Kartogenin-Incorporated Thermogel Supports Stem Cells for

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Kartogenin-Incorporated Thermogel Supports Stem Cells for Significant Cartilage Regeneration Xuezhou Li, Jianxun Ding, Zheng-Zheng Zhang, Modi Yang, Jia-Kuo Yu, JC Wang, Fei Chang, and Xuesi Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12212 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 8, 2016

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ACS Applied Materials & Interfaces

Kartogenin-Incorporated Thermogel Supports Stem Cells for Significant Cartilage Regeneration Xuezhou Li,† Jianxun Ding,*,‡ Zhengzheng Zhang,§ Modi Yang,† Jiakuo Yu,§ Jincheng Wang,*,† Fei Chang,*,† and Xuesi Chen*,‡

†

Department of Orthopaedics, The Second Hospital of Jilin University, Changchun 130041,

P. R. China ‡

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun 130022, P. R. China §

Institute of Sports Medicine, Peking University Third Hospital, Beijing 100191, P. R. China

ACS Paragon Plus Environment

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KEYWORDS: cartilage regeneration, kartogenin, mesenchymal stem cells, thermogel, tissue engineering

ABSTRACT: Recently, cartilage tissue engineering (CTE) attracts increasing attention in cartilage defect repair. In this work, kartogenin (KGN), an emerging chondroinductive non-protein

small

molecule,

was

poly(L-lactide-co-glycolide)−poly(ethylene

incorporated

into

a

thermogel

of

glycol)−poly(L-lactide-co-glycolide)

(PLGA−PEG−PLGA) to fabricate an appropriate microenvironment of bone marrow mesenchymal stem cells (BMSCs) for effective cartilage regeneration. More integrative and smoother repaired articular surface, more abundant characteristic glycosaminoglycans (GAGs) and collagen II (COL II), and less degeneration of normal cartilage were obtained in the KGN and BMSCs co-loaded thermogel group in vivo. In conclusion, the KGN-loaded PLGA−PEG−PLGA thermogel can be utilized as an alternative support for BMSCs to regenerate the damaged cartilage in vivo.


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1. INTRODUCTION The intrinsic properties of articular cartilage like avascularity and low cellularity restrict its spontaneously regeneration.1 The occurred cartilage defect always gradually gives rise to the degeneration of the entire joint, resulting in severe osteoarthritis and appealing for invasive intervenes, like arthroplasty. To prevent the progression of the aggressive lesion on articular cartilage, surgical interventions are required. Nowadays, several clinical managements are performed for cartilage repair, including microfracture (MF), mosaicplasty (MO), autologous chondrocyte implantation (ACI), and so on. Nevertheless, all these clinical strategies draw lots of controversies. The previous reports indicated that MF, MO, and ACI can obtain early release of the syndromes to a certain extent, while they are not linked with a long-term rehabilitation.2 With the development of tissue engineering (TE), it provides a promising therapeutic for cartilage regeneration. Given that scaffolds, growth factors, and cells are the three main TE elements, the researches of cartilage tissue engineering (CTE) always surround these aspects, and lots of progresses have been made.3 Scaffolds as a crucial ingredient of CTE serve as a spatial structure for cell proliferation and differentiation. In recent years, various scaffolds have been fabricated for cartilage regeneration, including natural or synthetic polymer three-dimensional (3D) porous materials, chemically or physically crosslinked hydrogels, etc.4 Among them, hydrogels, especially thermogels, have attracted increasing attention in many

TE

projects.5

The

thermogel

glycol)−poly(L-lactide-co-glycolide)

of

poly(L-lactide-co-glycolide)−poly(ethylene

(PLGA−PEG−PLGA)

with

appropriate

biodegradability and excellent biocompatibility can serve as a potential scaffold in CTE.6


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Thermo-sensitive hydrogel means it is in the sol status under the critical gelling temperature (CGT) and can encapsulate drugs and/or cells conveniently. Once the ambient temperature rises to above the CGT, the sol status can quickly transform into the gel status to control the release of drugs and support the growth of cells to achieve a better tissue regeneration.7 Besides, the sol−gel transition allows the thermogel to fill up almost all the shapes of cartilage defect in CTE. For CTE, chondrocytes and mesenchymal stem cells (MSCs) are the most commonly used seed cells to compensate for the low cellularity of repaired cartilage tissues.8 Compared to chondrocytes, several advantages of MSCs make them to be an ideal cell source for cartilage regeneration, such as facile extraction,9 differentiation into almost any end-stage lineage cells,10 and immunoregulation capacity.11 Beyond introducing MSCs into CTE, further strategies are implemented to promote cartilage reconstruction, and the addition of growth factors is one of the crucial steps. Tremendous studies demonstrate the continuous presence of transforming growth factor β (TGF-β) can induce the chondrogenesis of MSCs.12 Based on this, TGF-β has been recommended as a potential therapeutic agent to enhance the MSC-based articular cartilage repair for a long time.13 However, TGF-β possesses a very short half-life in vivo, which just lasts from minutes to hours,14 which means TGF-β cannot fully play its function for enough time in vivo; as 3 months are commonly needed to repair the defected cartilage. To reach a desired regeneration, a high therapeutic dose of TGF-β is needed, but as reported the intra-articular injection of a high dose of TGF-β is connected with osteophyte formation,15 synovitis, synovial fibrosis, joint swelling,16 and articular cartilage degradation.17 In addition,


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TGF-β as an exogenous protein easily denatures during storage and leads to in vivo immunogenicity. Kartogenin (KGN) as a non-protein small molecule chondrogenesis inducing agent was first reported by Johnson et al. in 2012.18 22,000 of structurally diverse and heterocyclic drug-like small molecules were screened, and KGN was revealed to significantly promote chondrocyte differentiation of human MSCs (hMSCs) in a dose-dependent manner without any toxicity. Most importantly, as a very stable small molecule, KGN can be stored and transported at room temperature. The obvious superiorities in comparison to protein growth factors make KGN to be a potential chondrogenesis promoter in CTE. Several studies of KGN on cartilage protective effects have been reported.19-20 Typically, Kang et al. revealed that the intra-articular injection of KGN-conjugated chitosan nano/microparticles could inhibit the aggressive degeneration of osteoarthritis (OA) in vivo.19 Another study of KGN on cartilage reconstruction was still managed by intra-articular injection, while an animal model with full-thickness cartilage defect was utilized.20 The results showed that KGN can promote the healing of cartilage defect. Intra-articular injection can deliver KGN into articular cavity, but it's not precise enough to deliver it into the defect area, and most parts of KGN are absorbed into the circulatory system. On the above background, if KGN can promote the chondrogenesis of MSC-encapsulated scaffolds as TGF-β does in CTE, it will be worth for preclinical and clinical exploitation. In this work, the KGN-incorporated PLGA−PEG−PLGA thermogel was employed to support bone marrow MSCs (BMSCs) for effective cartilage regeneration (Scheme 1). The KGN and BMSCs co-loaded thermogel achieve a better cartilage repair with smoother repair articular


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surface and more extracellular matrices (ECMs), such as characteristic glycosaminoglycans (GAGs) and collagen II (COL II), compared with the blank and KGN- or BMSC-encapsulated thermogel. The growth factor and stem cells co-incorporated thermogel was demonstrated to exhibit great potential in cartilage regeneration in vivo and even in clinic in the future.

Scheme 1. Synthesis pathways of PLGA−PEG−PLGA triblock copolymer (A) and KGN (B). Schematic illustration of cartilage regeneration with KGN and BMSCs co-loaded PLGA−PEG−PLGA thermogel (C). 2. MATERIALS AND METHODS


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2.1. Materials. PEG (number-average molecular weight (Mn) = 1000 g mol−1) and stannous octoate (Sn(Oct)2) were purchased from Sigma-Aldrich (Shanghai, P. R. China). L-Lactide (L-LA) and glycolide (GA) were obtained from Changchun SinoBiomaterials Co., Ltd. (Changchun, P. R. China) and recrystallized from ethyl acetate under nitrogen atmosphere before use. PLGA1650−PEG1000−PLGA1650 was synthesized through the ring-opening polymerization (ROP) of L-LA and GA with PEG as a macroinitiator and Sn(Oct)2 as a catalyst (Scheme 1A). 1000 and 1650 were the Mns of PEG and PLGA, respectively. In addition, the molar ratio of L-LA and GA in PLGA segment is 3:1. Both Mn and molar ratio were calculated from proton nuclear magnetic resonance (1H NMR). As shown in Scheme 1B, KGN was synthesized from phthalic anhydride and 4-aminobiphenyl according to the previously reported proposal.18 Antibiotics, trypsin-EDTA (0.05% trypsin and 0.02% EDTA) solution, low glucose Dulbecco's modified Eagle's medium (LG-DMEM) and fetal bovine serum (FBS) were bought from Gibco (Grand Island, NY, USA). Cholecystokinin octapeptide (CCK-8) kit for mammalian cells was obtained from Beyotime Co., Ltd. (Shanghai, P. R. China). Elastase and chymotrypsin were bought from Aladdin Reagent Co., Ltd. (Shanghai, P. R. China). Pepsin, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), and Alexa Fluor 488 phalloidin (Alexa 488) were purchased from Sigma-Aldrich (Shanghai, P. R. China). Primary antibody of COL II was purchased from Novus Biologicals (Littleton, CO, USA). GAGs assay kit was purchased from Antibodies-online Inc. (Shanghai, P. R. China). COL II enzyme-linked immunosorbent assay (ELISA) kit was purchased from Chondrex Inc. (Redmond, WA, USA). 2.2. Thermogel Fabrication and Characterizations


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2.2.1. Phase Diagram and Rheology Analyses. The sol−gel transition behavior of PLGA−PEG−PLGA thermogel in phosphate-buffered saline (PBS) was evaluated by a vial inverting test with a temperature increase of 1 °C per 5 minutes. The CGT was defined as that no flow of the system was observed within 30 seconds when the vial was inverted. The rheological study of PLGA−PEG−PLGA triblock copolymer solution in PBS of pH 7.4 with the optimized concentration of 20 wt.% was tested on a MCR 302 rheometer (Anton Paar, Graz, Austria). The test temperature was set to increase from 10 to 45 °C at a speed of 0.5 °C min−1. The storage modulus (G′) was detected under a controlled strain γ of 1% and a frequency of 10 rad s−1.21 Besides, the G′ of thermogel at 37 °C based on time was also tested. 2.2.2. Degradation of PLGA−PEG−PLGA Thermogel in Vitro. The proper copolymer concentration of 20 wt.% was selected from phase diagram for degradation assessments. The optimized copolymer solution was prepared and transferred into vials with an inner diameter of 16 mm at 4 °C, and then incubated at 37 °C for 10 minutes. All the obtained hydrogels were divided into three groups (n = 3): PBS, PBS with elastase (0.2 mg mL−1), and PBS with chymotrypsin (0.2 mg mL−1). The degradation media were added into the top of thermogels individually, and the buffer was collected at every predetermined time interval. Once the remaining thermogels were weighted, the fresh degradation media were supplemented. 2.2.3 KGN Release from Thermogel in Vitro. Because the concentration used for cartilage regeneration in vivo was too small to detect, the concentration of KGN was raised to 200.0 µg mL−1 to test the control release behavior of KGN in the thermogel. Once 1.0 mL


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of the KGN-contained thermogel was formed in vial with an inner diameter of 16 mm, 2.0 mL of PBS was added as release medium. At each predetermined time interval, the release medium was collected and tested by ultraviolet-visible (UV-vis) spectrophotometry (UV-1800, Shimadzu, Kyoto, Japan). 2.2.4. In Vivo Degradation and Biocompatibility of PLGA−PEG−PLGA Thermogel. The in vivo degradation and biocompatibility of thermogel were evaluated in Sprague-Dawley (SD) rats. In brief, once the aqueous solution of PLGA−PEG−PLGA copolymer at a concentration of 20 wt.% was obtained at 4 °C, 500.0 µL of the above solution was injected into the dorsal area of rat by a syringe with a 21-gauge needle subcutaneously. At defined time points (i.e., 0, 7, 14, 28, and 35 days), rats were sacrificed and the local sites of injected thermogel were photographed. In addition, to evaluate the inflammatory response to the thermogel in vivo, the skin around the thermogel was collected, fixed in 4% (W/V) PBS-buffered paraformaldehyde, and hematoxylin & eosin (H&E) staining was performed after 5 µm thick section of the skin was cut. 2.3. Isolation and Chondrogenesis of BMSCs 2.3.1. Isolation and Culture of BMSCs. BMSCs were isolated from the bone marrow of a 1 month-old New Zealand rabbit. Briefly, all the long bones, including tibia, fibula, femur, radius and ulna, and humerus, of which soft tissues were stripped, were harvested under aseptic environment after the rabbit was genteelly sacrificed. The ends of the long bones were cut by a pair of sharp scissors just the end of marrow cavity. A 23-gauge needle attached to a 10-mL syringe containing complete LG-DMEM was inserted into marrow cavity and repeatedly flushed the marrow out of the cut end of the bone with 2.0 mL of


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complete LG-DMEM, and the suspension was collected in a culture dish. The cell suspension was filtered through a 70-mm filter mesh. The obtained bone marrow cells were cultured in a 95-mm culture dish in 10.0 mL of complete LG-DMEM at a density of 2.5 × 107 cells mL−1. The dish was placed into an incubator without disturbance at 37 °C with 5% (V/V) carbon dioxide (CO2). After 24 hours incubation, the non-adherent cells that accumulated on the surface of the plate were removed by replacing the medium with fresh LG-DMEM.22 Culture was continued until the homogeneous adherent cells were left, and the passage 4 − 6 cells were used to evaluate the chondrogenesis ability of KGN and implanted into cartilage defect in vivo. 2.3.2. Chondrogenesis Ability of KGN on BMSCs. The pellet culture method was performed to induce the chondrogenesis of Passage 4 BMSCs. Briefly, 5 × 105 BMSCs in 1.0 mL of culture medium was centrifuged at 500 g for 15 minutes in a 15-mL centrifuge tube. All the pellets were grown in chondrogenic medium to induce chondrocyte differentiation. The chondrogenic medium without TGF-β consisted of LG-DMEM supplemented with 10% (V/V) FBS, 100.0 nM of dexamethasone, 50.0 µg mL−1 of ascorbic acid, 6.25 µg mL−1 of insulin, and antibiotic/antimycotic solution. Both 100 nM and 1 µM of KGN were used to induce the chondrogenesis of BMSCs in chondrogenic medium as previously reported.19 The culture medium was changed every 2 or 3 days. After culture for 3 weeks, the chondrogenesis promoting characteristics of KGN were tested by H&E staining, toluidine blue O (TBO) staining, COL II immunofluorescence analyses after the pellet was sectioned, and the real-time reverse transcriptase polymerase chain reaction (RT-PCR) of aggrecan (AGG) and COL II. The used RT-PCR primers were designed according to their


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gene sequences obtained from gene bank as showed in Table S1, Supporting Information. 2.3.3. H&E and TBO Staining, and COL II Immunofluorescence Analyses. The cell morphology in pellets was evaluated by H&E staining. TBO staining can specifically mark the basophilic proteoglycans secreted by the induced chondrocytes. It was performed after 3 weeks chondrogenic culture of BMSC pellets based on KGN. Briefly, at the pre-set time point, pellets were washed 2 times with PBS and fixed in 4% (W/V) paraformaldehyde for 15 minutes. And then, the pellets were dehydrated by graded ethanol and embedded by paraffin. Subsequently, 5 µm thick sections were obtained for staining and other tests. After the sections were dewaxed and immersed by a proper volume of TBO staining solution (0.5% (W/V)) at a concentration of 0.1 mg mL−1, the staining solution was washed out by PBS at 3 minutes later, and the staining status was observed and captured under a microscopy (Nikon Eclipse Ti, optical Apparatus Co., Ardmore, PA, USA). For COL II immunofluorescence analyses, all the procedures were performed according to the manufacturer's protocol. Typically, the sections of pellets were dewaxed, and antigen retrieval was carried out by a multiple digestive enzyme. After endogenous peroxidase block and goat serum incubation, the primary antibody solution of COL II with a proper concentration (2.0 µg mL−1) was covered on the surface of the cells for incubation overnight at 4 °C. Next day, the sections were nurtured with anti-mouse rabbit secondary antibody and labeled green with Alexa 488 at room temperature for 30 minutes after the uncombined primary antibodies were washed thoroughly. Then, cell nucleus was stained blue with DAPI for 3 minutes. Immunofluorescence of the sections was detected by a LSM 780 CLSM (Carl Zeiss, Jena, Germany) with 10 × eyepiece and 10 × objective.


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2.4. Animal Model Assessments 2.4.1. KGN and BMSC Dispersions in Thermogel, and Cell Proliferation Test. For KGN and BMSCs encapsulation, the proper concentrations of PLGA−PEG−PLGA triblock copolymer (20 wt.%) and KGN (1 µM) were mixed and dissolved by PBS in a vial at 4 °C. After homogeneous solution was obtained, Passage 4 BMSCs with a density of 1 × 106 cells mL−1 were mixed into the above solution. For proliferation test of BMSCs in thermogel in vitro, the copolymer solution without KGN was transited into a 24-well TCP plate and cultured by filled 2.0 mL of LG-DMEM, which was replaced every 2 or 3 days. At the pre-set time points of 3, 7, and 14 days, the culture medium was removed, and 2.0 mL of fresh medium and 20.0 µL of CCK-8 solution was supplemented. After 4 hours incubation, the supernatants were collected and tested by a Bio-Rad 680 microplate reader (Hercules, CA, USA) at 450 nm, and the absorbance at 600 nm was used for baseline correction. For in vivo implantation, the composite solutions of copolymer, KGN, and BMSCs were directly used. 2.4.2. Animal Surgery Procedure. Cartilage defect models of New Zealand white rabbits (2.7 − 3.0 kg, 5 − 6 months old) were carried out for the reconstruction experiment in vivo. All the procedures were approved by the Animal Care and Use Committee of Jilin University. To establish the animal model, the rabbit was first anesthetized by 3% (W/V) pentobarbital at a dosage of 50 mg kg−1. After carefully cleaning and draping the surgery site, the knee joint of rabbit was cut open. To fully expose the femur condyle, patellar was dislocated. Then a full-thickness osteochondral defect (4.5 mm diameter, 2.5 mm deep) was created on the trochlear groove by a drill. Subsequently, a standard microfracture procedure


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was performed to mobilize marrow blood. For thermogel-included groups, different solutions were filled into the osteochondral defects followed by 30 seconds infrared hyperthermia for gelation of the solutions. Finally, patella was reset, and the wound was sutured layer by layer. In this study, four experimental groups were included (n = 8): the control group (Control), only microfracture was applied without thermogel implantation; the thermogel group (Thermogel), only the thermogel was filled into the defect area following microfracture; the BMSC-encapsulated thermogel group (Gel/BMSCs), the thermogel loading with BMSCs was implanted into the defect site; the last group was KGN and BMSCs co-encapsulated thermogel group (Gel/KGN/BMSCs), which means that KGN companied with BMSCs was both loaded into the thermogel and implanted into the defect cartilage area. 2.4.3. Gross Observation of Repaired Cartilage. At the pre-set time points of 8 and 12 weeks, gross images of the harvested femur condyles were taken to evaluate the cartilage regeneration, and International Cartilage Repair Society (ICRS) macroscopic score was utilized (Table S2, Supporting Information). As a cartilage gross evaluation score, ICRS macroscopic score can systematically assess the regeneration results by evaluations of defect repair, integration into the border zone, and the macroscopic appearance.23 All the regenerations were scored by two independent observers blinded to the experimental groups. 2.4.4.

Histopathology

and

COL

II

Immunohistochemical

Analyses.

For

histopathological analyses, coronal sections of the femoral condyles from different groups were cut after fixation, decalcification, and embedded in paraffin. H&E and TBO staining were performed to do the evaluation. All the section was observed under microscopy. From


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the results of H&E staining, the ICRS visual histological assessment scale was used to evaluate the quality of repaired cartilage tissue (Table S3, Supporting Information).24 Besides, COL II in cartilage layer was also evaluated by immunohistochemical staining according to the manufacturer's protocol as mentioned above. 2.4.5. Biochemical Analyses of GAGs and COL II in Repaired Cartilages. The content of GAGs was assessed by a GAG assay kit. Briefly, the supernatant was collected after the repaired cartilage tissue was smashed by a homogenizer. The supernatant was diluted to a proper concentration, and GAGs content was tested by monitoring the absorbance of the above solution at 490 nm by a microplate reader. COL II content was analyzed with the COL II ELISA kit following to the manufacturer's protocol. Briefly, the repaired cartilage was harvested and digested with 0.1 mg mL−1 pepsin supplemented with 0.5 M acetic acid at 4 °C for 24 − 48 hours. The absorbance of solution samples was measured at 450 nm. The standard curve was calculated with standard COL II solution. 2.4.6. Nanoindentation Assessments of Repaired Cartilages. The mechanical property as one of the indicators for the evaluation of the repaired cartilage was measured by nanoindentation.25 Before the test, all samples were isolated from the repaired sites by a trepan and emerged into PBS solution to maintain their hydration at room temperature. All the indentation tests were performed by a TriboIndenter (Hysitron Inc., Minneapolis, MN, USA) with a 400-mm radius curvature conospherical diamond probe tip in five regions (anterior, posterior, central, medial, and lateral) on the samples. A trapezoidal load function was applied to each indent site with loading (10 s), hold (2 s), and unloading (10 s). Indentations were force-controlled to a maximum indentation depth of 500 nm. Meanwhile,


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micro-scanning apparatus was used to capture the microscopic geomorphology changes of the indentation zones. The hardness and reduced modulus were calculated using a linear fit to load-displacement data.26 2.5. Statistical Analyses. All the results were expressed as mean ± standard deviation (SD). All experiments were repeated at least three times. Differences between experimental groups were assessed by one-way analysis of variance with statistical software SPSS 17.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant, and P < 0.01 and P < 0.001 were considered highly significant. 3. RESULTS 3.1. Fabrication and Characterizations of PLGA−PEG−PLGA Thermogel. 3.1.1. Gelation and Rheological Properties. As shown in Figure 1A, the phase diagram was detected. Different concentrations of PLGA−PEG−PLGA triblock copolymer solution could transform into hydrogel at different temperatures, and the CGT reduced as the increase of solution concentration. The screened proper concentration of PLGA−PEG−PLGA copolymer for sol−gel transition was 20 wt.% with CGT of 31 °C, which was used in all the experiments of this work. The result of temperature-dependent rheological property of the copolymer aqueous solutions in PBS was showed in Figure 1B. As the temperature increased from 10.0 to 31.0 °C, the G′ of the thermogel increased to 800.0 Pa. And then, the G′ was 1210 Pa when the temperature reached up to 37 °C. Besides, as shown in Supplementary Figure S1, the average G′ of the thermogel at 37 °C was 1018.0 Pa for 1170 s.


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Figure 1. Temperature-dependent sol-gel phase transitions of PLGA−PEG−PLGA copolymer solutions in PBS at different concentrations (A). G′ of PLGA−PEG−PLGA copolymer in PBS at a concentration of 20 wt.% (B). In vitro degradation of PLGA−PEG−PLGA thermogel in PBS, or PBS with elastase or chymotrypsin (C). Proliferation behavior of BMSCs encapsulated in PLGA−PEG−PLGA thermogel (D). Data are presented as mean ± SD (n = 3; ***P < 0.001). 3.1.2. KGN Release Behavior of Thermogel in Vitro. The release profile of KGN from the thermogel in PBS was tested. The released KGN was detected by a standard curve method on an UV-vis spectrophotometry, and the absorption peak of KGN was at 278.4 nm. The result shown in Figure S2, Supporting Information, revealed that the thermogel got a well control release of KGN, and about 42.4% of the KGN was released from the thermogel


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at 196 h. 3.1.3. Degradation Behavior of Thermogel in Vitro. The degradation behaviors of thermogel in PBS without and with elastase or chymotrypsin were assessed for 30 days. Both the elastase- and chymotrypsin-incubated groups exhibited more rapid degradation rates than the PBS group (Figure 1C). The remaining thermogel of these two groups were 64.5 ± 2.1 and 46.6 ± 3.2 wt.%, respectively, after 30 days compared to that of the PBS group (90.2 ± 0.7 wt.%). In addition, chymotrypsin could catalyze the degradation of PLGA−PEG−PLGA thermogel more quickly than elastase with significant difference (P < 0.01). 3.1.4. Degradation and Biocompatibility of Thermogel in Vivo. To investigate the biodegradation and biocompatibility of PLGA−PEG−PLGA thermogel in vivo, the copolymer solution at a concentration of 20 wt.% was injected subcutaneously. The results showed that the thermogel degraded gradually in the subcutaneous layer in 4 weeks and were completely absorbed after 5 weeks (Figure S3, Supporting Information). The degradation of thermogel in vivo was faster than that in vitro as shown in Figure 1C. For inflammatory evaluation, H&E staining was conducted toward the skin section. As depicted in Figure S3, Supporting Information, the neutrophil granulocytes were aggregated at the surrounding skin tissue at the first week. More interestingly, with the extension of implantation time, the inflammation gradually diminished, which demonstrated that the thermogel possessed a good biocompatibility in vivo. 3.2. Isolation, Chondrogenic Differentiation, and Culture of BMSCs. BMSCs were successfully extracted and cultured. The proliferation test was carried out after BMSCs were


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encapsulated into the PLGA−PEG−PLGA thermogel. The result showed that BMSCs could normally proliferate in the thermogel (Figure 1D). Normalized to that on Day 3, after 7 and 14 days culture, the cells were 3.1 and 5.2 folds higher, respectively, with highly statistical significance (P < 0.001). After chondrogenesis culture of BMSC pellets based on the defined concentrations of KGN, that is, 100 nM and 1 µM, for 3 weeks, H&E, TBO staining, COL II immunofluorescence analyses, and RT-PCR of AGG and COL II were applied to assess the chondroinductive ability of KGN on BMSCs (Figures S4 and S5, Supporting Information). As shown in Supplementary Figure S4A, H&E staining showed that the pellets were well fabricated and cultured. TBO staining of the pellet sections indicated that KGN significantly promoted the secretion of basophilic proteoglycans in the KGN-incubated groups than that of the control group (Supplementary Figure S4A), and the immunofluorescence of COL II also revealed that KGN upregulated the COL II expression of induced chondrocytes (Figure S4B, Supporting Information). The RT-PCR analyses showed in Figure S5, Supporting Information. With the ascending concentration of KGN, the expression of AGG and COL II was accordingly increased. The 100 nM KGN group showed 1.1 and 1.4 folds higher expression of AGG and COL II than the control group, respectively. As expected, the 1 µM KGN group apparently possessed the highest AGG and COL II expression than the control and 100 nM KGN groups, which were 2.7 and 3.7 folds higher than the control group, respectively, with statistical difference (P < 0.01). All the results demonstrated that KGN could significantly induce the chondrogenesis of BMSCs.


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Figure 2. Gross evaluations of repaired cartilages. Macroscopic observation of repaired cartilages at 8 and 12 weeks (black arrow: degenerated cartilage) (A). Scale bar: 4 mm. ICRS macroscopic scores of repaired cartilages at 8 (B) and 12 weeks (C). Data are presented as mean ± SD (n = 5; *P < 0.05, **P < 0.01, ***P < 0.001). 3.3. Cartilage Repair Assessment of KGN- and BMSC-Incorporated Thermogel. 3.3.1. Animal Model Construction and Surgical Implantation. The cartilage defect model was successfully established, and all the procedures were conducted as designed in


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Scheme 1C and displayed in Figure S6, Supporting Information. At the predetermined time intervals of 8 and 12 weeks, the femurs condyles were harvested after the rabbits were gently sacrificed. Subsequently, various evaluations were performed to estimate the cartilage regeneration based on the composite implants. 3.3.2. Gross Evaluation. At the defined time points of 8 and 12 weeks, the rabbits were sacrificed and the femoral condyles of the four groups were harvested. For gross evaluation, images of the femoral condyles were taken and evaluated. As shown in Figure 2A, the Gel/KGN/BMSCs group exhibited the best regeneration of cartilage, and the defect was totally repaired after 12 weeks. Besides, the degeneration of normal cartilage caused by the defects was effectively inhibited due to the cartilage protective effect of KGN. As showed in the Control, Thermogel, and Gel/BMSCs groups, lots of osteophytes and cartilage wears can be seen macroscopically, which were indicated by black arrows in Figure 2A, while the smoother surface of articular cartilage was obtained in the Gel/KGN/BMSCs group. The ICRS macroscopic scores were calculated to evaluate the repaired cartilage as depicted in Figure 2B. The Gel/KGN/BMSCs group possessed significantly highest ICRS macroscopic scores of 8.4 ± 1.1 and 12.1 ± 0.8, which were 8.4 and 5.1 folds higher than the Control group at 8 and 12 weeks, respectively. Intuitively, the ICRS macroscopic scores of Gel/BMSCs group were 4.6 ± 1.1 and 6.8 ± 0.8 in 8 and 12 weeks respectively. The Thermogel group scored 2.2 ± 0.8 and 4.8 ± 0.8 in 8 and 12 weeks. The Control group exhibited the least ICRS macroscopic scores of 1.0 ± 0.7 and 2.4 ± 0.9 in 8 and 12 weeks respectively. All these ICRS macroscopic scores of the Control, Thermogel, and Gel/BMSCs groups were less than those of Gel/KGN/BMSCs group significantly (P < 0.001).


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Figure 3. Microscopic observations and histological evaluations of repaired cartilages. H&E staining of repaired cartilage at 8 and 12 weeks (A). Scale bar: 500 µm. ICRS visual histological scores for repaired cartilages at 8 (B) and 12 weeks (C). Data are presented as mean ± SD (n = 5; **P < 0.01 ***P < 0.001). 3.3.3. Histopathological Analyses of Regenerated Cartilages. The histopathological analyses of different groups of regenerated cartilages were represented in Figure 3. The Gel/KGN/BMSCs group showed the best reconstruction of cartilage with no doubt


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compared to all other groups no matter in 8 or 12 weeks (Figure 3A). The cartilage defect can hardly regenerate without intervenes, resulting in fibrous tissue filling in the Control group showed in Figure 3A. When the defect was filled with blank thermogel, cartilage tissue could be observed after 12 weeks, but it was not enough to full-fill the defect and the discontinued cartilage tissue generated. BMSCs as a kind of pluripotent stem cells can surely differentiate into chondrocytes as demonstrated above. So when the BMSCs-loaded thermogel was implanted into the defect, better cartilage regeneration was obtained. As shown in Figure 3B and 3C, the ICRS visual histological scores of Gel/KGN/BMSCs group were 10.0 ± 0.7 and 17.0 ± 0.8 in 8 and 12 weeks. The Gel/BMSCs group got scores of 5.8 ± 0.4 and 13.4 ± 1.1, respectively. The Thermogel group scored of 4.6 ± 0.5 and 12.8 ± 0.8 in 8 and 12 weeks, respectively. Finally, the Control group had the least scores of 2.2 ± 0.4 and 3.6 ± 0.9 in 8 and 12 weeks. The scores indicated that the Gel/KGN/BMSCs group represented the best histological regeneration among all the groups. 3.3.4. TBO Staining and COL II Immunohistochemical Analyses. Cartilage-specific staining was performed as TBO staining and immunohistochemical staining of COL II to evaluate the quality of repaired cartilage tissues as shown in Figures 4 and 5. As depicted in Figure 4, cartilage continuity of the Control group was interrupted, and the fibrous tissue appeared in the defect area. Although more cartilage tissues can be seen in the Thermogel group compared to the Control group, the repaired cartilage was still not integrative. When the thermogel encapsulated MSCs, the cartilage matrix was more abundant, but the surface was still rougher than that of the Gel/KGN/BMSCs group. On the other hand, the Gel/KGN/BMSCs group led to the significant enhancement of cartilage-like matrix


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production during the repair process of the defect. As depicted in Figure 5, the immunohistochemical staining results of COL II were consistent with those of TBO staining. The Gel/KGN/BMSCs group showed the most integrative cartilage tissue. All this results indicated that the superior quality and quantity of repaired cartilage in the Gel/KGN/BMSCs group were obtained without fibrotic or hypertrophic cartilage remodeling compared to those in the Control, Thermogel, and Gel/BMSCs groups.

Figure 4. TBO staining of repaired cartilages in different groups at 8 and 12 weeks. Scale bar: 500 µm. 3.3.5. GAGs and COL II Content of Repaired Cartilage. GAGs and COL II were two important characteristic components of hyaline cartilage with high specific expression. As the biochemical markers of cartilage regeneration, the contents of GAGs and COL II have been widely tested in many studies to evaluate the repaired quality of cartilage. In this study, GAGs and COL II contents were also tested by commercial ELISA detect kits. As showed in


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Figure 6A and 6B, the Gel/KGN/BMSCs group contained significantly more GAGs (1.8 ± 0.04 mg g−1) and COL II (17.5 ± 0.6 mg g−1) than any other groups, although they still lower compared to the normal cartilage (GAGs: 1.9 ± 0.4 mg g−1 and COL II: 19.2 ± 0.7 mg g−1). The integrity of cartilage in the Thermogel group was poor than that of the Gel/BMSCs group as showed in Figures 2 and 3, but there was no significant difference between the two groups of GAG and COL II Content (GAGs: 1.10 ± 0.005 vs 1.14 ± 0.05 mg g−1 and COL II: 12.8 ± 0.3 vs 14.1 ± 1.8 mg g−1) (P > 0.05).

Figure 5. Immunohistochemical staining of COL II of repaired cartilages in different groups at 8 and 12 weeks. Scale bar: 200 µm. 3.3.6. Mechanical Analyses. After regeneration for 3 months in vivo, the rabbits were gently sacrificed, and the nanoindentation test was performed to evaluate the mechanical


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properties of the repaired cartilage tissues. Figure 7A showed that the Gel/KGN/BMSCs group showed the most integrative and smoothest surface among all the test groups, although it was not as good as the normal cartilage. What's more, the cartilage surface of Thermogel group was similar to that of the Gel/BMSCs group, but the Control group was scraggier and rougher compared to the other groups.

Figure 6. After regeneration for 12 weeks, the biochemical contents of GAGs (A) and COL II (B) in the repaired cartilage. Data are presented as mean ± SD (n = 3; **P < 0.01, ***P < 0.001). To study the mechanical properties of repaired cartilage quantitatively, the mechanical parameters of reduced moduli and hardness were quantified according to the load-displacement curves in different groups. As depicted in Figure 7B and 7C, the reduced modulus and hardness of Gel/KGN/BMSCs group were 2.3 ± 0.09 GPa and 148.0 ± 8.5 kPa, respectively, which were 3.1 ± 0.2 GPa and 183.2 ± 15.7 kPa for normal cartilage, respectively. Although the reduced modulus and hardness of Gel/KGN/BMSCs group cannot catch up to that of native cartilage, it's significantly higher than those of Control (reduced


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modulus: 1.0 ± 0.1 GPa and hardness: 67.5 ± 12.0 kPa), Thermogel (reduced modulus: 1.6 ± 0.06 GPa and hardness: 82.0 ± 4.2 kPa), and Gel/BMSCs (reduced modulus: 1.7 ± 0.2 GPa and hardness 95.5 ± 6.4 kPa) groups. The Thermogel group showed no significant difference with the Gel/BMSCs group both in the two mechanical parameters.

Figure 7. Nanoindentation assessments of repaired cartilages. Surface microscopic geomorphology of repaired zone (A). Red scale bar: 4 µm. Reduced moduli (B) and hardness (C) of the repaired cartilages calculated based on loaded-displacement curves (n = 5; **P < 0.01, ***P < 0.001).

4. DISCUSSION With the development of CTE, the growth factor- and MSCs-contained hydrogel is one of the main composite scaffolds for cartilage regeneration.27-28 As spatial frames for MSCs to proliferate and differentiate, hydrogels can benefit cell transplantation. Hydrogels also allow the easy diffusions of nutrients and oxygen into their matrices and the efficient deposition of


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ECM secreted by the seed cells in them. Among the different types of hydrogels, thermogel plays an important role in cell encapsulation and drug delivery. Another merit of thermogel is that it can adapt to the varieties of defect shapes. The thermo-sensitive hydrogel from PLGA−PEG−PLGA triblock copolymer is the most common thermogel. As shown in Figure 1A, the aqueous solution of PLGA−PEG−PLGA copolymer at a concentration of 20 wt.% was in a sol status below the CGT of 31 °C, while the solution became to hydrogel when the surrounding temperature increased to above CGT. That's because the micelle with hydrophobic PLGA core and hydrophilic PEG shell is exhibited by self-assembly in aqueous solution below CGT. With the increase of temperature, the hydrophobic PLGA block in the PLGA−PEG−PLGA triblock copolymer will participate in different micelles, which causing extensive aggregation. And the abrupt increase in the number of aggregated micelles will induce the sol−gel transition.29 The gel formation can control the release of KGN, as showed in Figure S2, Supporting Information, the release content of KGN was only 42.4% at 196 hours. Therefore, KGN can exist and play its function in the local site of the defect cartilage for a prolonged time. As for biocompatibility, both PEG and PLGA have been approved by US Food and Drug Administration (FDA),30 and they can be totally tolerated in vivo by human body and cause less foreign body reaction. In this study, the results of proliferation test in vitro and compatibility evaluation in vivo all indicated that PLGA−PEG−PLGA thermogel exhibited excellent biocompatibility and was suitable for implantation (Figures 1D and S3, Supporting Information). The ideal scaffolds in TE should possess a property of biodegradation, which allows the regenerated tissues totally replaced the foreign implants. The PLGA−PEG−PLGA


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thermogel can totally degrade and be absorbed in vivo, which was also demonstrated in our study. As showed in Figures 1C and S3, Supporting Information, the thermogel degraded more quickly in vivo compared to that in vitro due to more complex microenvironment with various

enzymes

PLGA−PEG−PLGA

subcutaneously. thermogel,

the

Moreover,

along

accumulation

of

with

the

degradation

degradation

of

intermediates

subcutaneously caused a slightly acidic microenvironment, which also accelerates the biodegradation.31 Although the moduli of PLGA−PEG−PLGA thermogel is not as high as the normal cartilage, its gel status can be maintained after it is surrounded by hematoma in a very short time followed the microfracture. Once the stable hydrogel formed, it can serve as a drug vehicle and spatial structure of seed cells for cartilage regeneration. Typically, the thermogel-forming copolymer is easy to encapsulate growth factors and seed cells in a sol status. Once the solution of copolymer is obtained, the growth factor and seed cells can be dispersed in, and it can be implanted into cartilage defect region after the homogeneous mixture is obtained. After transforming into hydrogel at body temperature, the thermogel can serve as a spatial frame of seed cells and a control release vehicle of drug to promote cartilage regeneration. Based on these merits, thermogels have been explored as a promising platform in cartilage regeneration. As reported previously, the thermo-sensitive chitosan– Pluronic hydrogel can well support the proliferation of bovine chondrocytes in vitro.32 Recently, poly(ethylene glycol)−poly(L-alanine-co-L-phenyl alanine) thermogel has been tested as a scaffold to support the chondrogenesis of MSCs, which was demonstrated by the high expression of COL II and sulfated glycosaminoglycan.33 In addition, the


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chitosan/poly(vinyl alcohol) thermogel has been applied in cartilage regeneration in vivo, and it could effectively repair the rabbit articular cartilage defect as reported.34 All these studies showed the excellent application prospects of thermogels in CTE. For CTE, chondrocytes and MSCs are commonly encapsulated into hydrogels to promote cartilage regeneration.35-36 Although chondrocytes can be an alternative cell source, it cannot be widely applied in clinic before simple and noninvasive extraction strategies are accomplished. Exactly, easy and barely invasive extraction is one of the excellent properties of MSCs. MSCs also possess multilineage potential, and can differentiate into chondrocytes to participate in cartilage regeneration. Among various types of MSCs, BMSCs exhibit superior capacities of chondrogenesis under a standard differentiation protocol as reported.37 Therefore, BMSCs were utilized and encapsulated into the PLGA−PEG−PLGA thermogel as seed cells to regenerate the defected cartilage tissue in this study. As shown in Figure 1D, the BMSCs can well proliferate in the PLGA−PEG−PLGA thermogel. Although the pH may decrease during the degradation of PLGA, the buffering capability of culture medium can relieve the pH decrease. Once implanted into the body, there is no need to worry about the pH changes, because the buffer system of body fluid is more powerful and accurate than the culture medium. It is well known that TGF-β is a critical regulatory factor in the regulation of chondrocyte metabolism. It can drive lineage selection and cartilage differentiation of MSCs both in vitro 38

and in vivo.39 Besides, it can also inhibit the terminal hypertrophic differentiation of

chondrocytes.40 In recent years, TGF-β has been widely used in CTE to further facilitate the quality of repaired cartilage. As aforementioned, although TGF-β is considered as the main


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chondrogenesis inducing factor, limitations are also obvious, including short half-life, hard storage and transport, etc. The short half-life is the fatal defect of TGF-β in cartilage repair, since it commonly needs 3 − 6 months to reconstruct the damaged cartilage.14, 41 Besides, TGF-β as a bioactive agent can affect many physiological processes, safety data are poorly reported, and adverse events may have been underestimated.42 KGN used in this study is a small molecular agent screened by Johnson et al. in 2012 for chondrogenesis promotion.18 It is stable at room temperature and can effectively induce hMSCs into chondrocytes. Besides, several studies demonstrated KGN possessed excellent biocompatibility,43-44 the toxicity on cells was not observed even with a concentration of 100 µM.18 In this study, the concentration of KGN was selected according to the previous study.19 The differentiation results demonstrated that KGN could effectively induce the chondrogenesis of BMSCs in vitro, which was evaluated by TBO staining, COL II immunohistochemical staining, and the RT-PCR assessments of AGG and COL II (Figure S5, Supporting Information). All these properties make KGN possibly serve as an ideal enhancer to promote cartilage repair in vivo. Femoral condyle cartilage defect animal model of rabbit has become to be a classical model in CTE. It's easy to be managed and evaluated. Based on this classical animal model, lots of studies have been performed to explore the effects of TGF-β-loaded scaffolds on cartilage regeneration. As mentioned above, TGF-β suffers a short half-life time, which limits its clinical application. Considering that, some alternative strategies are utilized to evade this shortage for cartilage regeneration in vivo, like gene transfection of TGF-β in MSCs45 or loading plasmid-DNA nanoparticles in scaffolds.46 As aforementioned, KGN


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without a short half-life time can induce MSCs into chondrocytes as same as TGF-β does, the results in our study also demonstrated it (Figure S4, Supporting Information).18 How about its cartilage regeneration promoting ability in vivo? KGN serves as a cartilage repair promoter has been implanted into animal models for a few years. For examples, KGN can inhibit cartilage degeneration in OA animal models19 and also enhance the quality of full-thickness cartilage defect repair after microfracture20 both by intra-articular injection. The intra-articular injection of KGN has showed great effects on cartilage regeneration. However, the imprecise delivery of KGN into the defect area cannot strongly indicate that KGN can promote the local cartilage regeneration. Considering the TGF-β faced limitations, the KGN-incorporated thermogel was designed to support BMSCs in this study for in situ cartilage regeneration, when the implantation in vivo and the in situ cartilage defect based on KGN has not been studied. The in vivo cartilage repair after the implantation of KGN and BMSCs co-loaded thermogel was optimistic from the gross observation (Figure 2), histological evaluation (Figure 3), specific cartilage staining (Figures 4 and 5), and biochemical detection of GAGs and COL II (Figure 6). For gross evaluation, the Gel/KGN/BMSCs group already showed the best ICRS macroscopic score results than all the other groups even in 8 weeks (Figure 2B) (P < 0.001). More fascinatingly, the leading of Gel/KGN/BMSCs group was further expanded with an ICRS score of 12.1 ± 0.8 after 12 weeks (Figure 2C). The superiority of Gel/KGN/BMSCs group was also demonstrated by the ICRS visual score intuitively (Figures 3B and 3C). In detail, the Gel/KGN/BMSCs group exhibited the best cartilage integrity and most efficient cartilage matrix deposition demonstrated by the evaluation of


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H&E staining, TBO staining, and COL II immunohistochemical staining. Besides, the biochemical detection indicated the highest specific protein expression of Gel/KGN/BMSCs group compared to the Control, Thermogel, and Gel/BMSCs groups (Figure 6A and 6B). Mechanical property is another important aspect for assessing the quality of repaired cartilage. The more similar mechanical parameters the repaired cartilage exhibit to native cartilage, the better the repaired cartilage is. The nanoindentation results showed the KGN-contained PLGA−PEG−PLGA thermogel loading BMSCs can enhance the mechanical quality of repaired cartilage, which reflected on that the mechanical properties of the Gel/KGN/BMSCs group were drastically superior to those of all other groups both in reduced modulus and hardness. The superior was obvious, especially compared to the control group (Figure 7). In addition, ICRS visual histological scores, the biochemical levels, and mechanical properties of the Thermogel and Gel/BMSCs groups showed no significant differences (P > 0.05). But the gross observation showed the BMSCs-loaded group got a better ICRS macroscopic score. That's may be due to that the Thermogel group without BMSCs can't get plenty cell source for cartilage tissue repair, and then the sunk cartilage surface regenerated. Although the Gel/BMSCs group got a flat cartilage surface with BMSCs as seed cells to regenerate the defect cartilage tissues, the regenerated cartilage in the Gel/BMSCs group was partial of fibrous tissue compared to hyaline cartilage. That's also why the biochemical detections and mechanical studies showed no significant differences between the Thermogel and Gel/BMSCs groups. Based on above analysis, the Gel/KGN/BMSCs group showed the best results almost in every test, that's because the best quality of hyaline cartilage was


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regenerated with the chondrogenesis capacity of KGN. For local cartilage defect repair, the MSCs-encapsulated scaffolds are considered as a possible strategy. Beyond as the simple spatial structures for cell proliferation and differentiation, scaffolds are also used as vehicles to deliver the chondrogenesis promoter to the defect site in recently years. Due to its short half-life time, protein growth factor-based composite scaffolds suffer fetal limitation for extend inducing ability of chondrogenesis. Our composite thermogel system for CTE overcomes the limitations of protein growth factor-based scaffolds, and can reach a stable chondrogenesis inducing status for a long repair time. The in vivo animal study demonstrated it may serve as an appropriate strategy for cartilage defect repair by local delivering of the stable chondrogenesis promoter of KGN. 5. CONCLUSIONS In this study, the KGN-loaded PLGA−PEG−PLGA thermogel with appropriate biodegradation and good biocompatibility was fabricated to support BMSCs for cartilage regeneration in rabbit cartilage defect model. The Gel/KGN/BMSCs implantation demonstrated the most excellent apparent and histological cartilage repair, most efficient deposition of ECMs, and strongest mechanical property compared with those of blank thermogel or BMSC-loaded thermogel, which indicated the most effective cartilage regeneration in vivo. From this study, it can be concluded that KGN as a non-protein micro-molecular factor can effectively induce the chondrogenesis in vitro and in vivo, and the KGN and BMSCs co-loaded thermogel can be used to promote cartilage regeneration efficiently. In addition, KGN can be not only loaded into hydrogels but also encapsulated into other forms of scaffolds to promote cartilage regeneration. In short, the


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KGN-incorporated constructs exhibit great potential for clinical cartilage regeneration.

ASSOCIATED CONTENT Supporting Information G′ of thermogel at 37 °C; Release behavior of KGN from thermogel; Degradation and biocompatibility of PLGA−PEG−PLGA thermogel in vivo; H&E and TBO staining, COL II immunohistochemical staining of pellet sections; AGG and COL II expression; Diagrams of surgery for cartilage defect repair; Primers for RT-PCR, and ICRS macroscopic and visual histological evaluations of cartilage repair. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Authors *

E-mail:

[email protected]

(J.

Wang);

[email protected]

(J.

Ding);

[email protected] (F. Chang); [email protected] (X. Chen). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS


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This research was financially supported by National Natural Science Foundation of China (Nos. 81171681, 51303174, 51233004, 51390484, 51321062, 51473165, 51203153, and 51273196), National Science Foundation for Post-Doctoral Scientists of China (No. 2013M530990),

and

Scientific

Development

Program

of

Jilin

Province

(Nos.

20140520050JH, 20140309005GX, and 20130206058GX).


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REFERENCES (1) Li, X. Z.; Ding, J. X.; Wang, J. C.; Zhuang, X. L.; Chen, X. S. Biomimetic Biphasic Scaffolds for Osteochondral Defect Repair. Regener. Biomater. 2015, 2, 221−228. (2) Detterline, A. J.; Goldberg, S.; Bach, B. R., Jr.; Cole, B. J. Treatment Options for Articular Cartilage Defects of the Knee. Orthop. Nurs. 2005, 24, 361−366. (3) Mardones, R.; Jofre, C. M.; Minguell, J. J. Cell Therapy and Tissue Engineering Approaches for Cartilage Repair and/or Regeneration. Int. J. Stem Cells 2015, 8, 48−53. (4) Li, X. Z.; Ding, J. X.; Zhuang, X. L.; Chang, F.; Wang, J.C.; Chen, X. S. Chitosan-Based Scaffolds for Cartilage Regeneration. In Chitin and Chitosan for Regenerative Medicine; Dutta P. K., Eds.; Springer: 2016; Chapter 3, pp 61−82. (5) Drury, J. L.; Mooney, D. J. Hydrogels for Tissue Engineering: Scaffold Design Variables and Applications. Biomaterials 2003, 24, 4337−4351. (6) Zhang, Y. B.; Xu, W. G.; Wu, J.; Zhuang, X. L.; Chen, X. S. Biodegradable Thermogel as Culture Matrix of Bone Marrow Mesenchymal Stem Cells for Potential Cartilage Tissue Engineering. Chin. J. Polym. Sci. 2014, 32, 1590−1601. (7) Qiao, M. X.; Chen, D. W.; Ma, X. C.; Liu, Y. J. Injectable Biodegradable Temperature-Responsive PLGA−PEG−PLGA Copolymers: Synthesis and Effect of Copolymer Composition on the Drug Release from the Copolymer-Based Hydrogels. Int. J. Pharm. 2005, 294, 103−112. (8) Wakitani, S.; Goto, T.; Pineda, S. J.; Young, R. G.; Mansour, J. M.; Caplan, A. I.; Goldberg, V. M. Mesenchymal Cell-Based Repair of Large, Full-Thickness Defects of Articular Cartilage. J. Bone Joint Surg. Am. 1994, 76, 579−592.


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(9) Campagnoli, C.; Roberts, I. A.; Kumar, S.; Bennett, P. R.; Bellantuono, I.; Fisk, N. M. Identification of Mesenchymal Stem/Progenitor Cells in Human First-Trimester Fetal Blood, Liver, and Bone Marrow. Blood 2001, 98, 2396−2402. (10) Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.; Douglas, R.; Mosca, J. D.; Moorman, M. A.; Simonetti, D. W.; Craig, S.; Marshak, D. R. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999, 284, 143−147. (11) Frenette, P. S.; Pinho, S.; Lucas, D.; Scheiermann, C. Mesenchymal Stem Cell: Keystone of the Hematopoietic Stem Cell Niche and a Stepping-Stone for Regenerative Medicine. Annu. Rev. Immunol. 2013, 31, 285−316. (12) Mackay, A. M.; Beck, S. C.; Murphy, J. M.; Barry, F. P.; Chichester, C. O.; Pittenger, M. F. Chondrogenic Differentiation of Cultured Human Mesenchymal Stem Cells from Marrow. Tissue Eng. 1998, 4, 415−428. (13) Madry, H.; Cucchiarini, M. Tissue-Engineering Strategies to Repair Joint Tissue in Osteoarthritis: Nonviral Gene-Transfer Approaches. Curr. Rheumatol. Rep. 2014, 16, 450. (14) Stowers, R. S.; Drinnan, C. T.; Chung, E.; Suggs, L. J. Mesenchymal Stem Cell Response to TGF-β1 in Both 2D and 3D Environments. Biomater. Sci. 2013, 1, 860−869. (15) van der Kraan, P. M.; van den Berg, W. B. Osteophytes: Relevance and Biology. Osteoarthritis Cartilage 2007, 15, 237−244. (16) Elford, P. R.; Graeber, M.; Ohtsu, H.; Aeberhard, M.; Legendre, B.; Wishart, W. L.; MacKenzie, A. R. Induction of Swelling, Synovial Hyperplasia and Cartilage Proteoglycan Loss Upon Intra-Articular Injection of Transforming Growth Factor β-2 in the Rabbit. Cytokine 1992, 4, 232−238.


37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 44

(17) Mi, Z.; Ghivizzani, S. C.; Lechman, E.; Glorioso, J. C.; Evans, C. H.; Robbins, P. D. Adverse Effects of Adenovirus-Mediated Gene Transfer of Human Transforming Growth Factor β1 into Rabbit Knees. Arthritis Res. Ther. 2003, 5, R132−139. (18) 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. (19) Kang, M. L.; Ko, J. Y.; Kim, J. E.; Im, G. I. Intra-Articular Delivery of Kartogenin-Conjugated

Chitosan

Nano/Microparticles

for

Cartilage

Regeneration.

Biomaterials 2014, 35, 9984−9994. (20) Xu, X.; Shi, D.; Shen, Y.; Xu, Z.; Dai, J.; Chen, D.; Teng, H.; Jiang, Q. Full-Thickness Cartilage Defects Are Repaired Via a Microfracture Technique and Intraarticular Injection of the Small-Molecule Compound Kartogenin. Arthritis Res. Ther. 2015, 17, 20. (21) Zhang, L.; Shen, W.; Luan, J.; Yang, D.; Wei, G.; Yu, L.; Lu, W.; Ding, J. Sustained Intravitreal Delivery of Dexamethasone Using an Injectable and Biodegradable Thermogel. Acta Biomater. 2015, 23, 271−281. (22) Soleimani, M.; Nadri, S. A Protocol for Isolation and Culture of Mesenchymal Stem Cells from Mouse Bone Marrow. Nat. Protoc. 2009, 4, 102−106. (23) van den Borne, M. P.; Raijmakers, N. J.; Vanlauwe, J.; Victor, J.; de Jong, S. N.; Bellemans, J.; Saris, D. B. International Cartilage Repair Society (ICRS) and Oswestry Macroscopic Cartilage Evaluation Scores Validated for Use in Autologous Chondrocyte Implantation (ACI) and Microfracture. Osteoarthr. Cartilage 2007, 15, 1397−1402. (24) Mainil-Varlet, P.; Aigner, T.; Brittberg, M.; Bullough, P.; Hollander, A.; Hunziker, E.;


38

Page 39 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Kandel, R.; Nehrer, S.; Pritzker, K.; Roberts, S.; Stauffer, E. Histological Assessment of Cartilage Repair: A Report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J. Bone Joint Surg. Am. 2003, 85-A Suppl 2, 45−57. (25) Huang, H.; Zhang, X.; Hu, X.; Shao, Z.; Zhu, J.; Dai, L.; Man, Z.; Yuan, L.; Chen, H.; Zhou, C.; Ao, Y. A Functional Biphasic Biomaterial Homing Mesenchymal Stem Cells for In Vivo Cartilage Regeneration. Biomaterials 2014, 35, 9608−9619. (26) Oliver, W. C.; Pharr, G. M. An Improved Technique for Determining Hardness and Elastic Modulus Using Load and Displacement Sensing Indentation Experiments. J. Mater. Res. 1992, 7, 1564−1583. (27) Han, F.; Zhou, F.; Yang, X.; Zhao, J.; Zhao, Y.; Yuan, X. A Pilot Study of Conically Graded Chitosan-Gelatin Hydrogel/PLGA Scaffold with Dual-Delivery of TGF-β1 and BMP-2 for Regeneration of Cartilage-Bone Interface. J. Biomed. Mater. Res. B Appl. Biomater. 2015, 103, 1344−1353. (28) Kim, J.; Lin, B.; Kim, S.; Choi, B.; Evseenko, D.; Lee, M. TGF-β1 Conjugated Chitosan

Collagen

Hydrogels

Induce

Chondrogenic

Differentiation

of

Human

Synovium-Derived Stem Cells. J. Biol. Eng. 2015, 9, 1. (29) Lee, D. S.; Shim, M. S.; Kim, S. W.; Lee, H.; Park, I.; Chang, T. Y. Novel Thermoreversible Gelation of Biodegradable PLGA-block-PEO-block-PLGA Triblock Copolymers in Aqueous Solution. Macromol. Rapid Commun. 2001, 22, 587−592. (30) Swami, A.; Reagan, M. R.; Basto, P.; Mishima, Y.; Kamaly, N.; Glavey, S.; Zhang, S.; Moschetta, M.; Seevaratnam, D.; Zhang, Y.; Liu, J.; Memarzadeh, M.; Wu, J.; Manier, S.; Shi, J.; Bertrand, N.; Lu, Z. N.; Nagano, K.; Baron, R.; Sacco, A.; Roccaro, A. M.;


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 44

Farokhzad, O. C.; Ghobrial, I. M. Engineered Nanomedicine for Myeloma and Bone Microenvironment Targeting. Proc. Natl. Acad. Sci. 2014, 111, 10287−10292. (31) Wu,

L.;

Ding,

J.

In

Vitro

Degradation

of

Three-Dimensional

Porous

Poly(D,L-Lactide-Co-Glycolide) Scaffolds for Tissue Engineering. Biomaterials 2004, 25, 5821−5830. (32) Park, K. M.; Lee, S. Y.; Joung, Y. K.; Na, J. S.; Lee, M. C.; Park, K. D. Thermosensitive Chitosan-Pluronic Hydrogel as an Injectable Cell Delivery Carrier for Cartilage Regeneration. Acta Biomater. 2009, 5, 1956−1965. (33) Park, M. H.; Yu, Y.; Moon, H. J.; Ko du, Y.; Kim, H. S.; Lee, H.; Ryu, K. H.; Jeong, B. 3D

Culture

of

Tonsil-Derived

Mesenchymal

Stem

Cells

in

Poly(ethylene

glycol)-Poly(L-alanine-co-L-phenyl alanine) Thermogel. Adv. Healthc. Mater. 2014, 3, 1782−1791. (34) Qi, B. W.; Yu, A. X.; Zhu, S. B.; Zhou, M.; Wu, G. Chitosan/Poly(vinyl alcohol) Hydrogel Combined with Ad-hTGF-β1 Transfected Mesenchymal Stem Cells to Repair Rabbit Articular Cartilage Defects. Exp. Biol. Med. 2013, 238, 23−30. (35) Kontturi, L. S.; Järvinen, E.; Muhonen, V.; Collin, E.; Pandit, A.; Kiviranta, I.; Yliperttula, M.; Urtti, A. An Injectable, in Situ Forming Type II Collagen/Hyaluronic Acid Hydrogel Vehicle for Chondrocyte Delivery in Cartilage Tissue Engineering. Drug Delivery Transl. Res. 2014, 4, 149−158. (36) Snyder, T. N.; Madhavan, K.; Intrator, M.; Dregalla, R. C.; Park, D. A Fibrin/Hyaluronic Acid Hydrogel for the Delivery of Mesenchymal Stem Cells and Potential for Articular Cartilage Repair. J. Biol. Eng. 2014, 8, 10.


40

Page 41 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(37) Cavallo, C.; Cuomo, C.; Fantini, S.; Ricci, F.; Tazzari, P. L.; Lucarelli, E.; Donati, D.; Facchini, A.; Lisignoli, G.; Fornasari, P. M.; Grigolo, B.; Moroni, L. Comparison of Alternative Mesenchymal Stem Cell Sources for Cell Banking and Musculoskeletal Advanced Therapies. J. Cell Biochem. 2011, 112, 1418−1430. (38) Murphy, M. K.; Huey, D. J.; Hu, J. C.; Athanasiou, K. A. TGF-β1, GDF-5, and BMP-2 Stimulation Induces Chondrogenesis in Expanded Human Articular Chondrocytes and Marrow-Derived Stromal Cells. Stem Cells 2015, 33, 762−773. (39) Yin, F.; Cai, J.; Zen, W.; Wei, Y.; Zhou, W.; Yuan, F.; Singh, S. R.; Wei, Y. Cartilage Regeneration of Adipose-Derived Stem Cells in the TGF-β1-Immobilized PLGA-Gelatin Scaffold. Stem Cell Rev. 2015, 11, 453−459. (40) Yang, X.; Chen, L.; Xu, X.; Li, C.; Huang, C.; Deng, C. X. TGF-β/Smad3 Signals Repress Chondrocyte Hypertrophic Differentiation and Are Required for Maintaining Articular Cartilage. J. Cell Biol. 2001, 153, 35−46. (41) Rey-Rico, A.; Venkatesan, J. K.; Sohier, J.; Moroni, L.; Cucchiarini, M.; Madry, H. Adapted Chondrogenic Differentiation of Human Mesenchymal Stem Cells via Controlled Release of TGF-β1 from Poly(ethylene oxide)-Terephtalate/Poly(butylene terepthalate) Multiblock Scaffolds. J. Biomed. Mater. Res. A 2015, 103, 371−383. (42) Robson, M. C.; Phillip, L. G.; Cooper, D. M.; Lyle, W. G.; Robson, L. E.; Odom, L.; Hill, D. P.; Hanham, A. F.; Ksander, G. A. Safety and Effect of Transforming Growth Factor-β2 for Treatment of Venous Stasis Ulcers. Wound Repair Regen. 1995, 3, 157−167. (43) Liu, C.; Ma, X.; Li, T.; Zhang, Q. Kartogenin, Transforming Growth Factor-β1 and Bone Morphogenetic Protein-7 Coordinately Enhance Lubricin Accumulation in


41


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Bone-Derived Mesenchymal Stem Cells. Cell Biol. Int. 2015, 39, 1026−1035. (44) Zhang, J.; Wang, J. H. Kartogenin Induces Cartilage-Like Tissue Formation in Tendon-Bone Junction. Bone Res. 2014, 2, 14008. (45) Gelse, K.; von der Mark, K.; Aigner, T.; Park, J.; Schneider, H. Articular Cartilage Repair by Gene Therapy Using Growth Factor-Producing Mesenchymal Cells. Arthritis Rheum. 2003, 48, 430−441. (46) Lu, H.; Lv, L.; Dai, Y.; Wu, G.; Zhao, H.; Zhang, F. Porous Chitosan Scaffolds with Embedded Hyaluronic Acid/Chitosan/Plasmid-DNA Nanoparticles Encoding TGF-β1 Induce DNA Controlled Release, Transfected Chondrocytes, and Promoted Cell Proliferation. PLoS One 2013, 8, e69950.


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Table of Contents


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67x61mm (300 x 300 DPI)


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Kartogenin-Incorporated Thermogel Supports Stem Cells for

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