Biomaterials 52 (2015) 463e475

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Mechano growth factor (MGF) and transforming growth factor (TGF)b3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model Ziwei Luo a, b, 1, Li Jiang c, 1, Yan Xu b, Haibin Li a, Wei Xu a, Shuangchi Wu a, Yuanliang Wang a, Zhenyu Tang b, Yonggang Lv a, *, Li Yang a, ** a

Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400030, PR China SARI Center for Stem Cell and Nanomedicine, Chinese Academy of Science, Shanghai 201210, PR China c Department of Orthopaedics, Huashan Hospital, Fudan University, Shanghai 200040, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 August 2014 Accepted 6 January 2015 Available online 18 March 2015

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor b3 (TGF-b3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-b3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-b3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-b3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7days after subcutaneous implantation, TGF-b3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29þ/CD44 þ cells (P < 0.05). Similarly, more cartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-b3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (P < 0.05), indicating that MGF and TGF-b3 might be a better candidate for cartilage regeneration. This study demonstrated that TGF-b3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Mechano growth factor (MGF) Silk fibroin Stem cell recruitment Fibrocartilage Articular cartilage regeneration

1. Introduction Abbreviation: MSCs, mesenchymal stem cells; SF, silk fibroin; TGF-b3, transforming growth factor beta 3; MGF, mechano growth factor; TM, in vitro samples with TGF-b3 and MGF treatment; ST, TGF-b3 modified silk scaffolds; STM, TGF-b3 and MGF modified silk scaffolds; AGC, aggrecan; Collagen I, types I collagen; Collagen II, types II collagen. * Corresponding author. Tel./fax: þ86 23 65102507. ** Corresponding author. Tel.: þ86 23 65111802; fax: þ86 23 65102507. E-mail addresses: [email protected] (Y. Lv), [email protected] (L. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.01.001 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

The regeneration of damaged articular cartilage caused by disease or trauma is limited by poor ability to self-repair, and even small articular cartilage defects can progress to scar tissue mainly made up of premature fibrocartilage and finally lead to osteoarthritis (OA) over 2 years if left untreated [1]. Despite the high prevalence and morbidity of OA, there is a challenge of considerable appeal to researchers and clinicians [2]. Autologous chondrocyte implantation (ACI) has been developed as early as 1980s and used

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in trials for decades [3], but it is usually limited by a low yield of implanted chondrocytes and spontaneous dedifferentiation during in vitro expansion [4,5]. Adult endogenous MSCs or prechondrocytes resident in joint tissue provide a great potency for postnatal tissue repair [2,6e10]. These autologous multipotent cells, usually are quiescent in mature tissue or circulating blood, can be actived and recruited to injured sites in response to some specific signals, e.g. bioactive factors and/or mechanical factors [11,12]. Clinically, marrow stimulation techniques (MST), such as subchondral drilling and microfracture, are generally considered as preferred treatment for full-thickness cartilage lesions and have demonstrated good to excellent results in 60e80% of patients [13,14]. The aim of these surgical techniques to repair articular cartilage injuries is to achieve the regeneraiton of organized hyaline cartilage [15]. However, blood and mesenchymal cells from the underlying marrow cavity form clots in the defects that usually degenerates into scar and fibrocartilaginous tissue, especially in cases of large lesions [13,14]. An ideal scaffold plays key roles in regenerative medicine, in which host MSCs migrate to scaffolds and enhance healing without exogenous cells [16,17]. Silk fibroin (SF), derived from Bombyx mori cocoons, is a widely used natural protein polymer for biomaterial and biomedical applications. SF has remarkable mechanical properties, low immunological rejection, distinguished biocompatibility, and controllable degradation rates. Due to amount of hydrophilic groups, SF can be chemically modified to alter surface properties, or to immobilize growth factors for continuous release when exposed to alcohol to induce b-sheet crystalline conformation changes [18,19]. Although silk-based scaffolds had been designed for articular cartilage tissue engineering or regeneration and revealed excellent results [19e25], none scaffold was fabricated with pured silk solution. SF has demonstrated excellent properties for drug delivery, including antibiotic [26], protein or small molecule drugs [27]. Transforming growth factor beta 3 (TGF-b3) plays an indispensable role for chondrogenesis in embryonic development and postnatal tissue homeostasis. Other studies also demonstrated that TGF-b3 enhanced chondrogenesis and neocartilage regeneration both in vitro and in vivo [12,28,29], along with Collagen I expression at the trochlear sites. Importantly, a lot of studies have revealed that TGF-b could promote the migration and recruitment of MSCs [30e35]. Give its classical role in chondrogenesis, it is reasonable for TGF-b to serve as positive control in this study. Mechano growth factor (MGF), an alternative isoform of IGF-1 produced by stress, injury [36] or disease [37e39], has attracted increasing attentions because of its great potential in skeletal muscle, heart and neuron repair [36,40e42]. MGF is distinguished from IGF-1 by its specific C-terminal and has an independent effect on satellite cells [43], osteoblasts [44] and MSCs [45]. We and others have shown that MGF could not only directly stimulate human and rodent MSC migration [45e47], but also protect cardiomyocytes from hypertrophy and fibrosis in vivo [48]. However, whether MGF could influence the chondrogenic differentiation of MSCs is still unknown. In this study, we fabricated sponge-like silk fibroin scaffolds with incorporation of TGF-b3 and MGF to evaluate its effects on articular cartilage regeneration in New Zeland rabbits. We showed that TGF-b3 and MGF functionalized silk fibroin scaffolds (STM) could recruit mesenchynal multipotent cells for in situ articular cartilage regeneration and fibrosis suppression, thus providing a novel strategy for cartilage repair. 2. Materials and methods 2.1. Materials and reagents All reagents used for cell culture, including Dulbecco's Modified Eagle's Media (DMEM), fetal bovine serum (FBS), insulin, antibiotics and trypsine were purchased

from Life Technologies, Co., while recombinant basic fibroblasts growth factor (bFGF), epidermal growth factor (EGF) and transforming growth factor (TGF)-b3 were purchased from PeproTech Inc. (NJ, USA), except for mechano growth factor (MGF, 033-35) and Fluorescein isothiocyanate (FITC) labbled MGF (FITC-MGF, FG033-35B) were from Phoenix Pharmaceuticals, Inc. (AZ, USA). All drugs used for in vitro differentiation of hMSCs and isolated multipotent stem cells, including dexamethsome, b-glycerophosphate, L-ascorbic acid, indomethacin, isobutylmethylxanthine (IBMX), ITS þ 1, sodium pyruvate, L-proline, were purchased from SigmaeAldrich (CA, USA). Cytochemical staining dyes were from Shanghai Shenggong (Shanghai, China) and Beyontime (Shanghai, China). Primary and secondary antibodies used in this study were shown in Table 1. Quantification kits were all obtained from USCN, Inc. (Wuhan, China). 2.2. Cell isolation and cell culture 2.2.1. hMSCs culture hBMSCs were kindly gift from Dr. Song Li (UC Berkeley) and maintained as described before [49]. Briefly, Bone marrow aspirates from three healthy donors (ranging in age from 22 to 42 years old) were plated at a density of 10 ml of aspirate in a 10 cm diameter cell culture dish (Corning, NY) in low-glucose DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 U/ml), 0.1 mM nonessential amino acids, basic fibroblast growth factor (bFGF, 20 ng/mL) and epidermal growth factor (EGF, 20 ng/ml). Cells were maintained in a humidified incubator at 37 C with 5% CO2. Half of medium was replaced at 48 h of initial culture and every 2 or 3 days thereafter. When 70%e80% confluent, adherent cells were trypsinized with 0.05% trypsin-1 mM EDTA at 37 C for 2 min, harvested, and expanded in other dishes. A homogenous cell population was obtained after 2 weeks of culture. hMSCs at passage 2 or 3 cells were used for in vitro experiments in this study. 2.2.2. Multipotent stem cells isolation and identification from animals To isolate rabbit mesenchymal cells, four independent STM scaffolds from subcutaneous and defected joints implantation were harvested, washed three times with PBS supplemented with 1% penicillin/streptomycin (P/S), and cut into pieces for tissue explant culture. Culture medium was replaced twice a week as described above and cells were collected for further experiments. To character the isolated cells, Flow cytometry was used to detected surface antigens. When 70% confluent, cells were collected and non-specific sites were blocked with 1% BSA, incubating with 1ug of monoclonal primary antibodies (Table 1) for 30 min on ice. The supernatant was then centrifuged, removed and the sediment was rinsed with PBS to remove the excessive antibody. After resuspention, fluorescent-labeled secondary antibodies were added, incubating together for 30 min at room temperature. After rinsing with PBS, the cells were resuspended in 500 ml of 2% paraformaldehyde PBS solution and acquired on a BD FACSVerse™ flow cytometer and analyzed using FlowJo software (Tree Star). A non-specific antibody of the same isotype as the primary antibody was used as negative control. 2.3. In vitro effects of MGF and TGF-b3 on hMSCs 2.3.1. Proliferation assay To study the effects of MGF on cell growth, a density of 2000 cells per well of hMSCs were seeded into a 96-well plate as described [46]. MGF (20 ng/ml) and TGF-

Table 1 Primary and secondary antibodies used in this study.

Primary antibody Collagen II Collagen II Collagen II Collagen I Aggrecan (AGC) CD45 CD29 CD44 CD 68 Galectin-3 Secondary antibodies Donkey anti-mouse IgG Donkey anti-rabbit IgG Donkey anti-rat IgG Donkey anti-mouse IgG Donkey anti-rabbit IgG 40 , 6-diamidino-2phenylindole (DAPI)

Company

Catalog

Application/ dilution

Abcam Abcam Calbiochem Sigma Abcam AbD Serotec Abcam Abcam Abcam Abcam

ab79127 ab34712 CP-18 C2456 ab3773 MCA808GA ab78502 ab119335 ab955 ab2785

IF/1:200 IF/1:200 IF/1:200 IF/1:200 IF/1:200 FC/1:100 FC/1:100 FC/1:100 IF/1:200 IF/1:200

Life Life Life Life Life Life

A-21202 A-21206 A-21208 A-11056 A-10040 D3571

IF/1:1000 IF/1:1000 IF/1:1000 IF/1:1000 IF/1:1000 1ug/ml

technologies technologies technologies technologies technologies technologies

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b3 (10 ng/ml) were added into each well. Cell activities were determined by Cell Counting Kit-8 (CCK-8, Sigma) following the manufacturer's protocol. 2.3.2. Chemotaxis assay To compare the cell mobility between MGF and TGF-b3, migration assay was performed with Transwell system (Sigma) as previous study [50] following the manufacturer's protocol. Briefly, 2000 cells were seed at the bottom of upper chamber, and the lower chamber was added with 500 ul DMEM and 0.5% FBS, supplemented with MGF (20 ng/ml) or TGF-b3 (10 ng/ml). 8 h after seeding, cells were fixed with 4% PFA and stained with DAPI for 10 min at room temperature. An Olympas fluorenscence microscope was used for analysis of cell migration (n ¼ 3 doners). A tipcal wounds healing model was also introduced as described before [51]. A linear wound was introduced by scraping hMSCs cells with the same pipette tip, followed by extensive washing to remove cellular debris as described previously. To determine the repair rate, images were taken over a period of 0e24 h. The wound areas were quantified by image analysis software (cellSense Entry). Repair rate was equal to the reduced scratching area divided by the initial scratching area. 2.3.3. Chondrogenic differentiation Chondrogenic differentiation of hMSCs was performed as described [52]. Briefly, 2.5  105 cells were colleted and centrifuged in a 15-ml polypropylene conical tubes following a 15-day exposure to chondrogenic medium, containing 0.1 uM Dexamethsome, 50 ug/ml L-Ascorbic acid, 2.5% ITS þ 1, 100 ug/ml sodium pyruvate, 40 ug/ml L-proline, supplemented with TGF-b3 (10 ng/ml), or MGF (20 ng/ml), or the combination. Typically, TGF-b3 plays a key role in chondrogenic differentiation and is widely use. In this study TGF-b3 serve as positive control. MGF was added once a day because of its short term of half-life [53]. Pellets were collected for immunofluorescence and quantification of aggrecan, Collagen I and Collagen II. For qualitative analysis, cell pellets were frozen in OCT embedding and cut into 5 mm sections, followed by incubation of primary antibodies (Table 1) at 4 C overnight. A rabbit anti-collagen II (ab34712) was used for double staining for Collagen I and Collagen II. After incubation with specific secondary antibodies for 1 h and DAPI for 10 min (Table 1) at room temperature, sections were placed under an Olympas microscope and analyzed by cellSens software. For quantitative analysis, total protein were extracted and determined by ELISA kits following the manufacturer's guidence. 2.4. Preparation and characterization of MGF and TGF-b3 based silk fibroin scaffolds 2.4.1. 3D pured SF scaffold preparation 15e20% silk fibroin (SF) aqueous solution was prepared as before [18,54]. Sponglike 3D scaffolds were obtained after exposure to a vacuum freeze drier (Christ, Germany) for overnight. When TGF-b3 (10 ng/ml) was added to the same SF solution (3 ml), the spong-like scaffolds were marked with ST, as similar as to the STM (mixture of 10 ng/ml of TGF-b3 with 20 ng/ml of MGF, 3 ml). Scaffolds were collected for further application. 2.4.2. Fourier-transform infrared (FTIR) spectroscopy To investigate b-sheet conformation, SF scaffold was immersed in 90% methanol. The absorption peak files of different groups were obtained by a Nicolet spectrometer system (System 2000, PerkineElmer, USA) over a range of 4000e400 cm1. 2.4.3. Scanning electron microscopy (SEM) The morphology of SF scaffolds was characterized by field emission scanning electron microscopy (FESEM; Nova 400 NanoSEM, FEI, Germany) with an accelerating voltage of 10 kV after gold coating. Only SF scaffolds were characterized by SEM. 2.4.4. Mechanical properties The compressive modulus of SF scaffolds were determined by a tabletop uniaxial testing instrument (Instron, British) using a 50-N load cell under a speed of 1 mm per minute. All scaffolds were prepared in the form of cylinder shape with 3 mmdiameter and 3 mm-height. Five independent experiments were prepared. Native cartilage of rabbits were used for positive control. 2.4.5. Degradation rate of SF scaffolds SF scaffolds were immersed in a 6-well plate containing 5 ml PBS in each well and were incubated at 37 C for 40 days. At 0.5, 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, and 40 days, scaffolds were taken out and excess water were sucked, air dried in a 37 C incubator for 24 h, and subsequently turn to weight. Five independent SF scaffolds were used for degradation. 2.4.6. Drug release files To evaluate drug release potency of scaffolds, FITC-MGF and TGF-b3 were mixtured with SF solution and freeze dried as mentioned above. Spong-like scaffolds were immersed into a 48-well plate supplement with 200 ml PBS per well at room temperature in darkness for 30 days. The release medium was fully exchanged with fresh medium after 0.5, 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 days. Quantification of TGF-b3 was determined by an ELISA kit following the manufacturer's guidence. Quantification of

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FITC-MGF was calculated by fluorescence intensity. Five independent STM scaffolds were used for drug release assessment.

2.5. Animal models All rabbits uesed (16 week-old, weighing 3.0e3.5 kg) conformed to the Guiding Principles in the Care and Use of Animals and was approved by Chongqing University and Third Military Medical University Animal Care and Use Committee.

2.5.1. Subcutaneous implantation With isoflurane induced anesthesia, SF, ST and STM scaffolds in a shape of 3 mmdiameter and 3 mm-height cylinder were implanted into lateral subcutaneous pockets of rabbit for the evaluation of cell recruitment at 7 days and matrix secretion at 2 months. Two scaffolds of each group were implanted into the pockets. A total of six rabbits were prepared for subcutaneous implantation. Procaine penicillin was given intramuscularly preoperation and postoperation for prophylactic infection. All animals were allowed to have libitum access to food and water. 7 days later, three of the six rabbits were sacrificed and scaffolds were removed en bloc with the surrounding tissue naturally. Three scaffolds of each group were immersed into PBS containing 1% penicillin/streptomycin (P/S) and used for cell isolation as described above. Other scaffolds were fixed in 4% PFA for 24 h and processed for histology as described below. Samples at 2 months were as similar as we did at 7 days.

2.5.2. Articular joint drilling surgery Articular surgery was carried out as previously described [55]. 24 rabbits (six rabbits per group) were divided into 4 groups, blank, SF, ST, and STM. Briefly, the rabbits were anaesthetised with pentobarbital sodium (30 mg/kg), and maintained with 3% isoflurane. Knee joint of the rabbits was revealed through lateral incision, and the patella was dislocated. An osteochondral drilling defect of 3 mm-diameter and 3 mm-depth was created in the trochlear groove of the femur, followed by blank (empty control), SF, ST or STM scaffold implanted by press-fitting (Fig. S5A). Finally, the joint was closed with suture, and penicillin was given intramuscularly for prophylactic infection. After the operation, rabbits were allowed to move freely in their single cages and fed with standard food and water. 7 days and 2 months later, rabbits were sacrificed and scaffolds were treated as methods in section 2.5.1.

2.6. Histology and immunohistological analysis Histological analysis of articular samples was performed as we did before [56]. Briefly, rabbits were sacrificed 3 months postsurgery and the dissected distal femurs were fixed in 4% PFA for 48 h, decalcified with 15% EDTA (pH 7.2e7.4) and were paraffin embedded. 5 mm-thickness sections were cut and stained with haematoxylin and eosin (H&E), safranin O or masson's trichrome staining to assess proteoglycans and collagen in the matrix after a series of deparaffin and alcohol. For quantification of recruiting stem cells, antibodies against CD29 and CD44 were incubated with sections after deparaffin and antigen retrieval. Cell number of CD29þ/CD44þcells was randomly counted in ten microscopic views and showed by average. Five independent samples of each group were analyzed (Mean ± SD). Meanwhile, antibodies against CD68 and Galectin 3 (Mac-2) were used for assessment of actived macrophages. To evaluate the regenerative cartilage in the defects, immunoflourescence for aggrecan, Collagen I and Collagen II was introduced. Tissue sections were washed with 0.5% Triton X and incubated with monoclonal antibodies at 4 C overnight. Before AGC antibody application, chondroitinase ABC (Sigma) was used for digestion and disclosure of the specific antigen for 1 h at 37 C as described [57]. After 1 h incubation of secondary antibodies, images were taken from an Olympas microscope and analyzed by cellSense software.

2.7. Scoring evaluation of histological architecture Quantitative scoring of histological sections (stained with Safranin O and Masson's trichrome staining) was performed using a modified O0 Driscoll 24-point scoring system (see Table S1) that is commonly used for evaluating the quality of new tissue formation in articular cartilage defects in vivo [58e60]. The major categories scored in this system include assessment of cellular morphology, Safranin O staining, surface regularity, structural integrity, thickness, bonding to adjacent cartilage, hypocellularity, chondrocyte clustering, and freedom from degenerative changes in adjacent cartilage. All measurements were separately finished by 5 histologists in a blind manner.

2.8. Statistical analysis Results are represented as means ± standard deviations. Statistical analysis was performed using Student's t-test as well as one-way analysis of variance (ANOVA) followed by the Tukey HSD test for post hoc comparison (OriginLabOriginV8.0 Software). Difference was considered significant when P < 0.05.

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3. Results 3.1. Effects on cell migration in vitro Fluorescence microscopy revealed that green fluorescence located at the center of MSCs, indicating incorporation of FITC labeled MGF into MSCs (Fig. S1B, green arrows), in contrast with an earlier study with primary chondrocytes [61]. However, MGF alone did not stimulate cell proliferation (Fig. S1C), which was in consistent with another previous study [45]. To verify the effect of MGF on hMSCs mobility, we introduced Transwell system and wounds healing assay in vitro. 8 h after MGF stimulation in Transwell system, cell number was 2-fold higher than that in control group without factors, and 1.3-fold higher than TGF-b3. Combination of MGF and TGF-b3 induced a 2.7-fold increase in cell number than control (Fig. 1B). Similar to Transwell assay, both MGF and TGF-b3 increased the repair rate. Moreover, the combination showed the best repair rate of scratching at 24 h (Fig. 1C). These results confirmed that MGF peptide did not enhance hMSCs proliferation, but increased cell mobility [45]. 3.2. In vitro effects of MGF on TGFb3-induced chondrogenic differentiation of hMSCs Since MGF has little effects on the proliferation of hMSCs, we then investigated whether MGF has effects on the differentiation. After a period of 15-day exposure to differentiate media, Collagen II and aggrecan were detected in TGF-b3 group (Fig. 2A), but along with certain Collagen I. However, MGF alone failed to induce the chondrogenic differentiation (Fig. 2), causing the exclusion of MGF alone group in further study. Surprisingly, combination of TGF-b3 and MGF (TM) leaded to a prompt increase of Collagen II and aggrecan expression, and decreased Collagen I expression (Fig. 2A).

Our double-staining for Collagen I and Collagen II confirmed that TM treatment enhanced Collagen II expression and reduced Collagen I expression (Fig. S2A). Similar to the immunofluorescense staining, quantitative results showed that the content of Collagen II and aggrecan in TM group was significantly higher than those in TGF-b3 group (Fig. 2B and C), and Collagen I was significantly lower (Fig. 2D) than TGF-b3. These results suggested MGF acted synergetically with TGF-b3 for in vitro chondrogenic differentiation. 3.3. In vitro characterization of silk fibroin scaffolds 8 ~ 10% of silk fibroin solution was exposed to a vacuum freeze drier, resulting in sponge-like scaffolds (Fig. 3B). To induce b-sheet formulation for growth factors retention, SF scaffolds were immersed into 90% methanol and assessed by using FTIR [19]. Methanol treatment leaded to amide band shifts from 1653 cm1 to 1627 cm1, 1537 cm1 to 1529 cm1, and 1235 cm1 to 1254 cm1, indicating b-sheet conformation in the amide I, II and III region, respectively (Fig. 3C). The surface and inside structure of SF scaffolds were characterized by SEM as shown in Fig. 3D and E, indicating the three-dimensional interconnecting microchannels (200e500 mm diameter) as conduits to promote cell infiltration and nutrient supplies. However, the pore size was out of control during fabrication. 30 min after immersion into methonal, compressive modulus of scaffolds was increased up to 4.9 MPa, which was quite similar to native articular cartilages from rabbits (5.5 MPa) (Fig. 3F), suggesting structure conformation increased compressive modulus. Moreover, methonal treated scaffolds degraded more slowly, even as much as 60% of the initial mass left after 40 days immersion into PBS (Fig. 3G). To analyze the drug release potency of scaffolds, total cumulative release for 30 days of TGF-b3 was detected by ELISA and FITC-conjugated MGF was examined by flourence intensity. The scaffolds released a cumulative rate about

Fig. 1. Effects of MGF on the migration of hMSCs. (A) DAPI staining images for migrating cells. Bar ¼ 50 mm. (B) Quantification of hMSCs for Transwell system. (C) The wounds healing rate of hMSCs upon different treatment. *p < 0.05 vs. control; #p < 0.05 vs. TGF-b3.

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Fig. 2. Effects of MGF on TGF-b3-induced chondrogenesis of hMSCs. (A) Immunostaining images of AGC, Collagen II and Collagen I for chondrogenic differentiation of hMSCs. Bar ¼ 50 mm (BeD) Quantification of AGC (B), Collagen II (C), and Collagen I (D) for the chondrogenic differentiation (n ¼ 3). *p < 0.05 vs. control, #p < 0.05 vs. TGF-b3.

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Fig. 3. Characterization of scaffolds. (A) Schematic description of b-sheet conformation for growth factors incorporation into silk fibroin scaffolds. (B) Macroscopic observation of one silk scaffold. (C) Fourier Transform Infrared Spectroscopy (FTIR) profiles of scaffolds before and after methanol treatment. (DeE) SEM images of porous scaffolds for sagittal (D) and transverse plane (E). (F) Compressive modulus of silk scaffolds vs. time acquired for methanol treatment (n ¼ 6). (G) Degradation rate of scaffolds for 40days. (H) Cumulative release curves of TGF-b3 and FITC-MGF for 28 days.

65% for 15 days, and still increased up to about 75% for 30days (Fig. 3H). These results suggested silk scaffolds could be a good candidate for in situ regenerative medicine.

Moreover, these cells could be successfully induced towards osteogenic, adipogenic and chondrogenic differentiation (Fig. S3D). Taken together, STM scaffolds could promote multipotent stem cells infiltration when submitted to subcutaneous implantation.

3.4. Identification of multipotent stem cells infiltration into subcutaneously implanted scaffolds

3.5. Cartilage-like matrix formation by silk scaffolds

7 days after subcutaneous implantation, most cells aggregated at the surface of scaffolds in SF group (Fig. 4A), but few cells and CD29þ/ CD44þ cells infiltrated into the central area (Fig. 4B). By contrast, ST group showed increasing cells up to 220 cells per mm2 at surface and 55 cells per mm2 at center (Fig. 4C), 55.8% and 66.5% of which the cells were identified as CD29þ/CD44þ cells (Fig. 4D), respectively. However, total cell number in STM group was 400 cells per mm2 at the surface and 130 cells per mm2 at center, 73.2% and 87.5% of which the cells were CD29þ/CD44þ cells at surface and center. These results were confirmed by flow cytometric analysis (Fig. S3C).

To evalute the chondrogenic efficacy of scaffolds in vivo, SF, or ST or STM scaffolds were subcutaneously implanted in rabbits. Two months later, SF scaffolds disappeared, indicating completely degradation of SF scaffolds in subcutaneous pockets. Nevertheless, despite the macroscopic view of ST and STM scaffolds (Fig. S4A) was quite similar, the dry weight of STM was higher than ST group (Fig. S4B). Safranin O staining, which is specific for cartilage extracellular matrix (ECM) deposition, confirmed the deposition in STM scaffolds was more than that in ST scaffolds at both surface and central areas (Fig. S4D). As MGF could reduced type I collagen

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Fig. 4. Identification of stem cells at 7-day after subcutaneous implantation. (A) Immunostaining images represented for stem cells identification at surface. Bar ¼ 50 mm. (B) Immunostaining images represented for stem cells identification at central area. Bar ¼ 50 mm. (C) Quantification of cell number of infiltration into scaffolds (n ¼ 6). (H) Quantification of percentage of stem cells based on CD29þ/CD44 þ double positive cells (n ¼ 6). *p < 0.05 vs. SF, #p < 0.05 vs. ST. Dotted lines indicated the boundary between scaffold and native surrounding tissue.

secretion in vitro (Fig. 2, Fig. S2), we speculated that MGF may also decreased collagen I secretion when responsed to chondrogenic environment in vivo. To this end, Masson's trichrome staining was introduced and the results demonstrated that, total fibrillar collagen decreased in STM scaffolds, compared with native subcutaneous tissue and ST scaffolds (Fig. S4E). However, whether the total decrease of fibrillar collagen was due to the reduced secretion of either type I or type II collagen, or both, was still unclear. To address this question, immunostaining for collage I and collagen II was introduced (Fig. 5). Similarly, there were more chondrocytes and higher content of collagen II in STM group than that in ST group at surface and central area (Fig. 5A and B). However, the secretion of collagen I, one main component of scar or fibrous tissue, was much less in STM group than that in ST scaffolds (Fig. 5C and D), these results demonstrated that MGF not only enhanced chondrogenesis, but meanwhile reduced Collagen I secretion under chondrogenic environment, suggesting STM scaffolds facilitated cartilage-like ECM deposition in vivo.

4. Identification of stem cells infiltration into implanted scaffolds in joints Autologous stem cells or chondrocytes transplantation has been used for articular regeneration, but limited by cell sources. Endogenous stem cells in adult tissues provide a good choice for

damaged tissue repair, but it is difficult to recruit enough cells to injured sites, resulting in fibrosis or scar formation. To address this problem, STM scaffolds were evaluated in an osteochondral defect model in rabbit (Fig. S5A). At 7days after implantation, there were about 230 cells per mm2 and 250 cells per mm2 at the surface and central areas in STM scaffolds, 2.1 times and 5 times higher than those in ST scaffolds (Fig. 6A and B). Similar to the results from subcutaneous implantation, over 50% of the cells were CD29þ/ CD44þ multipotent stem cells (Fig. 6C, D and Fig. S3). At 7 days after implantation, no immunorejective effect was detected in the drilling sites (Fig. S5D and E), as evidenced by actived macrophages markers CD68 and Mac-2. It is worth noting that the percentage of stem cells at central areas in all scaffolds was higher than those at surface areas (Fig. 6D).

4.1. Effects of silk scaffolds on articular cartilage repair As STM scaffolds can enhance stem cell migration, infiltration and chondrogenesis in vitro and in vivo, we speculated that these scaffolds could facilitate cartilage repair in articular joints. In order to evaluate the repair efficacy, SF, ST or STM scaffolds were implanted into the drilling hole (3 mm in diameter) in rabbit joints for 90 days. Then the rabbits were anesthetic and sacrificed for further histological analysis. In this study, none of rabbits in any of the groups developed grossly apparent degeneration or synovial

470 Z. Luo et al. / Biomaterials 52 (2015) 463e475 Fig. 5. Lineage commitment of multipotent cells at 2-month after subcutaneous implantation. (A) Immunostaining images for Collagen II at the surface (left) and central (right) area of scaffolds. Bar ¼ 100 mm. (B) Quantification of fluorescence intensity of Collagen II (n ¼ 6). (C) Immunostaining images for Collagen I at the surface (left) and central area of scaffolds. Bar ¼ 100 mm. (D) Quantification of fluorescence intensity of Collagen I (n ¼ 6). S: undegradable scaffolds. Native: native tissue as control. Arrows indicated Collagen II, or Collagen I-positive cells. Dotted lines indicated the boundary between scaffold and native tissue. *p < 0.05 vs. ST. **p < 0.05 vs. ST.

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Fig. 6. Cell infiltration and multipotent stem cell identification at 7-day after implantation in articular joint. (A) Total view of scaffolds from rabbits. Bar ¼ 500 mm. The edges between native cartilage and scaffold were enlarged in Image 1, 3 and 5. The central area of scaffold was shown in Image 2, 4 and 6. Bar ¼ 100 mm. (B) Quantification of cell number of cell infiltration (n ¼ 10). (C) Immunostaining images of CD44, CD29 antibodies for stem cells identification (white arrows). Bar ¼ 50 mm. (D) Quantification of percentage of stem cells of each scaffolds (n ¼ 10). *p < 0.05 vs. SF, #p < 0.05 vs. ST.

hypertrophy of the joint 3 months postsurgical operation. Despite macroscopic observation of defects revealed no obvious differences between all groups (Fig. S5B), osteochondral defects in the blank group were still empty with little fibrillar tissue at the trochlear surface (Fig. S5E). We concluded that little spontaneous osteochondral repair happened in this study, so that it was reasonable to exclude the empty control in further analysis.

Histological evaluation revealed that neotissues in SF and ST groups were likely to be connective or fibrocartilage-like tissue with abnormal cell density, having fibroblast-like cells, and disordered structure organization as evidenced by Safranin O and Masson's trichrome staining. In contrast, defects treated with STM scaffolds showed nearly complete tissue filled, and the neotissue was similar in color and architecture to the native cartilage.

Fig. 7. Representative images of rabbit articular at 3 months after implantation. (A) Safranin O staining for cartilage extracellular matrix glycosaminoglycans. Bar ¼ 200 mm tThe repaired status of incised edges and central regions were respectively represented at middle and right column. Bar ¼ 100 mm. (B) Masson's trichrome staining for collagen organization. Bar ¼ 200 mm. The architecture of collagen of incised edges and central regions were respectively represented in middle and right column. Bar ¼ 100 mm.

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Fig. 8. Regeneration of rabbit articular cartilage. (A) Immunostaining images of native cartilage (Nc) for positive and negative control. (B) Immunostaining images of the edges and center of wound for the evalution of cartilage regeneration. Bar ¼ 200 mm.

Additionally, cells were organized in a columnar architecture, and resembled chondrocyte-like morphology (rounded and in lacunae). Cell density and clusters were quiete similar to the surrounding undamaged native cartilage (Fig. 7). In most cases of these groups, the incised edges could rarely be distinguished from native cartilage, indicating excellent integration of the regenerated tissue to the surrounding native cartilage. Specific immunostaining with AGC antibody nearly matched the intensity and quality of Safranin O staining and Masson's trichrome staining (Fig. 8). However, there were significant differences in Collagen II and type collagen I expression among the SF, ST and STM groups. Although the intensity of collagen II at the injured sites in ST scaffolds was similar to native cartilage, little collagen II and a large amount of collagen I were detected in the central regions of the trochlear surface. By contrast in STM scaffolds, expression of Collagen II at both injured edges and central regions was as much as native tissue, with little collagen I expression (Fig. 8). These results indicated that MGF and TGF-b3 could enhance in situ articular hyaline cartilage regeneration.

each category and total scores were presented in Table 2. There were significant differences in histological scoring between groups treated with SF (6.2 ± 1.6), ST (12.4 ± 1.5) and STM (21 ± 1.6) scaffolds. These results indicated that the reparative tissue in STM group is similar to the surrounding cartilage at three months after implantation. 5. Discussion In this study we reported the successful use of silk fibroin for regeneration of articular cartilage defects in rabbits. Our results Table 2 Scoring for histological evaluation. Score (Mean ± SD)

Cellular morphology Safranin O staining of the matrix Surface regularity Structural integrity Thickness Bonding to the adjacent cartilage Hypocellularity Chondrocyte clustering Adjacent cartilage degenerative joint disease

0.6 1.0 0.4 0.2 0.4 1.2 1.0 0.4 1.0

Total

6.2 ± 1.6

SF

4.2. Evaluation of histological scoring Quantitative scoring of histological sections (stained with Safranin O and Masson's trichrome staining) was performed using a modified O0 Driscoll 24-point scoring system (Table S1) that is commonly used for evaluating the quality of new tissue formation in articular cartilage defects in vivo [58e60]. The average scores in

O'Driscoll category

*

#

¼ p < 0.05 vs. SF, ¼ p < 0.05 vs. ST.

ST ± 0.6 ± 0.0 ± 0.5 ± 0.4 ± 0.5 ± 0.4 ± 0.0 þ 0.6 ± 0.0

1.4 1.0 1.0 1.0 1.0 2.0 2.4 1.0 1.6

STM ± ± ± ± ± ± ± ± ±

1.8a 0.0 0.0 0.0a 0.0 0.0 0.5a 0.0 0.5a

12.4 ± 1.5a

3.0 3.0 3.0 1.8 1.8 1.8 2.8 1.4 2.8

± ± ± ± ± ± ± ± ±

0.0a 0.0a# 0.0a# 0.4a# 0.4a# 0.4 0.4a 0.5a# 0.4a#

21 ± 1.6a#

Z. Luo et al. / Biomaterials 52 (2015) 463e475

demonstrated that STM scaffolds, without exogenous cells seeding, could recruit endogenous multipotent stem cells for chondrogenesis and facilitate hyaline cartilage regeneration in vivo accompanied with fibrocartilaginous tissue suppression. As others demonstrated that MGF was nucleus located [62,63], the green fluorescence showed the corperation of FITC labeled MGF into hMSCs (Fig. S1), in contrast to primary chondrocytes [61]. However, whether the corperation was due to pinocytosis or endocytosis is still unknown. Fluorescence intensity gradually weaken and finally disappeared in 30 min, consistent with the conclusion of short half-life of the MGF peptide [36]. In consistent with others, migration of hMSCs was increased in response to MGF treatment [45,46], but a synergistic effect was also displayed when combinated with TGF-b3, indicating a chemotactic role of MGF. To our knowledge, the effect of MGF on differentiation of MSCs is unclear. Here we showed that MGF alone had no effects on chondrogenesis of hMSCs, but enhanced the TGF-b3 induced chondrogenesis of hMSCs. Moreover, MGF downregulated collagen I secretion during the chondrogenesis (Fig. 2 and Fig. S3). These results were particularly interesting for fibrocartilage prevention for damaged articular cartilage regeneration and repair. Articular cartilage is a connective tissue that functions hydrodynamically to bear loads and provide almost friction-free movement of diarthrodial joints [64], and degenerates with aging, displaying a limited self-repair capacity once damaged. At present, subchondral drilling or microfracture has been used for cartilage repair, frequently leading to scar or fibrous tissue at the trochlear surface. Fibrocartilage, mainly composed of collage I, is characterized by bundles of tough collagen that are clearly viewable under microscope. Although fibrocartilage is able to fill in articular cartilage defects, its structure is significantly different from hyaline cartilage; it is much denser and it cannot withstand the resistance to both compression and shearing forces required [65]. In the case of Collagen I suppression by MGF, it was likely to introduce the chemokine for cartilage regeneration. Silk fibroin, a natural protein, is widely used for cartilage tissue engineering [19,21,23,24,29,66]. Nevertheless, those scaffolds fabricated in these researches were all from silk-based blends, either with IGF-1 or TGF-b3 supplement. In an earlier study, SF was also designed for cartilage repair in rabbit model [55], but placentaderived mesenchymal stem cells were seeded and showed some reparative capacity. More recently, Singh et al. identified a specific scaffold mixture by different ratios of silk to cellulose without growth factors that successfully directed chondrogenesis of MSCs in vitro [25]. However this blend still needed further investigation in vivo. Here we fabricated STM scaffolds for in situ evalution of cartilage regeneration. Stem cell-based transplantation has been considered as a hopeful strategy for tissue fibrosis and other disease [67e72], of which the trophic effect is widely accepted, but limited by low expansion and short term of survival after transplantation. MGF, may act as a novel chemokine, was introduced to recruit host endogenous multipotent stem cells to injured sites. MGF is an alternative splicing isoform of IGF-1 transcripts, but distinguished from its C-terminal peptide. However, MGF acted independently from IGF-1 receptor [42,73], and showed greater potential for tissue repair [74]. MGF has been regarded as an autocrine growth factor [36]. Once released, MGF probably served as a stem cell homing factor, activated quiescent stem cells and “kick-starts” cell migration to defects. As expected, MGF recruited a larger number of stem cells than TGF-b3 did. Moreover, MGF had been proved to provide cell replenishment [36] and cell survival protection [40,75]. The in vivo experiments revealed that STM scaffolds showed similar safranin O and Masson's staining, neocartilage archetecture organization, collagen I and collagen II to the surrounding native

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cartilage (Fig. 7 and Fig. 8). Despite lack of related devices for mechanical analysis of regenerated cartilage, it was credible to conclude that more hyaling cartilage and less fibrocartilage were formed in STM treated group. Meanwhile, the average scores of STM group in histological scoring system were quite close to that of normal cartilage (Table 1), indicating the excellent reparative efficacy of STM scaffolds. 6. Conclusion Silk fibroin-based scaffolds and TGF-b3 have been reported to be effective for articular cartilage regeneration, but undesired fibrocartilage was also formed. In this study, we prepared STM scaffolds and evaluated the potential of articular cartilage repair in rabbit osteochondral defect model. Our results demonstrated that MGF acted synergistically with TGF-b3 to recruit endogenous multipotent stem cells for chondrogenesis, enhanced directed differentiation of endogenous stem cells and inhibited fibrosis, indicating the facilitation of cartilage regeneration. This study provides a new therapeutic strategy for cartilage repair. Disclosure statement No competing financial interests exist. Acknowledgments We sincerely thank Dr. Song Li (UC Berkeley, USA) for the kindly gift of human bone marrow mesenchymal stem cells, Dr. Qiaolin Liu (SARI Center for Stem Cell and Nanomedicine, CAS, Shanghai, China) for the help of histological analysis. This work was supported by grants from Innovation and Attracting Talents Program for College and University (“111” Project) (B06023), National Natural Science Foundation of China (11032012, 10902130, and 30870608) and Fundamental Research Funds for the Central Universities (CQDXWL-2014-007). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.01.001 References [1] Wang Y, Ding C, Wluka AE, Davis S, Ebeling PR, Jones G, et al. Factors affecting progression of knee cartilage defects in normal subjects over 2 years. Rheumatol Oxf 2006;45:79e84. [2] Noth U, Steinert AF, Tuan RS. Technology insight: adult mesenchymal stem cells for osteoarthritis therapy. Nat Clin Pract Rheumatol 2008;4:371e80. [3] Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889e95. [4] Lim CT, Ren X, Afizah MH, Tarigan-Panjaitan S, Yang Z, Wu Y, et al. Repair of osteochondral defects with rehydrated freeze-dried oligo[poly(ethylene glycol) fumarate] hydrogels seeded with bone marrow mesenchymal stem cells in a porcine model. Tissue Eng Part A 2013;19:1852e61. [5] Holtzer H, Abbott J, Lash J, Holtzer S. The loss of phenotypic traits by differentiated cells in vitro, I. Dedifferentiation of cartilage cells. Proc Natl Acad Sci U S A 1960;46:1533. €rbling M, Estrov Z. Adult stem cells for tissue repairda new therapeutic [6] Ko concept? N Engl J Med 2003;349:570e82. [7] Wu L, Bluguermann C, Kyupelyan L, Latour B, Gonzalez S, Shah S, et al. Human developmental chondrogenesis as a basis for engineering chondrocytes from pluripotent stem cells. Stem Cell Rep 2013;1:575e89. [8] Bianco P, Cao X, Frenette PS, Mao JJ, Robey PG, Simmons PJ, et al. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med 2013;19:35e42. [9] Sabelstrom H, Stenudd M, Reu P, Dias DO, Elfineh M, Zdunek S, et al. Resident neural stem cells restrict tissue damage and neuronal loss after spinal cord injury in mice. Science 2013;342:637e40.

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