Surface modification on polycaprolactone electrospun mesh and human decalcified bone scaffold with synovium-derived mesenchymal stem cells-affinity peptide for tissue engineering Zhenxing Shao,1 Xin Zhang,1 Yanbin Pi,1 Ling Yin,2 La Li,1 Haifeng Chen,2 Chunyan Zhou,3 Yingfang Ao1 1

Institute of Sports Medicine, Peking University Third Hospital, Haidian District, Beijing 100191, People’s Republic of China Department of Biomedical Engineering, Peking University College of Engineering, Haidian District, Beijing 100871, People’s Republic of China 3 Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, Haidian District, Beijing 100191, People’s Republic of China 2

Received 26 January 2014; revised 11 March 2014; accepted 21 March 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35177 Abstract: Synovium-derived mesenchymal stem cells (SMSC) have been studied for over a decade since first being successfully isolated in 2001. These cells demonstrate the most promising therapeutic efficacy for musculoskeletal regeneration of the MSC family, particularly for cartilage regeneration. However, the mobilization and transfer of MSCs to defective or damaged tissues and organs in vivo with high accuracy and efficiency has been a major problem in tissue engineering (TE). In the present study, we identified a seven amino acid peptide sequence [SMSCs-affinity peptide (LTHPRWP; L7)] through phage display technology that has a high specific affinity to SMSCs. Our analysis suggested that L7 efficiently and specifically interacted with SMSCs without any species specificity. Thereafter, L7 was covalently conjugated onto both polycaprolactone (PCL) electrospun meshes and human decalcified bone scaffolds (hDBSc) to investigate its TE applications. After 24 h coculture with human SMSCs

(hSMSCs), L7-conjugated PCL electrospun meshes had significantly more adherent hSMSCs than the control group, and the cells expanded well. Similar results were obtained using hDBSs. These results suggest that the novel L7 peptide sequence has a high specific affinity to SMSCs. Covalently conjugating this peptide to either artificial polymer material (PCL mesh) or natural material (hDBS) significantly enhances the adhesion of SMSCs. This method is applicable to a wide range of potential SMSC-based TE applications, particularly to cartilage regeneration, via surface modification on various C 2014 Wiley Periodicals, Inc. J Biomed Mater type of materials. V Res Part A: 00A:000–000, 2014.

Key Words: synovium-derived mesenchymal stem cell, phage display, affinity peptide, tissue engineering, surface modification

How to cite this article: Shao Z, Zhang X, Pi Y, Yin L, Li H, Chen C, Zhou Y, Ao Y. 2014. Surface modification on polycaprolactone electrospun mesh and human decalcified bone scaffold with synovium-derived mesenchymal stem cells-affinity peptide for tissue engineering. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

Synovium-derived mesenchymal stem cells (SMSC) were first successfully isolated and identified by De Bari et al.1 in 2001. They have proven to be the most promising therapeutic cell from the MSC family for musculoskeletal regeneration, particularly for cartilage regeneration, due to their high expansion and chondrogenic potentials.1–6 Also, the multipotency of SMSCs does not appear to be affected by cell passaging or donor age.1,2 Many studies on the tissue engineering (TE) repair of articular cartilage using SMSCs with various approaches and scaffolds show good results.7–12

Currently, the inability to deliver MSCs to tissues of interest with high efficiency and engraftment is a significant barrier for MSC-based TE strategies.13,14 Most studies report that only a small percentage of stem cells remains after several weeks of engraftment.15,16 In addition, many studies have used endogenous, rather than exogenous, stem cells for repair by in vivo cell homing to minimize the expenses and difficulties associated with MSC cell culture, storage, and distribution, as well as to minimize the immune response.17–19 In 2011, De Bari and coworkers20 provided the first evidence for the existence of resident MSCs in the

Additional Supporting Information may be found in the online version of this article. Correspondence to: Y. Ao; e-mail: [email protected], C. Zhou; e-mail: [email protected], and H. Chen; e-mail: [email protected] Contract grant sponsor: National Natural Science Foundation of China (NSFC); contract grant numbers: 81071474; 81171726 Contract grant sponsor: Specialized Research Fund for the Doctoral Program of Higher Education; contract grant number: 20110001130001 Contract grant sponsor: National Basic Research Program of China; contract grant number: 2012CB933903

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knee joint synovium that undergo proliferation and chondrogenic differentiation in vivo following injury. Synovial membrane also contributes to the repair of partial-thickness cartilage defects in rabbit knee joints.21 These results support the existence of SMSCs and their contributions to articular cartilage repair, which also suggests the possibility of using endogenous SMSCs for in situ cartilage regeneration. In a previous study, we successfully identified a bone marrow-derived MSC-affinity peptide, E7, through phage display technology, and covalently conjugated it onto polycaprolactone (PCL) electrospun meshes to construct a “MSChoming device” for the recruitment of MSCs.22 In the present study, we focus on SMSCs, which have shown better chondrogenic potential. We identified an SMSC-specific affinity peptide, SMSCs-affinity peptide (LTHPRWP; L7), through phage display technology as previously described.22,23 This peptide was then covalently conjugated onto PCL electrospun meshes to evaluate its TE applications. Furthermore, we conjugated this peptide onto the surface of a natural, porous scaffold material, and human decalcified bone scaffold (hDBS), which we found is beneficial for native hyaline articular cartilage regeneration.24 We also investigated the adhesion of SMSCs onto these two fundamentally different materials after surface modification by SMSCs-affinity peptide L7. MATERIALS AND METHODS

All animals and experimental procedures were approved by the local Institutional Animal Care and Use Committee complying with the “Guide for the Care and Use of Laboratory Animals” published by the National Academy Press (NIH Publication No. 85-23, revised 1996). Cell cultures Human SMSCs (hSMSCs) and human bone marrow-derived MSCs (hBMSCs) were isolated from eight patients (three males and five females, aged 60.38 6 6.28 years with an age range of 50–73 years) who underwent total knee arthroplasty. A written informed consent was obtained according to the guidelines of the Peking University Third Medical School Committee on the use of human subjects in research. hBMSCs were collected following a previously described procedure.22 hSMSCs were collected as follows: after dissection, the synovial tissues were repeatedly washed with sterile phosphate buffered saline (PBS) plus 1% penicillin– streptomycin, cut into 1 mm3 pieces, and digested with 0.2% collagenase Type I (GIBCO Invitrogen, Carlsbad, CA) for 2 h at 37 C. The digested pieces were plated onto 10 cm2 flasks and cultured in DMEM with 10% FBS at 37 C in 5% CO2 and allowed to attach for 4–5 days. Nonadherent cells were removed by changing the medium. Identification of the characteristics of hSMSCs The specific cell surface antigen markers of SMSCs were examined via flow cytometry (FCM). The antibodies for positive markers included CD44 (ab51037), CD90 (ab23894), and CD105 (ab11414), whereas the negative markers

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included CD34 (ab6330) and CD45 (ab33533) (Abcam, HKSTP, N.T Hong Kong).1,6,25–27 A trilineage-induced differentiation experiment was also performed to identify the multiple differentiation potential of hSMSCs, which included adipogenesis, osteogenesis, and chondrogenesis. The hSMSCs of passage 2 were used in all experiments. Briefly, the hSMSCs were incubated in a sixwell plate at a density of 105 cells/well with Human MSC Adipogenic or Osteogenic Differentiation Medium (Cyagen Biosciences, Sunnyvale, CA) for adipogenesis or osteogenesis induction, respectively. The cells were examined for adipogenesis through oil red O staining after a week of culture, or for osteogenesis through alkaline phosphatase and alizarin red staining after 2 weeks of culture. For chondrogenesis, pellet culture was performed. Briefly, the hSMSCs were digested with trypsin, and resuspended in DMEM in a 15 mL polypropylene centrifuge tube. A total of 1 3106 cells/ tube were washed with DMEM twice at 150 g for 5 min, resuspended in 0.5 mL of Human MSC Chondrogenic Differentiation Medium (Cyagen Biosciences), and centrifuged at 150g for 5 min. The pellet was incubated at the bottom of the tube with the supernatant at 37 C in 5% CO2 for 24 h, and the tube was gently flicked to ensure the pellet was free-floating. The medium was changed every 2–3 days. After 3 weeks of incubation, the pellet was fixed in 4% paraformaldehyde, and embedded in paraffin. Alcian Blue staining was then performed to assess the glycosaminoglycan formation in the extracellular matrix of the pellet. Immunohistochemical staining for type II collagen was performed to test if the cells functioned as chondrocytes. Phage display biopanning Phage display biopanning, an affinity selection technique that selects peptides binding to a given target, was conducted following a previously described procedure with modifications.22,23 A peptide phage display library was commercially constructed (Ph.D.-7 phage display library; NEB, Beverly, MA) and used in the following experiments. The hSMSCs and fibroblasts were harvested from the total knee arthroplasty procedure and used at passage 2. In the negative selection procedure, the Ph.D.-7 phage display library [1 3 1012phage forming unit (PFU)] was incubated with the fibroblasts (1 3 106) for 1 h to exclude the fibroblast affinity phage clones. The supernatant was then collected and incubated with hSMSCs (1 3 106) for 1 h to allow hSMSCs internalization. Subsequently, the hSMSCs were washed with 1 mL of 0.2 M glycine–HCl (pH 2.2) (Sigma-Aldrich, St. Louis, MO) coupled with 1 mg/mL of BSA (albumin, bovine fraction V; Merck, Darmstadt, Germany) for 10 min to remove the poorly binding phage clones. The hSMSCs were neutralized with 150 mL of 1 M Tris–HCl (pH 9.1), and lysed in 20% NP-40 (Fluka Chemie, Buchs, Switzerland) for 30 min. The phage clones from the lysed hSMSCs were collected and amplified in Escherichia coli ER2738 (NEB), and then titrated and purified according to the manufacturer’s standard protocol. Additional three to four rounds of selection were performed until the optimal results were obtained. The experiments were duplicated for verification.

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The recovery efficiencies of the specific phage clones were calculated as the output phage titer divided by the input phage titer. The results were expressed as PFU. Synthesis of peptides and peptide-affinity assay A specific peptide was identified in the hSMSCs-affinity clones and designated as L7. A peptide with the same amino acids as L7 in a scrabbled order was used as the negative control and designated as scrambled peptide (PRHLPTW; P7). A peptide comprising three amino acids (arginine, glycine, and aspartic acid) was used as the positive control and designated as Arg-Gly-Asp peptide (RGD).28–30 All the peptides were synthesized through solid-phase peptide synthesis using Fmoc Chemistry (Scilight-Peptide, Beijing, China) based on a previous study.23 An extra cysteine was linked at the carboxyl (C) terminus of all peptides to facilitate PCL polymer and decalcified bone conjugation and fluorescein-5isothiocyanate (FITC; Fanbo Biochemicals Co., Beijing, China) labeling. The FITC-labeled peptides were purified on a Merck C18 column (Merck) at a wavelength of 220 nm, lyophilized in liquid nitrogen, and stored in 220 C. The peptides were dissolved in DMSO to yield a 2-mg/mL concentration before usage. The peptide-affinity assay was performed according to a previously mentioned method.22 The hSMSCs at passage 2 were incubated with 100 nM FITC-labeled peptides for 1 h at 37 C to allow cell binding and internalization. The cells were washed thrice with PBS, detached with 0.2% trypsin, and then centrifuged. The cell pellets were resuspended and fixed in FACS buffer (PBS supplemented with 1% paraformaldehyde, 2% glucose, 0.1% NaN3), and stored at 4 C for no more than 6 h before measurement. Rat and rabbitSMSCs were also used to investigate the species specificity of the identified peptide. The SMSCs affinity properties of the peptides were analyzed quantitatively through FCM (BD FACS CaliburTM Flow Cytometer) at a wavelength of 488 nm. The cells were cultured on 3.5 mm glass bottom culture dishes (Wuxi Nest Biotechnology, Wuxi, China) until 70– 90% confluence was achieved, and incubated with 100 nM FITC-labeled peptide for 1 h at 37 C. The hSMSCs were incubated with rhodamine phalloidin (Cytoskeleton, Denver, CO) for 1 h at 37 C to show the cytoskeleton of cells. The nuclei were counterstained with Hoechst 33258 (Fanbo Biochemicals Co), and examined under a confocal microscope. For further investigation on the specificity of this peptide, FITC-labeled L7 was incubated with hBMSCs and fibroblasts, respectively, following the same procedure above, comparing to hSMSCs. And the results were also analyzed quantitatively through FCM. Fabrication of the PCL electrospun mesh and hDBS Nonwoven PCL fibrous meshes were prepared from a 10% PCL solution. In a typical procedure, 1 g of PCL (MW 43,000–50,000) was dissolved in 10 mL of CH2Cl2. After stirring for 3 h until PCL was completely dissolved, 1 mL of the solution was loaded into a glass syringe with a bluntended stainless steel needle. The feeding rate, collection distance, and applied voltage were controlled at 0.5 mL/h, 10

cm, and 8 kV, respectively. The electrospun fibers were collected on a flat plate covered by aluminum foil. Finally, the fibrous meshes were removed from the foil, and dried under vacuum for 3 h. Human cortical cancellous blocks were directly purchased from the Shanxi Provincial Tissue Bank (Shanxi OsteoRad Biomaterial Co., Shanxi, China). The commercial human cortical cancellous bone was demineralized by soaking in ethylene diamine tetraacetic acid (EDTA, 0.5 M, pH 5 8.3) according to a previously mentioned method to obtain the natural 3D DBS (see related results in Supporting Information Fig. S1).24 At last, the human decalcified bone was made into a cylinder 2 mm in diameter and 4 mm in height by a corneal trephine [Fig. 7(a)]. Peptide conjugation with PCL meshes and hDBS and characteristics The PCL meshes and hDBS were immersed into a 10% (w/v) solution of 1,6-hexanediamine (Sigma) prepared in isopropanol at 37 C for 1 h. After the exposure, the PCL meshes were thoroughly washed in ultrapure water and dried under vacuum at room temperature. For the conjugation of L7, P7, and RGD peptides to the surface of the aminated PCL meshes, the aminated PCL meshes were washed thrice with activation buffer (0.1 M PBS containing 0.15 M NaCl, pH 7.2) before treatment, and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC; Thermo Fisher Scientific, Rockford, IL) was used as a crosslinker. A total of 500 mL (2 mg/mL) sulfo-SMCC solution was added onto the aminated PCL meshes, incubated for 1 h at room temperature, and washed with conjugation buffer (activation buffer containing 0.1 M EDTA, pH 7.0). The peptides were dissolved in conjugation buffer at a concentration of 0.1 mM. Up to 500 mL of the peptide solution was applied onto the sulfo-SMCC-treated PCL scaffold and incubated overnight at 4 C. The peptideconjugated PCL meshes were thoroughly washed thrice with ultrapure water, and dried under vacuum at room temperature. After the conjugation, the meshes were observed under a scanning electron microscope (SEM) and a confocal microscope. The immobilization of the peptides on the PCL meshes was measured via X-ray photoelectron spectroscopy (XPS).31 The chemical composition of the scaffolds was examined by XPS (Thermo K-Alpha XPS; Thermo Fisher Scientific, West Palm Beach, FL). The instrument was equipped with a mono chromatic Al Ka X-ray source (hm 5 1468.6eV) and spectra were collected using an X-ray spot size of 100 mm and a pass energy of 200 eV, with 1 eV increments, at 55 takeoff angle. To eliminate the influence of the amination process and sulfo-SMCC, aminated PCL mesh processed with sulfo-SMCC was used as negative control. The hDBS was processed in the similar way as PCL mesh to be conjugated with FITC-labeled L7. After the conjugation, the scaffold was vertically embedded in optimal cutting temperature, and immediately frozen in liquid nitrogen. The frozen scaffold was cut into 5 lm sections using a Leica CM3050 Microtome-Cryostat (Leica CM 3050, Nussloch, Germany),

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FIGURE 1. The characteristics of hSMSCs were examined through immunohistochemistry and flow cytometry analysis. The cells at passage 2 showed a homogeneous phenotype (A). The adipogenesis was checked through oil red O staining (B), and the osteogenesis potential was examined using alizarin red staining (C) and alkaline phosphatase staining (D). Type II collagen (E) and alcian blue staining (F) were used to assess the ability of chondrogenesis. Specific cell surface antigen markers of hSMSCs were tested thrice via flow cytometry analysis, and the representative results are shown in (a–c). Bar 5 300 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

mounted on 3-aminopropyltriethoxysilane-treated slides (Zhongshan Goldenbridge, Beijing, China), fixed with 4% paraformaldehyde, and rinsed in PBS with 1% (v/v) TritonX100 (Sigma-Aldrich). The scaffold was observed under SEM, and the cryosections were observed under a confocal microscope. Cell behavior on the affinity peptide-conjugated PCL meshes and hDBS in vitro The hSMSCs at passage 2 were seeded onto PCL meshes and hDBS, respectively, to investigate the adhesion of the cells on the peptide-modified PCL meshes and hDBS. hBMSCs and fibroblasts at passage 2 were also seeded onto L7-conjugated PCL meshes, respectively, comparing to hSMSCs. All PCL meshes and DBS were sterilized through ultraviolet exposure for 4 h in a superclean bench before use. PCL meshes. After 24 h of incubation, the PCL meshes were washed thoroughly three to five times with PBS, and fixed with 4% paraformaldehyde. The meshes were stained with rhodamine phalloidin for 1 h at 37 C to reveal the cytoskeleton of the cells. The nuclei were then counterstained with Hoechst 33258 and examined under a confocal microscope. All the meshes were also observed under SEM. The total DNA/dry weight ratio of every PCL mesh was also calculated for the quantitative analysis. All meshes were weighed first using an analytical balance. The total DNA of the cells on the meshes was extracted with a DNAzol Reagent (Invitrogen) according to the manufacturer’s protocol. The DNA content was measured using a UV-2800H ultraviolet spectrophotometer (UNICO, Shanghai, China). Three meshes in each group were randomly selected and measured, and the data were expressed as mean 6 standard variation. The P7conjugated and pure PCL meshes in the same scale were used as the negative controls, whereas the RGD-conjugated

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PCL meshes were used as the positive control. In order to further investigate the specific recruitment of the material conjugated with L7 toward SMSCs, hBMSCs and fibroblasts at passage 2 were also seeded onto L7-conjugated PCL meshes, respectively, comparing to hSMSCs. Human DBS. The hSMSCs at passage 2 were seeded onto the preshaped L7-conjugated hDBS, and incubated for 24 h. The scaffolds were then stained in the same protocol as the PCL meshes, and were observed under a SEM and a confocal microscope. The pure and P7-conjugated scaffolds in the same scale were used as the negative controls, whereas the RGD-conjugated scaffolds were used as positive control. The total DNA/dry weight ratio of every hDBS was also calculated for the quantitative analysis in the same way as PCL meshes. Statistical analysis For statistical analysis, Student’s t-test were performed for comparison of two groups and analysis of variance (ANOVA) for comparison of multiple groups, after testing the data for normality; p < 0.05 was considered to indicate statistical significance. All the results were expressed as mean 6 standard deviation. All experiments were performed and analyzed in triplicate. Data analysis was performed with SPSS. RESULTS

hSMSCs primary culture and characterization All characterizations were performed on cells at passage 2. After isolation and expansion, the hSMSCs began to show a homogeneous phenotype [Fig. 1(A)]. FCM results showed that the hSMSCs consisted of a single phenotypic population positive for CD44 (98.56 6 0.98%), CD90 (99.25 6 0.96%), and CD105 (98.61 6 1.32%). By contrast, the cells were negative for other markers of the hematopoietic lineage,

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TABLE I. Recovery Efficiencies of Phages in Each Panning Input Titer (PFU) Round Round Round Round

1 2 3 4

1.0 1.0 1.0 1.0

3 3 3 3

Output Titer (PFU)

12

10 1011 1011 1011

1.0 5.3 1.2 1.5

3 3 3 3

Recovery Efficiency

3

10 104 106 105

1.0 5.3 1.2 1.5

3 3 3 3

29

10 1027 1025 1025

Fold Increase 1 530 12,000 15,000

Four rounds of biopanning were performed to identify the hSMSCs-affinity phage clones. For each round of biopanning, the recovery efficiency was calculated as the output titer divided by the input titer. The best recovery efficiency was achieved in the fourth round, which was 15,000-fold higher than that in the first round (set as 1).

including the lipopolysaccharide receptor CD34 (3.37 6 1.06%) and the leukocyte common antigen CD45 [2.38 6 0.41%; Fig. 1(a–c)]. The trilineage differentiation experiment was performed to identify the multiple differentiation potential of the hSMSCs. The results showed that the hSMSCs were successfully differentiated into adipocytes [Fig. 1(B)], osteocytes [Fig. 1(C,D)], and chondrocytes [Fig. 1(E,F)]. Recovery efficiency after biopanning and peptide sequencing After biopanning, the recovery efficiencies of the specific phage clones were calculated as the output titer divided by the input titer of the phages. The input titer of the phages was 1 3 1012 PFU in the first round of biopanning, and 1 3 1011 for the remaining three rounds. As shown in Table I, the best recovery efficiency was achieved in the fourth round, which was 15,000-fold higher than that in the first round. The remaining phage clones in the last three rounds of biopanning were selected for peptide sequencing. The hSMSCs affinity phage clones were isolated after four rounds of biopanning. In every round of biopanning, 20 phage clones were randomly selected. In addition, LTHPRWP (L7), a sequence predominant in the fourth round, was found. In the fourth round, L7 phage clones

were found to possess over 50% in all clones. L7 showed a high affinity to the hSMSCs, as demonstrated in the subsequent experiments (Table II). hSMSCs affinity and specificity of the identified peptide The identified peptide (LTHPRWP) was designated as an hSMSCs-affinity peptide (L7). PRHLPTW (P7), a randomly scrambled peptide, was used as the negative control, whereas RGD, a peptide consisting of three amino acids (arginine, glycine, and aspartic acid), was used as the positive control. The hSMSCs were incubated for 1 h with FITClabeled L7, RGD, or P7, and measured via FCM. The average fluorescence intensity was 126.2 6 13.0 for the hSMSCs incubated with FITC-L7, 118.1 6 17.3 for the hSMSCs incubated with FITC-RGD, and 5.4 6 2.2 for the hSMSCs incubated with FITC-P7 [n 5 6, p < 0.05; Fig. 2(a,b)]. The cells were also observed under a confocal microscope. Strong fluorescent signals were observed in the cells incubated with FITC-L7 and FITC-RGD [Fig. 2(G,K)], whereas a weak fluorescence signal was observed in the cells incubated with FITC-P7 only [Fig. 2(C)]. To investigate the species specificity of the identified peptide, rat and rabbit SMSCs were also cultured and incubated with FITC-L7, RGD, or P7, and the fluorescent intensities were measured via FCM. The average fluorescence intensities for the rat SMSCs incubated

TABLE II. Sequences of the Peptides Identified by Phage Display Peptide Sequences Round 1 Not sequencing

Round 2

Round 3

Round 4

LTHPRWP(2/20) TTYNSPP(2/20) ARPLEIT(2/20) TDRLHFL(2/20) HPSRSLD TFAKSAY TYSTLGY EQFSAPI QHHPTYM HSHYSLK MSLSHIT FTHPRRR SMYGSYN SLDALLS TQLLEPT ARPRNIT

LTHPRWP(3/20) YSIPKSS(3/20) SILPYPY(2/20) TFAKSAY(2/20) ASLVRMM(2/20) IVLPYPI HSHYSLK DFKLPAS SETATHP SWHFVVS EQFSAPI VPPSMRP Empty phage

LTHPRWP(11/20) SILPYPY(2/20) SHPASHD TFAKSAY TDRLHFL VRPHTSS SAWTYEY DFKLPAS Empty phage

Twenty phage clones were randomly selected for sequencing in each round. A specific peptide sequence (LTHPRWP) appeared 11 times in the fourth round, occupying over 50% of all the selected phage clones. (LTHPRWP is showed in boldface, which means LTHPRWP becomes predominant in round 4).

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FIGURE 2. Affinity of the peptide were investigated via flow cytometry and immunofluorescence staining. The affinity of the peptides was analyzed quantitatively via flow cytometry after the incubation of FITC-labeled L7 (FITCL7), RGD (FITC-RGD), and P7 (FITC-P7) with hSMSCs (a). The average fluorescence intensity of six independent experiments are shown in b (ANOVA, n 5 6, *p < 0.05). The hSMSCs incubated with FITC-L7, FITC-RGD, and FITC-P7 were also observed under a confocal microscope. Strong FITC signals were observed in cells incubated with FITC-L7 and FITC-RGD (G and K), whereas very weak signals were observed in FITCP7-incubated cells (C). The nuclei were stained with Hoechst 33258 (A, E, and I). The cytoskeleton was stained with rhodamine phalloidin (B, F, and J). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

with FITC-L7, RGD, and P7 were 108.5 6 5.0, 107.9 6 11.1, and 5.1 6 1.2, respectively [n 5 6, p < 0.05; Supporting Information Fig. S2(A,B)], whereas those for the rabbit SMSCs incubated with FITC-L7, RGD, and P7 were 86.2 6 10.1, 106.8 6 8.6, and 4.8 6 1.5, respectively [Fig. S2(C,D)]. The average fluorescence intensities of L7 were 23.4-, 21.3-, and 18.0-fold higher compared with those of P7 in the human, rat, and rabbit SMSCs, respectively. This result suggests that L7 has high affinity and specificity for SMSCs and can bind with human, rat, and rabbit SMSCs without species specificity. To further investigate the specificity of this affinity peptide, FITC-labeled L7 was incubated with hBMSCs and fibroblasts, respectively, comparing to hSMSCs. The average fluorescence intensity was 10.2 6 3.5 for L7 incubated with hBMSCs, comparing 126.1 6 18.4 for L7 incubated with hSMSCs [Fig. 3(A,B)]; and 6.1 6 2.4 for L7 incubated with fibroblasts, comparing 110.1 6 3.9 for L7 incubated with hSMSCs [Fig. 3(C,D); n 5 6, p < 0.05].

PCL-mesh and hDBS-conjugated peptides and their characteristics The L7, P7, and RGD peptides were covalently conjugated with the PCL meshes for surface modification on the PCL electrospun fibers following a previously described procedure22 using sulfo-SMCC as a crosslinker [Fig. 4(A)]. After conjugation, the PCL fibers showed a homogeneous green fluorescence because the peptides were prelabeled with

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FITC [Fig. 4(D)], indicating a homogeneous conjugation of the peptides on the PCL surface. XPS showed that elemental nitrogen (N), which exist in peptides, on the surface of PCL fibers significantly increased after conjugation with L7 and RGD [Fig. 5(A–C) and Table III]. This confirmed that the peptides were indeed successfully conjugated on the surface of the PCL fibers. hDBS was processed in the similar way as PCL mesh to be conjugated with FITC-labeled L7. The cryosections also showed a homogeneous green fluorescence, indicating a homogeneous conjugation of the peptides on the hDBS surface [Fig. 7(B)]. Cell behavior on peptide-conjugated PCL mesh and hDBS in vitro PCL meshes. After 24 h of in vitro culture on the L7conjugated PCL meshes, the hSMSCs were fully attached to the fibers. These cells bridged between several fibers and integrated with the surrounding mesh to form a threedimensional network, indicating that the L7-conjugated PCL fiber has a good property for hSMSCs attachment and adhesion [Fig. 6(a,b)]. The SEM results indicated that L7 and RGD-conjugated PCL meshes had plenty of hSMSCs adhered and grown well on the surface of the meshes [Fig. 6(A,E) and (C,G)]. In contrast, P7-conjugated and pure PCL meshes seemed not so suitable for hSMSCs adhesion and growth, hSMSCs were hardly observed in the meshes [Fig. 6(B,F) and (D,H)]. The total DNA/dry weight ratio of the PCL

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FIGURE 3. Specificity of the peptide were investigated via flow cytometry. FITC-labeled L7 was incubated with hBMSCs and fibroblasts, respectively, comparing to hSMSCs. The results were also analyzed quantitatively through FCM. The average fluorescence intensity was 10.2 6 3.5 for L7 incubated with hBMSCs, comparing 126.1 6 18.4 for L7 incubated with hSMSCs (A and B) and 6.1 6 2.4 for L7 incubated with fibroblasts, comparing 110.1 6 3.9 for L7 incubated with hSMSCs (C and D; Student’s t-test, n 5 6, *p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

mesh was also calculated to obtain a quantitative analysis. The results showed a similar trend as the immunofluorescence staining results [L7, 150.23 6 10.30 and RGD, 125.75 6 25.58 vs. P7, 22.15 6 7.74 and pure, 16.59 6 5.46, n 5 3, p < 0.05; Fig. 6(e)]. Comparing to hSMSCs [Supporting Information Fig. S3(A)], after 24 h of in vitro culture on the L7-conjugated PCL meshes, hBMSCs spreaded not so well and much fewer adhered cells were observed [Supporting Information Fig. S3(B)]; and fibroblasts were hardly observed on the L7-conjugated PCL meshes [Supporting Information Fig. S3(C)]. The total DNA/dry weight ratio of the PCL mesh showed a similar trend as the SEM results [Supporting Information Fig. S3(A); hSMSCs, 172 6 9.61 vs. hBMSCs, 58.48 6 15.2 vs. fibroblasts, 15.33 6 3.34, n 5 3, p < 0.05]. Human DBS. After 24 h of in vitro culture on the L7conjugated hDBS, the hSMSCs also showed good adhesion and growth morphology on the surface of the scaffold [Fig. 7(C,D)]. Most of the hSMSCs spreaded well on the surface of the hDBS with L7 and RGD modification [Fig. 7(E,G)], whereas cells could barely spread on P7-conjugated and

pure hDBS surface and still stayed in round morphology [Fig. 7(F,H)]. The total DNA/dry weight ratio of the hDBS was also calculated to obtain a quantitative analysis. The results showed a similar trend as the SEM results [L7, 8.60 6 1.18 and RGD, 7.59 6 0.92 vs. P7, 3.51 6 0.54 and pure, 0.84 6 0.26, n 5 3, p < 0.05; Fig. 7(I)]. DISCUSSION

As we previously described,22 one of the most common strategies currently in TE is the combination of a biodegradable matrix (scaffold), exogenous or endogenous living cells, and/or biologically active molecules to form a construct that promotes tissue repair and regeneration.32 Promoting the accurate and efficient homing and entrapment of stem cells into damaged tissues to form “stem cell nests” should be first considered in TE. Such processes will also help to build regenerative “stem cell niches” in damaged sites, since MSCs can perform many trophic and immune-modulatory activities.15,33,34 Unfortunately, several reported studies show that only a small percentage of stem cells remain engrafted after a few weeks.15,16 In

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FIGURE 4. FITC-labeled peptides were covalently conjugated with PCL meshes, and were characterized via SEM. The morphology (B) and microstructure (C) of PCL electrospun meshes. Sulfo-SMCC was used as a crosslinker between the FITC-labeled peptides and the PCL electrospun fibers (A). After conjugation, homogeneous green fluorescence on PCL fibers was visualized under a confocal microscope (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

addition, endogenous stem cells are being used more frequently due to certain advantages compared to exogenous stem cells.17–19 Thus, the low efficiency and engraftment of MSCs becomes more acute in endogenous stem cellbased TE.

SMSCs are considered a good cell source for cartilage regeneration because of their high proliferation rates and chondrogenic potential, as demonstrated by many studies.1–5,7–10,12,20,35–37 Although several studies show that SMSCs possess some capacity to migrate toward

FIGURE 5. The immobilization of the peptides on PCL meshes was evaluated via XPS. Elemental nitrogen (N), which exists in peptides, on the surface of PCL fibers significantly increased after conjugation with L7 (B) and RGD (C) compared to aminated PCL mesh processed with sulfoSMCC (A). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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TABLE III. XPS Test of Peptide-Conjugated PCL Mesh Atomic Concentration (%)

PCL PCL 1 L7 PCL 1 RGD

C 1s

O 1s

46.68 6 1.33 43.37 6 2.47 47.37 6 0.98

36.46 6 2.88 29.11 6 1.26 32.49 6 1.79

a

N 1s

1.24 6 0.17 7.83 6 0.22 5.08 6 0.15

a

S 2s

0 0.93 6 0.11 0.38 6 0.06

a

S 2p

0 0.56 6 0.02 0.68 6 0.08

The immobilization of peptides on the PCL meshes was measured via X-ray photoelectron spectroscopy (XPS). Elemental nitrogen (N), which exists in peptides, on the surface of PCL fibers significantly increased after conjugation with L7 and RGD (N 1s: 7.83 6 0.22 and 5.08 6 0.15 vs. 1.24 6 0.17; ANOVA, n 5 3). a p < 0.05.

damaged sites,1,38,39 the problems of homing, engraftment, and function remain when adopting SMSCs as a cell source for cartilage regeneration. Abundant SMSCs need to home to and be entrapped within the site of tissue damage to build a “stem cell niche” and allow SMSCs to constantly facilitate repair during the entire regeneration process.

To address the problems above and improve cartilage regeneration based on SMSCs, we identified a seven amino acid peptide sequence (L7) through phage display technology that has a specific affinity to SMSCs. The recovery efficiency increased 15,000-fold after four rounds of screening. This result indicates that L7 has a high affinity to SMSCs, which was supported by the in vitro studies. L7 was then

FIGURE 6. The hSMSCs on FITC-labeled L7-conjugated PCL meshes were characterized based on their adhesion and spreading through immunofluorescence staining under a confocal microscope and SEM. After 24 h of culture with the FITC-L7-conjugated PCL meshes, the cells became fully attached to the fibers and expanded well (a and b). FITC-RGD-conjugated PCL meshes, as positive control, have the similar results as FITCL7-conjugated PCL meshes (d), whereas hSMSCs were hardly observed in the FITC-L7-conjugated PCL meshes (c; the nuclei were stained with Hoechst 33258, and the cytoskeleton was stained with rhodamine phalloidin). The SEM results indicated that L7 and RGD-conjugated PCL meshes had plenty of hSMSCs adhered and grown well on the surface of the meshes (A, E and C, G). In contrast, P7-conjugated and pure PCL meshes seemed not so suitable for hSMSCs adhesion and growth, hSMSCs were hardly observed in the meshes (B, F and D, H). The total DNA of the cells on the meshes was measured using a UV-2800H ultraviolet spectrophotometer, and divided by the dry weight of the meshes. The results are expressed as total DNA/dry weight ratio, and the data are expressed as mean 6 standard variation (e; ANOVA, n 5 3, *p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 7. The hSMSCs on FITC-labeled L7-conjugated human decalcified bone scaffolds were characterized based on their adhesion and spreading through immunofluorescence staining under a confocal microscope and SEM. After demineralized, the scaffolds were made into a cylinder 2 mm in diameter and 4 mm in height by a corneal trephine (a) and conjugated with FITC-labeled L7 (A). The cryosections of L7conjugated human decalcified bone scaffolds showed a homogeneous green fluorescence under a confocal microscope (B). After 24 h of in vitro culture, the hSMSCs showed good adhesion and growth morphology on the surface of the L7-conjugated human decalcified scaffold (C and D). Most of the hSMSCs spreaded well on the surface of the hDBS with L7 and RGD modification (E and G), whereas cells could barely spread on P7-conjugated and pure hDBS surface and still stayed in round morphology (F and H). The total DNA of the cells on the meshes was measured using a UV-2800H ultraviolet spectrophotometer, and divided by the dry weight of the meshes. The results are expressed as total DNA/dry weight ratio, and the data are expressed as mean 6 standard variation (I; ANOVA, n 5 3, *p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

covalently conjugated onto PCL electrospun meshes, which are a popular synthetic polymer material being widely used in TE,40,41 and hDBS, which is a natural scaffold with advantages for native hyaline articular cartilage regeneration,24 to investigate its effect on SMSC adherence on materials used for TE. The results show that L7 conjugation of both kinds of materials significantly improved SMSC adhesion after 24 h culture. SMSCs also spread well on the L7-modified materials surfaces. This result demonstrates that surface modification with L7 is promising for TE applications using SMSCs, particularly in cartilage regeneration because of the chondrogenic potential of SMSCs. In the present study, RGD was used as a positive control because it is best known for cell adhesion on synthetic material surfaces.28–30 However, as we mentioned in our previous study,22 this peptide lacks specificity for the general existence of fibronectin in all cells. This could lead to a more significant inflammatory response when RGD is used for surface modification of materials for tissue regeneration because cartilage damage is often followed by a massive release of inflammatory cells and cytokines. According to recent studies, nonspecific protein adsorption can sometimes be harmful to tissue regeneration because the proteins may trigger the immune system, causing severe nonspecific inflammation and obstructing the regeneration process.42 In our previous study, we confirmed that RGD-conjugated PCL meshes indeed caused more significant inflammatory responses after implantation in a rat knee cartilage defect.22 Compared to RGD, the L7 peptide that we identified through phage display in the present study has excellent specific affinity toward SMSCs. In our biopanning procedure, fibroblasts were used for negative selection to obtain better specificity. All these properties will help minimize the inflammatory response caused by the application of this peptide. Inefficient homing of systemically delivered MSCs is a major limitation of existing MSC-based therapeutic approaches, particularly for endogenous MSCs since their

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number is limited.13,14 Less than 1% of infused MSCs ultimately reach the target tissue.43,44 To address this problem, different strategies have been used, including cell surface engineering13 and chemotaxis-induced cell homing via cytokines, such as stromal cell-derived factor 112,45,46 and tumor growth factor b3.19 However, the use of adhesive peptides identified by phage display is more promising because a short peptide is more stable, cost-effective, and easier to control than functional proteins, such as growth factors. In previous studies, we have successfully identified a chondrocyte-homing peptide CAP23 and a bone marrowderived MSC-affinity peptide E7.22 Furthermore, we conjugated E7 onto PCL electrospun meshes to construct an “MSC-homing device”, and verified increased MSC adhesion, spreading and proliferation on it.22 In the present study, we focused on SMSCs, which are a recently reported MSC with high chondrogenic potential. We identified an SMSC-affinity peptide (L7) through phage display and successfully conjugated L7 onto the surface of two fundamentally different materials: PCL electrospun meshes and hDBS. Both materials demonstrated high SMSC adhesion. The number of MSCs in human synovial fluid increases in the knee with cartilage degeneration and osteoarthritis,47,48 and they may be derived from synovium. Other studies have verified the existence of these SMSCs in vivo and their contribution to articular cartilage repair.20,21 Based on these results, an appropriate procedure for mobilization of endogenous SMSCs could significantly improve cartilage regeneration through endogenous SMSCs with L7-conjugated scaffolds. In addition, these scaffolds with L7 surface modification will minimize the inflammatory response because L7 has excellent specific affinity toward SMSCs. Although this study is just an initial step toward constructing an ideal bioscaffold for cartilage TE and regeneration, it still offers a promising method to improve TE techniques, particularly for cartilage regeneration. Using this method, we can further improve scaffolds for cartilage

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regeneration in the future. For example, the scaffold could be hierarchically modified with appropriate affinity/mimic peptides to reconstruct the depth-dependent architecture of normal cartilage. To achieve this, the exact mechanism of the peptide should be further investigated to make better use of the peptide. CONCLUSION

In the present study, we identified an SMSC-affinity peptide (L7) through phage display and successfully conjugated it onto the surface of two fundamentally different materials: PCL electrospun meshes and hDBS. After conjugation with L7, both PCL electrospun meshes and hDBS demonstrated increased SMSC adhesion and well spreading. The present study offers a promising way to improve SMSC-based TE techniques, particularly for cartilage regeneration, via surface modification on various type of materials. REFERENCES 1. De Bari C, Dell’Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001;44:1928–1942. 2. Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source. Arthritis Rheum 2005;52: 2521–2529. 3. Nimura A, Muneta T, Koga H, Mochizuki T, Suzuki K, Makino H, Umezawa A, Sekiya I. Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: Comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum. Arthritis Rheum 2008;58:501–510. 4. Suzuki S, Muneta T, Tsuji K, Ichinose S, Makino H, Umezawa A, Sekiya I. Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration. Arthritis Res Ther 2012;14:R136. 5. Fan J, Varshney RR, Ren L, Cai D, Wang DA. Synovium-derived mesenchymal stem cells: A new cell source for musculoskeletal regeneration. Tissue Eng Part B Rev 2009;15:75–86. 6. Futami I, Ishijima M, Kaneko H, Tsuji K, Ichikawa-Tomikawa N, Sadatsuki R, Muneta T, Arikawa-Hirasawa E, Sekiya I, Kaneko K. Isolation and characterization of multipotential mesenchymal cells from the mouse synovium. PLoS ONE 2012;7:e45517. 7. Lee J-C, Min HJ, Park HJ, Lee S, Seong SC, Lee MC. Synovial membrane-derived mesenchymal stem cells supported by platelet-rich plasma can repair osteochondral defects in a rabbit model. Arthroscopy 2013;29:1034–1046. 8. Fan J, Gong Y, Ren L, Varshney RR, Cai D, Wang D-A. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater 2010;6:1178– 1185. 9. Varshney RR, Zhou R, Hao J, Yeo SS, Chooi WH, Fan J, Wang DA. Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system. Biomaterials 2010;31: 6876–6891. 10. Lee J-C, Lee SY, Min HJ, Han SA, Jang J, Lee S, Seong SC, Lee MC. Synovium-derived mesenchymal stem cells encapsulated in a novel injectable gel can repair osteochondral defects in a rabbit model. Tissue Eng Part A 2012;18:2173–2186. 11. Nakamura T, Sekiya I, Muneta T, Hatsushika D, Horie M, Tsuji K, Kawarasaki T, Watanabe A, Hishikawa S, Fujimoto Y, Tanaka H, Kobayashi E. Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs. Cytotherapy 2012;14:327–338. 12. Zhang W, Chen J, Tao J, Jiang Y, Hu C, Huang L, Ji J, Ouyang HW. The use of type 1 collagen scaffold containing stromal cellderived factor-1 to create a matrix environment conducive to

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