Materials Chemistry B

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1 Journal of Materials Chemistry B PAPER Cite this: J. Mater. Chem. B, 2018, 6, 675 TGF-b1 affinity peptides incorporated within a chitosan sponge scaffold can significantly enhance cartilage regeneration Jiaqing Chen, a Yijiang Li, a Bin Wang, b Jiabei Yang, a Boon Chin Heng, c Zheng Yang, d Zigang Ge * ab and Jianhao Lin* b Received 9th August 2017, Accepted 7th January 2018 DOI: /c7tb02132a rsc.li/materials-b Growth factors, such as TGF-b and BMPs, play key roles in the chondrogenic differentiation of mesenchymal stem cells (MSCs) and cartilage regeneration in vivo. Nevertheless, there are some technical challenges in delivering exogenous growth factors in vivo, such as burst release and loss of bioactivity. In this study, TGF-b1 affinity peptides were incorporated within porous chitosan scaffolds to enhance cartilage regeneration. Significant upregulation of gene expression levels of Sox9, Col II and AGG during chondrogenic differentiation of MSCs in vitro, were positively correlated with increasing amounts of TGF-b1 affinity peptides incorporated within the chitosan scaffolds. The results of ectopic implantation of scaffolds in nude mice showed that incorporation of TGF-b1 affinity peptides and preloading of TGF-b1 synergistically enhanced ectopic cartilage formation at both high and low cell densities. Furthermore, in a rabbit osteochondral defect model, implantation of chitosan scaffolds incorporated with TGF-b1 affinity peptides (CHI-PEP) could significantly promote cartilage regeneration, even in the absence of exogenous growth factors and seeded cells. Notably, inflammation and cartilage degeneration were markedly alleviated in the CHI-PEP group. Hence, incorporation of TGF-b1 affinity peptide within the chitosan sponge scaffold significantly enhanced articular cartilage regeneration. 1. Introduction Articular cartilage has limited capacity for self-regeneration after injury, due to lack of blood vessels, nerves, and lymphatic system. Growth factors promote regeneration of articular cartilage by enhancing recruitment and differentiation of mesenchymal stem cells in vivo. 1 3 Transforming growth factor b (TGF-b), which plays a key role during embryonic development of cartilage, has been shown to enhance in vitro chondrogenic differentiation of stem cells, as well as full-thickness cartilage regeneration in vivo. 1 However, controlled delivery of TGF-b is challenging, regardless of the methods utilized, i.e. absorption, 1,4 encapsulation, 5,6 or covalent binding, 7 with technical challenges such as burst release of growth factors and loss of bioactivity over time. 8 Moreover, TGF-b, with two pairs of b-sheets and a long a-helix, 9 a Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, , P. R. China. gez@pku.edu.cn b Arthritis Clinic and Research Center, Peking University People s Hospital, Beijing, , P. R. China. linjianhao@pkuph.edu.cn c Faculty of Dentistry, Department of Endodontology, The University of Hong Kong, Pokfulam, Hong Kong SAR, P. R. China d Tissue Engineering Program, Life Sciences Institute, National University of Singapore, 27 Medical Drive, Singapore , Singapore Electronic supplementary information (ESI) available. See DOI: /c7tb02132a is difficult to chemically incorporate within scaffolds, with loss of conformation and bioactivity often occurring during the immobilization procedure. 10 Peptides with specific affinity to growth factors could potentially circumvent the aforementioned challenges, by recruiting endogenous growth factors with functional conformation and bioactivity. Peptides with specific affinity to individual growth factors have been widely utilized in diverse tissue regeneration and therapeutic applications, such as BMP-2 affinity peptides for spinal arthrodesis, 11 peptides with collagen-binding sequence for targeting injured vasculature, 12 and peptides with specific affinity for MSCs or endothelial cells Implantation of scaffold materials incorporated with heparin-binding proteins that bind TGF-b can efficiently lead to cartilage formation and regeneration by fine-tuning controlled release of TGF-b. Exogenous TGF-b is usually loaded onto the scaffolds in vitro before implantation into the body. 16,17 However, the specificity affinity to growth factors is important when the scaffolds are implanted in vivo without initial loading of exogenous growth factors. In a previous study, TGF-b1 affinity peptide (HSNGLPL) physically mixed with hydrogel enhanced rabbit cartilage regeneration by recruiting endogenous TGF-b1. 18 Furthermore, this TGF-b1 affinity peptide sequence ameliorated inflammation caused by the scaffold materials upon implantation in mouse muscles, probably through recruitment This journal is The Royal Society of Chemistry 2018 J. Mater. Chem. B, 2018, 6,

2 Fig. 1 Schematic diagram of experimental design. of TGFs. 19,20 However, physical entrapment within diffusible hydrogels can cause loss of peptides due to the limited mechanical strength of the scaffold matrix. With respect to cartilage regeneration, little is known about the possibility and effects of immobilizing this affinity peptide on stiffer scaffold biomaterials. We hereby postulate that an optimal dosage of TGF-b1-affinity peptides immobilized on an implanted scaffold can enhance cartilage regeneration. In this study, TGF-b1 affinity peptides were incorporated within porous chitosan sponges, and the optimal concentrations of affinity peptides were determined by in vitro chondrogenic differentiation of mesenchymal stem cells (MSC). The effects of three key factors for cartilage regeneration: affinity peptides, TGF-b1 and cell density, were evaluated by ectopic cartilage formation in nude mice. The efficacy of TGF-b1 affinity peptides in promoting orthotopic cartilage regeneration were evaluated in a rabbit cartilage defect model. The degradation of the implanted scaffold material and the associated immune response were also investigated. A schematic diagram of the experimental design is illustrated in Fig Experimental section 2.1 Preparation of TGF-b1 affinity chitosan scaffold Chitosan was purchased from Sigma-Aldrich (St. Louis, MO, USA). The peptide HSNGLPL (TGF-b1 affinity) was commercially purchased from Kangbei Biotechnology Co., China (purity 498%), and was custom-synthesized via solid phase peptide synthesis (SPSS) by CPC Scientific. Chitosan sponge was fabricated as described previously. 21 The chitosan sponge was modified with carboxylic groups using succinic anhydride (Guoyao, P. R. China), before being incorporated with peptide HSNGLPL via the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/n-hydroxysuccinimide (EDC/NHS) method. Chitosan sponges containing the following mass ratios of acetylated chitosan to peptide were prepared (10 : 0, 10 : 1, 10 : 2, 10 : 3), and were labeled as CHI, CHI-PEP 10%, CHI-PEP 20% and CHI-PEP 30%, respectively. For example, CHI-PEP 30% was prepared by incubating 0.16 g chitosan sponge in MES buffer solution containing 5mgml 1 of EDC and 5 mg ml 1 NHS solution for 30 minutes. After adjusting ph to 7.2, 0.05 g affinity peptide was added to react for 3 hours at room temperature. The sponges were then washed and freeze-dried. 2.2 Characterization of the chitosan sponges The morphology and pore sizes of the scaffolds were evaluated by SEM. The samples were mounted and sputter coated with gold palladium, before being examined with scanning electron microscopy (SEM; Quanta 200FEG, FEI, USA) at an accelerating voltage of 5 kv. Pore size was analyzed using the Image-Pro Plus (IPP) software with 13 repeated samples. Unconfined compression tests were carried out with an Instron 5843 mechanical testing system (Instron Corporation, America). All samples were preloaded three times to a 10% strain before constant loading was applied at a displacement of 0.5 mm min 1 until 40% of strain was reached under dry conditions. Young s modulus (E = s/e, where s and e denote the stress and strain of the sample, respectively) was determined automatically. This process was repeated on three specimens for each group. Characterization of the CHI and CHI-PEP (10%, 20% and 30%) scaffolds were performed with a Fourier-transform infrared spectrometer (FTIR, Nicolet is50, ThermoFisher, USA). 2.3 In vitro release of TGF-b1 To analyze the TGF-b1 release dynamics of the different scaffolds, CHI-PEP 30% and CHI sponges were first loaded with TGF-b1 by soaking in TGF-b1 solution (100 ng ml 1 ) for 3 h at room temperature. Then the samples were washed with PBS solution three times to remove the unbound growth factors. The sponges were then incubated in PBS and the amount of TGF-b1 released into PBS at 2 h, 6 h, 12 h, 24 h, 72 h, 120 h and 168 h was determined by ELISA (Ebioscience, ). The total amount of TGF-b1 incorporated in chitosan scaffolds were also determined by ELISA by dissolving the scaffold in 0.1 N HCl solution. 2.4 Cell isolation and culture After getting approval from Fuwai Hospital Chinese Academy of Medical Sciences ( GZR), MSCs were isolated from the 676 J. Mater. Chem. B, 2018, 6, This journal is The Royal Society of Chemistry 2018

3 bone marrow of pigs (Yorkshire, months). The MSCs were then washed with PBS for 5 minutes before being cultured in growth medium composed of Dulbecco s modified Eagle s medium (DMEM, Gibco-Invitrogen, China), 10% (v/v) of fetal bovine serum (FBS) (Gibco-Invitrogen), 100 mg ml 1 of streptomycin, and 100 mg ml 1 of penicillin at 37 1C in a humidified 5% CO 2 atmosphere. Non-adherent cells were washed out after 72 h. When cells reached 80% confluence, they were detached using 0.05% of trypsin (Gibco-Invitrogen) for further passage. 2.5 Chondrogenic differentiation of MSCs To screen for the optimal incorporation ratio of peptides to promote MSC chondrogenesis, in vitro chondrogenic differentiation was performed in differentiation medium composed of high glucose DMEM (HyClone-Thermo Fisher, China), 10 7 Mof dexamethasone, 50 mg ml 1 of ascorbic acid, 1 mm of sodium pyruvate (Sigma-Aldrich, China), 4 mm of proline (Sigma- Aldrich, China) and 1% of ITS + premix. The chitosan sponges (CHI and CHI-PEP 10%, 20% and 30%) were preloaded with 10 ml of 100 ng ml 1 TGF-b1 for 3 h, and then washed with PBS to remove any unbound growth factors prior to cell culture in the experimental group, where the chondogenic media did not contain any TGF-b1. Fifty microliter medium with 10 6 MSCs were loaded onto individual sponges (3 3 2 mm) and allowed to adhere for 3 h, before additional growth factor-free differentiation medium was added. The positive control was included, in which MSCs seeded CHI sponges were cultured in chondrogenic media supplemented with TGF-b1. The samples were cultured in the differentiation medium for 7 days or 21 days with medium change every three days. 2.6 F-actin fluorescence staining F-Actin fluorescence staining was used to investigate MSC morphology in the sponges. Chitosan sponges in the CHI and CHI-PEP 30% groups were immersed in 100 ng ml 1 TGF-b1 solution for 3 h before being co-cultured with MSCs in chondrogenic media without TGF-b1. MSCs seeded on CHI sponges, were cultured in chondrogenic media supplemented with TGF-b1, which served as the positive control. Samples on day 3 and day 7 were rinsed, fixed with 4% (w/v) of paraformaldehyde and permeabilized with 0.1% (w/v) of Triton-X 100. F-Actin were stained with rhodamine-conjugated phalloidin (Sigma). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Images were captured under confocal microscopy at a depth of 300 mm in the scaffold (CLSM-LSM510; Zeiss, Germany) (n = 3). 2.7 Quantitative real-time PCR RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer s instructions, as described previously. 22 RNA concentration was quantitated with the Nano-Drop instrument (Nano-Drop Technologies, DE, USA). The cdna was synthesized with the iscript TM cdna synthesis kit (Bio-Rad, CA, USA). Real time PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) on the Applied Biosystems 7500 RT-PCR System (Applied Biosystems). Cycling condition was as follows: 95 1C for 15 min, followed by 40 cycles of denaturation for 15 s at 94 1C, 30 s of annealing at 55 1C, and 30 s of elongation at 72 1C. The target genes were normalized to the reference gene glyceraldehydes-3- phosphate dehydrogenase (GAPDH). The primer sequences used are listed in Table S1 (ESI ). Five replicates samples were analyzed for each gene (n = 5). 2.8 Quantification of glycosaminoglycan (GAG) The GAG content was measured using DMMB assay. In brief, the cell scaffold compounds were digested in proteinase K (50 mg ml 1, Calbiochem) at 56 1C overnight, then heated in 90 1C for 10 minutes to stop the reaction. After filtration, the solution was conjugated with DMMB and agitated for 30 minutes. After conjunction, the solution was centrifuged at rpm for 10 minutes. The sediment was dissolved in a decomplexation and the absorbance was measured at 630 nm. 2.9 Ectopic implantation of scaffold constructs in nude mice Approval for the nude mice study was obtained from the IACUC of Peking University (COE-GeZ-3). Eight experimental groups (n = 3) were set up according to three parameters: sponges (3 mm 3mm2 mm) incorporated with/without TGF-b1 affinity peptides (CHI/CHI-PEP); with or without loading with TGF-b1 (10 ml of 100 ng ml 1 TGF-b1 solution for 3 h) (); low or high cell seeding density (MSCs were constituted to a titer of and cells per ml, and each scaffold was seeded with 100 ml of cell suspension), as described in Section 2.4. After cell seeding, the scaffolds were incubated in the differentiation medium without TGF-b1 at371c in5%co 2 for 7 days, before implantation in 6-week-old male athymic nude BALB/c mice. Briefly, the mice were tranquilized by ketamine (10 mg per 100 g of body weight) before 5 cm incisions were made in the middle-line of the back of nude mice. Scaffolds were inserted into subcutaneous pockets on the back. The mice were sacrificed at 4 weeks after implantation, and the implants were harvested together with the surrounding tissues. The tissue specimens were fixed with 4% (w/v) of paraformaldehyde at 4 1C for24hand embedded in paraffin. The specimens were then sectioned (5 mm thick) and processed for Safranin-O staining and immunohistochemical staining of collagen I, collagen II and collagen X Rabbit full thickness articular cartilage defect study All experiments were conducted according to the protocols established by the Animal Research Committee of the People s Hospital of Peking University. The rabbits were assigned into three groups (n = 8, 24 male New Zealand White rabbits at 3 months of age); blank group (defect left empty without implantation), CHI group and CHI-PEP (CHI-PEP 30%) group. After anesthesia with chloral hydrate, a midline incision was made in front of the knee joint. Defects with 4 mm in diameter and 4 mm in depth were created at the middle of the femoral trochlear of the knee joint. Sponges (4 mm in diameter, 4 mm in height) were pressed-fit into the defects of the experimental groups. The rabbits were sacrificed at 3 months and 6 months (n = 4 per time point), while the implants were harvested This journal is The Royal Society of Chemistry 2018 J. Mater. Chem. B, 2018, 6,

4 together with the surrounding tissues (Two rabbits belonging to the Blank group and CHI-PEP group with otodectes cynotis were sacrificed at 2 months and 4 months respectively. The final number of samples for statistical analysis were 3 for each group). The harvested tissue specimens were fixed in 4% (w/v) paraformaldehyde at 4 1C for 24 h and decalcified for 3 weeks before being embedded in paraffin Histology and immunohistochemical analysis For H&E staining, tissue sections were stained in Harris hematoxylin solution for 20 minutes, and then counterstained in eosin phloxine solution. For Safranin O staining, tissue sections were subjected to aqueous Safranin O (0.1%, w/v) and Fast green solution (0.001%, w/v). For the immunohistochemical staining of mice samples, tissue sections were incubated with hydrogen peroxide to block endogenous peroxidase, followed by pepsin treatment for 20 min. Monoclonal antibodies against collagen I (dilution, 1 : 100, ab23446, Abcam, UK), collagen II (dilution, 1 : 200, ab34712, Abcam, UK), and collagen X (dilution, 1 : 2000, ab49945, Abcam, UK) were then added and incubated at 4 1C overnight, followed by addition of a biotinylated goat antimouse IgG antibody (ab64259, Abcam, UK). After incubation with streptavidin peroxidase, the sections were stained with 3,3 0 - diaminobenzidine as the chromogenic agent. Gill s hematoxylin was applied for the cell nuclei counterstaining. The stained tissues were imaged under a light microscope (DM6000M, Leica, Germany) and analyzed by Image-Pro Plus. For immunohistochemical staining of rabbit samples, the tissue sections were treated with hydrogen peroxide and pepsin as described above. Then monoclonal antibodies against collagen I (dilution, 1 : 200, ab90395, Abcam, UK), collagen II (dilution, 1 : 100, CP-18, Calbiochem, USA), and macrophages (RAM-11, dilution, 1 : 200, M063301, Dako, Denmark) were added and the mixture was incubated at 4 1C overnight, followed by addition of a biotinylated goat anti-mouse IgG antibody, streptavidin peroxidase, 3,3 0 -diaminobenzidine and Gill s hematoxylin as described previously. The stained tissues were scanned by Aperio epathology (Leica, Germany) and scored by ICRS Macroscopic score assessment and ICRS Visual Histological Assessment Scale 23 (Tables S2 and S3, ESI, n = 3, each sample judged by two individuals) Statistical analysis Statistical analysis was performed using SPSS V17.0 (one-way ANOVA, (*) for P o 0.05, (**) for P o 0.01, and (***) for P o 0.001) for characterization of the sponge scaffolds, PCR analysis, immunohistochemical staining analysis of the nude mice model and macroscopic evaluation of the rabbit cartilage repair. The data are expressed as mean standard deviation. 3. Results 3.1 Characterization of the chitosan sponge and controlled release of TGF-b1 A series of chitosan scaffolds with different mass ratios of TGF-b1 affinity peptides were designed and fabricated to induce chondrogenic differentiation of MSCs. TGF-b1 affinity chitosan scaffolds were prepared by introducing carboxylic groups to chitosan using succinic anhydride. Then the scaffolds were grafted with peptide HSNGLPL via the 1-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride/n-hydroxysuccinimide (EDC/NHS) method (Fig. 2A). SEM images showed that the chitosan sponges were porous with good connectivity (Fig. 2B). The average pore sizes of CHI-PEP sponges increased from 54.5 mm (CHI) to mm with increased ratio of peptide-incorporated chitosan (no significant differences with different ratios of incorporated peptide, Fig. 2C). Sponges incorporated with TGF affinity peptide with a ratio of 30% had relatively lower Young s modulus ( kpa) than the unconjugated chitosan sponges ( kpa; mean standard deviation, Fig. 2D). FTIR was used to determine whether the TGF affinity peptides had been successfully incorporated into the chitosan sponge scaffold. The stretching band of the NH bond from amide in 3349 and 3286 cm 1 and the CQO bond from amide in 1647 and 1552 cm 1 were obviously higher in CHI-PEP 30%, thus suggesting successful incorporation of TGF affinity peptides (Fig. 2E). As compared with the CHI group, TGF-b1 released from the CHI-PEP 30% sponges presented a slower release curve without initial burst release of TGF-b1 at the early time-points (Fig. 2F and Fig. S1, ESI ), suggesting successful binding between TGF-b1 and TGF affinity peptides and the positive effects of the affinity peptide on the controlled release of growth factors (Fig. 2F). 3.2 Inducement of MSC chondrogenesis in CHI-PEP sponges preloaded with TGF-b1 To investigate the chondrogenic capacity of CHI-PEP bound with TGF-b1, TGF-b1 were loaded onto the sponges, before the MSCs were seeded. The scaffold constructs were cultured in growth factor-free chondrogenic media. After 7 days of in vitro culture, the gene expression levels of Sox 9, Col II and aggrecan in the CHI-PEP 30% scaffolds were the highest compared with other groups (Fig. 3A). The gene expression profiles exhibited similar trend at 21 days, with higher expression levels of Col II and aggrecan in the CHI-PEP 30% group (7.8 and 4.5 folds). Although Col I was upregulated in the CHI-PEP 30% group on day 7, this was not significantly different compared with other groups on day 21. Col X remained low at both time points in the CHI-PEP group (Fig. 3A). The ratios of Col II/Col I and Col II/Col X have also been calculated. The highest ratio of Col II/Col I showed up at the CHI-PEP 10% groups, while the highest level of Col II/Col X appeared at the CHI-PEP 30% group at day 7. On day 21, groups of CHI-PEP 30% had the highest level of Col II/ Col I, and the groups of CHI-PEP 10% and CHI-PEP 30% had relatively high level of Col II/Col X. The comparisons of Col II/ Col I and Col II/Col X showed that the CHI-PEP 10% and 30% groups had better results. Except for Col II/Col I on day 7, the groups of CHI-PEP 30% exhibited high levels of Col II/Col I and Col II/Col X, with significant increases being observed at day 7. The expression levels of upregulated chondrogenic genes (Sox 9, Col II and AGG) in the CHI-PEP 30% group were significantly higher compared to the positive control, in which MSC in the 678 J. Mater. Chem. B, 2018, 6, This journal is The Royal Society of Chemistry 2018

5 Fig. 2 Fabrication and characterization of chitosan sponge. (A) Schematic illustration of synthesis of the chitosan scaffolds incorporated with TGF-b1 affinity peptides. (B) SEM images of chitosan sponges crosslinked acetylated chitosan scaffolds (CHI), 10% peptide grafted scaffolds (CHI-PEP 10%), 20% peptide grafted scaffolds (CHI-PEP 20%), 30% peptide grafted scaffolds (CHI-PEP 30%), respectively. All ratios refer to the mass ratio (peptide : chitosan). (C) Pore sizes of sponges with different composition (n =13,*Po0.05). (D) Compressive Young s modulus of sponges with different composition (n =5, *P o 0.05). (E) FTIR results of CHI and CHI-PEP (10%, 20% and 30%). * Higher peak appearing after grafting with affinity peptide with a mass ratio of 30%. (F) Release dynamics of TGF-b1 from the CHI and CHI-PEP 30% in vitro (n = 3, mean values SD). CHI sponges were cultured in chondrogenic media supplemented with TGF-b1. These results on MSC differentiation in vitro showed that the scaffolds incorporated with the TGF affinity peptide with a ratio of 30% had distinctly better effects in inducing MSC chondrogenic differentiation. Hence, the CHI-PEP 30% scaffold was chosen for subsequent experiments. In the following experiment, the groups, with peptide incorporated within chitosan sponges with a mass ratio of 30%, was denoted as CHI-PEP. This journal is The Royal Society of Chemistry 2018 J. Mater. Chem. B, 2018, 6,

6 Fig. 3 Chondrogenic differentiation of MSCs and cell morphology. (A) Gene expression (fold change, normalized to GAPDH) of selected chondrogenic and hypertrophic markers in BMSCs-seeded chitosan sponge scaffolds after 7 or 21 days of in vitro culture (n= 5, mean values SD, *P o 0.05). The positive control group refers to MSCs cultured in CHI sponges with chondrogenic media containing TGF-b1. Other groups (CHI and CHI-PEP 10%, 20%, 30%) were cultured in chondrogenic media without TGF-b1. (B) The morphology of clustered MSCs in the CHI, positive control and CHI-PEP groups. Red: F-actin; blue: nuclear (DAPI). F-Actin was stained with rhodamine phalloidin after 3 days and 7 days in culture. CHI-PEP here and after means peptide grafted on chitosan sponges at a mass ratio of 30%. (C) The morphology of single MSCs seeded on the CHI, positive control and CHI-PEP groups. F-Actin was stained with rhodamine phalloidin after 3 days and 7 days in culture. 3.3 MSC morphology in CHI-PEP sponges To investigate the effects of CHI-PEP bound with TGF-b1 on MSC morphology, we utilized rhodamine phalloidin to stain for F-actin, so as to observe cytoskeletal morphological changes. The results showed that the MSCs seeded in CHI sponges were slightly stretched and displayed fibroblast-like morphology in isolated or clustered cell colonies, persisting up to day 7 (Fig. 3B and 3C). Non-clustered MSCs seeded in CHI-PEP sponges adopted rounder morphology as early as day 3, while clustered cells aggregated and showed apparent condensation. Morphology of MSCs in the CHI-PEP sponge was very similar to cells in the positive control (CHI + TGF-b1) groups (Fig. 3B and C). 3.4 Chitosan sponges incorporated with TGF-b1 affinity peptide and preloaded with TGF-b1 significantly enhanced ectopic cartilage formation in nude mice To evaluate the chondrogenic capacity of CHI-PEP in vivo and the effects of various factors including cell density, affinity peptide and growth factor, a series of scaffolds seeded with MSCs were implanted subcutaneously on the back of nude mice for 4 weeks. The Safranin-O staining were darker in scaffolds incorporated with affinity peptide and preloaded with TGF-b1 (Fig. 4A). Semi-quantitative analysis of staining optical density showed the same mean density trend (IOD sum/area, Fig. 4B). Upon immunohistochemical staining of collagen II, the most intense Col II staining was also detected in scaffolds incorporated 680 J. Mater. Chem. B, 2018, 6, This journal is The Royal Society of Chemistry 2018

7 Fig. 4 Implants in nude mice model at 4 weeks post-surgery with a seeding density of 106 cells per sponge. (A) Results of Safranin-O staining and immunohistochemistry staining for detection of collagen II, I and X, with a seeding density of 106 cells per sponge. (B) GAG content in sponges. (C) Collagen II content in sponges. (D) Collagen I content in sponges. (E) Collagen X content in sponges (n = 9, mean values SD, *P o 0.05, **P o 0.01, ***P o 0.001). Scale bar represents 50 mm. with affinity peptide and preloaded with TGF-b1 (Fig. 4A and C), which was consistent with the Safranin-O staining results. Staining of collagen type I revealed that groups incorporated with TGF-b1 affinity peptide and preloaded with TGF-b1 had This journal is The Royal Society of Chemistry 2018 significantly lower mean density of collagen I (Fig. 4A and D). Staining of collagen X, regarded as the indicator of hypertrophy, also revealed that groups incorporated with TGF-b1 affinity peptide and preloaded with TGF-b1 had relatively lower mean J. Mater. Chem. B, 2018, 6,

8 density than other groups (Fig. 4A and E). These results indicated that incorporation with the affinity peptide and preloading with TGF-b1 could promote chondrogenic differentiation of MSC, as well as ameliorate hypertrophy. The results with scaffolds at a cell seeding density of cells per sponge showed a similar trend (Fig. S2A E, ESI ). 3.5 Chitosan sponges incorporated with TGF-b1 affinity peptides promote cartilage regeneration in a rabbit osteochondral defect model The capacity of chitosan sponges with and without incorporated TGF-b1 affinity peptide in promoting host tissue orthotropic cartilage regeneration was investigated in a rabbit osteochondral defect model. The cartilage defect was implanted with chitosan sponges without exogenous TGF-b1 or cells. Microscopic examination indicated that more than 75% of the defect was filled in both the CHI-PEP groups, while less than 50% of the defect areas were filled in the Blank group after 3 months of cartilage regeneration (Fig. 5A). After 6 months of cartilage regeneration, the gross appearance revealed good filling of the chondral defects with a smooth surface in the CHI-PEP sponge group compared with the Blank and CHI groups, in which tissue indentation at the defect site was obvious (Fig. 5A). The ICRS macroscopic score was highest in the CHI-PEP group at both 3 and 6 months of regeneration ( and ), followed by the Blank group ( and ) and CHI group ( and ) (Fig. 5B, total points were 12). H&E staining was performed to assess the cell distribution and tissue regeneration. After 3 months of regeneration, the remaining sponges in both the CHI group and CHI-PEP group were found within the marrow, and sponges in the CHI-PEP groups appeared to be more degraded compared with the CHI sponges (Fig. 5C, 4 mm in diameter, 4 mm in height when implanted). No sponges were observed in the CHI-PEP group, but there were still significant remaining residues in the CHI group after 6 months of regeneration (Fig. 5D, gray arrow in H&E). Compared with the Blank and CHI groups, the cells in the CHI-PEP groups appeared to exhibit a more column-like distribution at both 3 and 6 months post-surgery (Fig. 5C and D). After 6 months of regeneration, only a sparse number of cells could be observed in the defect regions of the CHI groups, in contrast to abundant cell distribution in the CHI-PEP groups, as compared with the surrounding healthy cartilage tissues (Fig. 5D). Although one joint had some connective fibrous tissues and minor inflammation, the regenerated surface was still smooth, indicating successful cartilage regeneration (Fig. S3, ESI ). To evaluate cartilage regeneration, different regions of the defect including the lateral, intermediate and interior regions of the defect sites were stained with Safranin-O and then ICRS Fig. 5 Cartilage regeneration in rabbits at 3 months and 6 months post-surgery. (A) Macroscopic appearance of the articular cartilage. (B) ICRS macroscopic score of regenerated tissues (n = 3, mean values SD, *P o 0.05). (C) H&E staining of cartilage after 3 months of regeneration. (D) H&E staining of cartilage after 6 months of regeneration (n = 3). 682 J. Mater. Chem. B, 2018, 6, This journal is The Royal Society of Chemistry 2018

9 visual histology score assessment was carried out (an illustration is presented in Fig. S4, ESI ). Compared with the Blank and CHI groups, the cartilage surface in CHI-PEP was smooth and continuous at both 3 and 6 months post-surgery. And cartilage regeneration in the lateral area was the best in almost all groups at both 3 and 6 months post-surgery (Fig. 6A and C). In addition, new tide mark was observed in the defect area in the CHI-PEP groups after 3 months regeneration in all regions, while the blank and CHI groups did not show the same results. After 6 months of regeneration, in the CHI-PEP groups, new tideline formation was observed which was continuous with the original tideline in all regions (Fig. 6A and C). In summary, after 3 months of regeneration, the CHI group showed no improvement, except for the lateral region, getting even worse when compared to the Blank control group (Fig. 6A and B). The CHI-PEP group had the highest score indicating improved repair (average scores were , , , from the lateral to interior part). For the CHI and CHI-PEP groups, regenerated tissues in the lateral area exhibited the best outcome while the interior area displayed the worst (Fig. 6B and Table 1). Both the CHI group and Blank control group showed little GAG deposition whereas the CHI-PEP group had significantly higher GAG content in the defects (Fig. 6A(c, f and i)). After 6 months of regeneration, the score of the CHI group ( , , ) was the lowest, while the CHI-PEP group (12.7 3, , ) had the highest scores (Fig. 6D and Table 2). Meanwhile, the CHI group displayed higher collagen type I deposition, filling the unrepaired defect, while the CHI-PEP group showed the highest collagen type II deposition at both 3 and 6 months post-surgery (Fig. 7A and B). Immunohistochemical staining demonstrated the presence of macrophages at the defect sites of the CHI group (Fig. 7D, black arrows), indicating inflammation elicited by the pure chitosan scaffold. Except CHI-PEP at 6 months postimplantation, infiltrated macrophages could be detected within the scaffolds in all groups (Fig. 7C and D). After 3 months regeneration, CHI-PEP exhibited better regeneration and significantly less macrophage were detected in the defect, as compared with the CHI groups. After 6 months regeneration, a large number of infiltrated macrophages could still be detected in the CHI group, but not in CHI-PEP. All these results thus Fig. 6 Safranin-O staining and scoring at 3 months and 6 months post-surgery. (A) Safranin-O staining from the lateral to interior region of the defects at 3 months post-surgery. (B) ICRS visual histological score at 3 months post-surgery (n = 3, mean values SD). (C) Safranin-O staining from the lateral to the interior region of the defects at 6 months post-surgery. (D) ICRS visual histological score at 6 months post-surgery (n = 3, mean values SD). Scale bar represents 2 mm. This journal is The Royal Society of Chemistry 2018 J. Mater. Chem. B, 2018, 6,

10 Table 1 ICRS itemized rating score after 3 months of regeneration Blank CHI CHI-PEP Lateral Intermediate Interior Lateral Intermediate Interior Lateral Intermediate Interior 3 months Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Surface Matrix Cell distribution Cell population viability Subchondral Cartilage mineralization Table 2 ICRS itemized rating score after 6 months of regeneration Blank CHI CHI-PEP Lateral Intermediate Interior Lateral Intermediate Interior Lateral Intermediate Interior 6 months Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Surface Matrix Cell distribution Cell population viability Subchondral Cartilage mineralization indicate that chitosan sponge incorporated with the TGF-b1 affinity peptide is bioactive and chondro-inductive, and can efficiently promote cartilage regeneration. 4. Discussion Temporal delivery of TGF-b is critical for chondrogenic differentiation, particularly during the first week or slightly later, when receptors of TGF-b are abundantly expressed. 24,25 On the other hand, uncontrolled continuous exposure to TGF-b1 has been reported to produce adverse side effects on the joints, resulting in osteophyte formation, swelling and synovial hyperplasia. 26 Compared with absorption, encapsulation and covalent binding, peptides with specific affinity to TGF-b provide strong binding without interfering with its bioactivity. 8 In this study, TGF-b1 binding to affinity peptides released from the CHI-PEP scaffolds exhibited a slower release curve without initial burst release of TGF-b1 during the early time-points, as compared to TGF-b1 adsorbed on CHI sponges. The incorporation of TGF-b1 affinity peptides on the CHI sponge scaffold, led to higher expression levels of chondrogenic related genes by MSCs in the CHI-PEP groups. Incorporation with TGF-b1 affinity peptides and preloading of TGF-b1 synergistically enhanced ectopic cartilage formation. At both high and low seeding densities, either incorporation with TGF-b affinity peptides or preloading of growth factors had the capacity to enhance ectopic cartilage formation (Fig. 4 and Fig. S1, ESI ). However, a combination of these two factors resulted in much stronger enhancement of the chondrogenic differentiation of MSCs. This could be because the pre-loaded incorporation of TGF-b affinity peptides resulted in a slower release of TGF-b that enhanced chondrogenic differentiation of the MSCs, instead of being exhausted quickly by burst release. 18 Because the TGF concentration is relatively low in subcutaneous tissue, delivery of exogenous TGF is still necessary. 27 Scaffolds incorporated with TGF-b1 affinity peptides could significantly enhance regeneration of articular cartilage and ameliorate inflammation. The TGF-b1 affinity peptide could bind TGF-b1 from the synovial fluid or bone marrow, where TGF-b1 is known to be abundant, particularly during injuryinduced inflammation. 28,29 TGF-b1, a chemoattractant, can recruit MSCs from the bone marrow and synovium to migrate to the injury site. 30,31 Final differentiation of MSCs towards the chondrocyte lineage under induction by TGF-b1 would repair the defect and regenerate cartilage tissues. In this study, chitosan scaffolds incorporated with TGF-affinity peptides significantly ameliorated inflammation at 6 months post-implantation, possibly through recruitment and differentiation of progenitor/stem cells, as well as by modulating the immune-responses against the implanted biomaterials. The effective utilization of biomaterials in cartilage regeneration is ambiguous, as biomaterials could elicit unexpected side effects, particularly through uncontrolled degradation. 32 An increase in the degree of acetylation of chitosan scaffolds could induce a more intense inflammatory response upon implantation in vivo. 33 The recruited TGF-b, undifferentiated MSCs and MSCs undergoing chondrogenic differentiation, could be involved in this process, either individually or synergistically, through the secretion of immunomodulatory anti-inflammatory cytokines, which could in turn enhance cell survival Though regeneration in the lateral regions of the CHI group was better than that in the Blank groups at 3 months post-implantation, this difference became undiscernible at 6 months post-implantation. In general, there was poor regeneration in both the CHI and blank groups. 684 J. Mater. Chem. B, 2018, 6, This journal is The Royal Society of Chemistry 2018

11 Fig. 7 Immunohistochemical staining for detection of collagen I, II, and macrophages within the rabbit cartilage defect. (A) Immunohistochemical staining for detection of collagen II after 3 or 6 months of cartilage regeneration. (B) Immunohistochemical staining for detection of collagen I after 3 or 6 months of cartilage regeneration. (C) Immunohistochemical staining for detection of macrophages after 3 months of cartilage regeneration. (D) Immunohistochemical staining for detection of macrophages after 6 months of cartilage regeneration. As fewer cells could penetrate into the interior of the scaffolds because of relatively small pore sizes and lack of enough chemoattractant (in this case, TGF-b1), the regeneration of the interior area was therefore inhibited. As time progressed, some regenerated cartilage was eroded by inflammation caused by chitosan degradation, since chitosan could provoke inflammation through activation of the complement system after implantation Conclusion Chitosan sponges incorporated with TGF-b1 affinity peptides significantly enhanced chondrogenic differentiation of MSCs in vitro and cartilage regeneration in vivo within a rabbit osteochondral defect model. In the absence of exogenous bioactive molecules, these scaffolds provide an economical, safe and effective strategy for clinical cartilage regeneration. Conflicts of interest There are no conflicts of interest to declare. Acknowledgements The authors would like to thank Prof. B. P. Chan for manuscript proofreading and constructive advice. The authors would also like to acknowledge support from the National Natural Science Foundation of China ( and ). References 1 C. H. Lee, et al., Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study, Lancet, 2010, 376(9739), This journal is The Royal Society of Chemistry 2018 J. Mater. Chem. B, 2018, 6,

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