced Therapeutic Efficacy. Adv. Funct. Mater. 2014, 24, 2295−2304.

H

DOI: 10.1021/acsnano.5b06663 ACS Nano XXXX, XXX, XXX−XXX