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Synthesis of E7 peptide-modified biodegradable polyester with the improving affinity to mesenchymal stem cells
Materials Science and Engineering C 73 (2017) 562–568
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of E7 peptide-modified biodegradable polyester with the improving affinity to mesenchymal stem cells Qian Li, Dongming Xing, Lie Ma ⁎, Changyou Gao MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e
i n f o
Article history: Received 17 August 2016 Received in revised form 30 November 2016 Accepted 17 December 2016 Available online 22 December 2016 Keywords: Maleimide Biodegradable polyester E7 peptide Affinity BMSCs
a b s t r a c t As the most promising stem cell, bone marrow-derived mesenchymal stem cells (BMSCs) has attracted many attentions and applied widely in regenerative medicine. A biodegradable polyester with tunable affinity to BMSCs plays critical role in determining the properties of the BMSCs-based constructs. In this study, maleimide functionalized biodegradable polyester (P(MTMC-LA)) was synthesized through ring-opening copolymerization between L-lactide (LA) and furan-maleimide functionalized trimethylene carbonate (FMTMC) and a subsequent retro Diels-Alder reaction. P(MTMC-LA) was modified by different amounts of BMSCs specific affinity peptide (EPLQLKM, E7) through click-chemistry to investigate the effect on BMSCs. The E7 peptide modified P(MTMCLA) was casted into films on glass slides and BMSCs were seeded onto the films. In vitro study showed that E7 peptide modified P(MTMC-LA) films supported BMSCs adhesion and proliferation compared to unmodified P(MTMC-LA) film. Besides, the adhesion and proliferation were enhanced by the increasing peptide grafting ratio. These results indicated that the novel biodegradable polyester can serve as a biomaterial with great potential application in tissue engineering and regenerative medicine. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Synthetic biodegradable polyesters such as polylactide (PLA), poly (lactide-co-glycolide) (PLGA) and polycaprolactone (PCL) have widespread applications in biomaterial field [1–4]. However, those conventional polyesters are challenged by their hydrophobicity and the absence of reactive groups, which results in their limited capacity to combine with biomolecules [5]. Recently, the efficient synthesis and post-functionalization of reactive biodegradable polymers have been a powerful strategy to fabricate biomaterials with enhanced bioactivity [6]. In particular, functional polyesters that are biodegradable, biocompatible, and easy-designed have gained increasing attention and hold immense promise in the application of tissue engineering, regenerative medicine, and novel drug delivery system [7,8]. Ring-opening copolymerization is one of the most common method to synthesize functional biodegradable polyesters [9]. Functional biodegradable polyesters that possess hydroxyl, carboxyl, and amine pendant groups have been synthesized through ring-opening copolymerization [10–12]. Versatile polymers based on the bio-functionalization of those functional polyesters have already been used to investigate their effect on cell behaviors [13]. As biomolecules can be easily degraded, the process for the post functionalization of biodegradable polyester should be mild. “Click
⁎ Corresponding author. E-mail address: [email protected] (L. Ma).
http://dx.doi.org/10.1016/j.msec.2016.12.088 0928-4931/© 2016 Elsevier B.V. All rights reserved.
chemistry” is an effective and potential method to modify functional biodegradable polyesters under mild conditions [14]. Wang et al. prepared novel vinyl sulfone-functionalized polyesters which can be synthesized by ring-opening copolymerization and modified by different molecules to control cell adhesion [15]. Poly(ester-carbonate) copolymer with maleimide group has been developed and bio-functionalized through the “click reactions” by Xing et al. [16] The laminin-derived peptide modified biodegradable polyester supported the proliferation and neurite outgrowth of PC12. As one of the key elements in tissue engineering, seed cells paly great role in determining the properties of the repaired tissues. Among them, stem cells especially bone marrow-derived mesenchymal stem cells (BMSCs) have been proved to be promising cells for tissue engineering due to easy isolation, rapid proliferation, self-renew, and multiple differentiation potential [17]. Typically, stem cells should be combined with biomaterial matrix for their application in tissue engineering and regenerative medicine. Thus, the effective adhesion of stem cells on the biomaterial is a vital process to realize its function [18,19]. Cell adhesion is closely related to the surface properties of biomaterial such as wettability, stiffness and topography [20–22]. To enhance the affinity between cells and biomaterial, surface modification is a widely accepted method [23]. A number of studies have fabricated versatile biomaterial system to promote the adhesion of stem cells for tissue regeneration. For example, protein-modified polyethylene terephthalate (PET) and polyurethane (PU) facilitate rats MSC adhesion,
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proliferation and viability [24]. Herein, the functional peptide play the function to establish the ECM mimicking environment for controlling cell behavior because it is more stable, cost-effective, and controllable than functional proteins. For example, functional mimicking peptide has been used to modify materials to establish the ECM mimicking environment for cell behavior because peptide is more stable, cost-effective, and easy to control than functional proteins, such as growth factors. Among these functional peptides, arginine-glycine-aspartic acid (RGD), a peptide derived from fibronectin in extracellular matrix (ECM) have been used widely to promote cell-biomaterial interaction, the functional biomaterials modified by which have been reported by many studies [25]. Recently, a novel peptide with the amino acid sequence of “EPLQLKM” (E7) with highly specific affinity to BMSCs has been identified and applied to specifically enhance the adhesion and proliferation of BMSCs [26]. The E7 modified PCL mesh showed the significant improvement on promoting the attachment and recruitment of BMSCs in vivo. Besides, it is reported that the collagen scaffold modified with E7 accelerated wound healing in acute full-thickness skin wounds [27]. In this study, to design novel biodegradable polyester with enhanced affinity to BMSCs specifically, the maleimide-functionalized poly (carbonate ester) was synthesized through ring-opening copolymerization between furan-maleimide functionalized trimethylene carbonate (FMTMC) and L-lactide. Then, different amounts of E7 peptides were conjugated to the polyester by “click reaction” to enhance the bioactivity. The affinity of BMSCs on the E7-modified polyester was evaluated by in vitro BMSCs culture test. 2. Experimental sections 2.1. Material 2,2-Bis(hydroxymethyl) propionic acid, 2,2-dimethoxypropane, ptoluenesulfonic acid monohydrate, and N,N′-dicyclohexylcarbodiimide (DCC), 4-Dimethylamiopryidine (DMAP) were purchased from Aladdin Industrial Inc. (China). Exo-3, 6-epoxy-1, 2, 3, 6-tetrahydrophthalic anhydride and Dowex H+ 50WX2 were purchased from Alfa Aesar (USA). L-Lactide (LA) was purchased from GLACO Ltd. (China). 1, 8diazabicyclo [5.4.0] undec-7-ene (DBU) was purchased from Sigma-Aldrich (USA). Ethyl chloroformate was purchased from Jinan Dacheng Chemical Co., Ltd. (China). Dichloride methane, tetrahydrofuran (THF), and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd. (SCRC, China) and purified over CaH2 before use. Other agents were purchased from SCRC without purification. E7 peptide was customized using a solid-state peptide synthesis method from GL Biochem Ltd. (China).
2.2. Synthesis of P(MTMC-LA) Maleimide-functionalized poly(carbonate ester) (P(MTMC-LA)) was synthesized through the following steps. Firstly, furan-maleimide functionalized trimethylene carbonate (FMTMC) was synthesized according to previous report [16]. Secondly, furan-maleimide functionalized poly(carbonate ester) (P(FMTMC-LA)) was obtained by copolymerization between FMTMC and LA. Typically, 0.533 g FMTMC (1.5 mmol) and 1.224 g L-lactide (8.5 mmol) were added to a glass vial. After evacuated and charged with nitrogen gas three times, chloroform was added through a syringe under nitrogen atmosphere followed by 21.9 mL (0.15 mmol) of DBU. The vial was sealed and the reaction mixture was stirred at 30 °C for 48 h. The resulting polymer was precipitated and purified in cold methanol. The products were dried under vacuum at room temperature. Finally, maleimide-functionalized polyester (P(MTMC-LA)) was obtained via the retro Diels-Alder reaction of P(FMTMC-LA) at 100 °C in toluene under nitrogen atmosphere for 12 h. 1H NMR, 13C NMR (CDCl3, 99.8 at.% D, Bruker DMX-500
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spectrometer, Switzerland) and Mass spectra (Bruker Esquire 3000plus ion trap mass spectrometer, Germany) were used to characterize FMTMC. The chemical structures of P(FMTMC-LA) and P(MTMC-LA) were characterized by 1H NMR (CDCl3, 99.8 at.% D). Gel permeation chromatography system (GPC, Waters 1515 Isocratic HPLC, USA) was used to determine the molecular weight and molecular weight distribution of P(FMTMC-LA) and P(MTMC-LA). 2.3. Synthesis and characterization of E7-modified P(MTMC-LA) To synthesize the E7-modified P(MTMC-LA), namely P(MTMC-LA)E7, cysteine was linked at the carboxyl terminus of E7 peptide in order to be reacted with P(MTMC-LA). Typically, P(MTMC-LA), E7 peptide, and pyridine were dissolved in DMF. The reaction was carried out under nitrogen atmosphere at room temperature for 24 h. The products was precipitated and purified from cold diethyl ether. Molar ratio of maleimide: -SH: pyridine = 1:0.15:0.15, 1:0.25:0.25, 1:0.5:0.5 were used to synthesize three kinds of P(MTMC-LA)-E7 with different E7 grafting ratio, which were noted as P(MTMC-LA)-E7-15, P(MTMC-LA)E7-25, P(MTMC-LA)-E7-50, respectively. 2.4. Preparation of P(MTMC-LA)-E7 films P(MTMC-LA)-E7 was dissolved in DMF for 24 h to form a solution with 10% mass ratio. P(MTMC-LA)-E7 films were prepared by spincoating on glass slides. The films were washed by ethanol and dried under vacuum at room temperature. X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD, Japan) was used to character the surface element of the P(MTMC-LA)-E7 films. The morphology and wettability of the films were analyzed by scanning electron microscopy (SEM, Hitachi S-4800, Japan) and static water contact angle measurement (Kruss DSA100, Germany). P(MTMC-LA) film was chosen as control. 2.5. Cell culture BMSCs were isolated from Sprague-Dawley male rats (6–8 weeks old) according to the methods reported previously [28]. The procedures were performed in accordance with the “Guidelines for Animal Experimentation” by the Institutional Animal Care and Use Committee, Zhejiang University. Briefly, the marrow was extracted from the femoral shafts using alpha minimum essential medium (α-MEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL streptomycin and 100 U/mL penicillin. The cells from the marrow were cultured in cell dishes in a humidified atmosphere with 5% CO2 at 37 °C. The medium was changed every 3 days. Cells were detached and passaged at 80% confluence. BMSCs at passage 3 were used for further experiments. The films were placed in 24 well plates and sterilized in 75% ethanol for 30 min, followed by three times washes in phosphate buffered saline (PBS, pH 7.2). BMSCs were seeded onto the films with the density of 2 × 104 cells per well. 2.6. Cell morphology Fluorescent staining of F-actin and cell nucleus were carried out to reveal the morphology of BMSCs. Briefly, after being cultured for 1 day, the attached BMSCs were washed with PBS 3 times to remove the suspended BMSCs and then fixed with 4% formaldehyde solution for 30 min at room temperature. The fixed BMSCs were treated with 0.5% Triton/PBS solution at 4 °C for 10 min. After being rinsed with PBS 3 times, the samples were treated with 1% BSA/PBS solution to block nonspecific adsorptions for 2 h. Finally, the BMSCs were stained with DAPI (Sigma, USA) for nucleus and rhodamine phalloidin (Invitrogen, USA) for cytoskeleton at 37 °C for 1 h. After 3 time washes in PBS, the morphology of the BMSCs were observed under a confocal laser scanning microscope (CLSM, LSM510, Carl Zeiss, Germany). The
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BMSCs were stained with fluorescein diacetate (FDA, Sigma, USA) and the images were taken under a fluorescence microscope (IX81, Olympus, Japan) for cell area assay.
ANOVA (SPSS 16.0) using SPSS software (SPSS Inc., Chicago, USA). The significant level was set as p b 0.05. 3. Results and discussion
2.7. Cell adhesion force
3.1. Synthesis and characterization of P(MTMC-LA)
The adhesion force of BMSCs on different P(MTMC-LA)-E7 films were measured according to Reyes's work [29]. Briefly, at day 1, the P(MTMC-LA)-E7 films were washed with PBS to remove the suspended cells, the BMSCs were stained with fluorescein diacetate (FDA) and the number of BMSCs on the films was counted under a fluorescence microscope (Zeiss Axiovert 200). Then the films were placed vertically at the bottom of tubes filled with PBS. The number of BMSCs remained on the films were counted after being centrifuged at 600 and 1000 rpm for 5 min respectively. Then the ratio of BMSCs adhered on the films before and after centrifugation was calculated, based on which the adhesion force can be calculated according to the theory described in Reyes's work. 2.8. Cell viability Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide staining (MTT) assay. Briefly, BMSCs were cultured on the films for 1 day, 3 days and 5 days, respectively. At each time interval, the culture medium was removed and the fresh medium containing 0.5 mg/mL MTT was added, and then BMSCs were incubated for another 4 h at 37 °C. The dark blue formazan crystals generated by mitochondrial dehydrogenase in living cells were dissolved in dimethyl sulfoxide (DMSO). The absorbance at 570 nm was measured by a microplate reader (Biorad Model 550, USA). 2.9. Statistical analysis At least three independent experiments were carried out if not specifically stated. Experimental data were expressed as mean ± standard deviation (SD) and the significant difference were analyzed by one way
FMTMC was synthesized through the cyclization reaction between furan-maleimide diol and ethyl chloroformate at the presence of triethylamine in THF (Scheme S1). The 1H, 13C NMR spectra (Fig. S1) and the Mass spectrum (Fig. S2) results proved that FMTMC was synthesized eventually. As shown in Scheme 1, P(FMTMC-LA) was synthesized through the copolymerization between FMTMC and LA in dry chloroform using DBU as catalyst under N2 atmosphere at 30 °C and P(MTMC-LA) was obtained through retro Diels-Alder reaction of P(FMTMC-LA) at 100 °C in toluene for 12 h. The reason for choosing DBU over Sn(Oct)2 is that the condition of ring-opening copolymerization catalyzed by DBU is mild while the temperature for Sn(Oct)2 is N100 °C which could lead to the crosslink between maleimide groups. All the chemical shift of hydrogen protons of P(FMTMC-LA) can be found in the 1H NMR spectra (Fig. 1), which proved the chemical structure of P(FMTMC-LA). After the retro Diels-Alder reaction, the peaks at δ 5.21 and 2.80 ppm vanished and a new peak at δ 6.67 ppm appeared which is assigned to the vinyl protons of maleimide groups in P(MTMC-LA) (Fig. 1). The change of the characteristic peaks confirmed the successful synthesis of P(MTMC-LA). Besides, through 1H NMR spectra, the actual ratio of MTMC in P(MTMC-LA) was calculated as 14.2% less than theoretical value of 15%, which could be attributed to the lower polymerization activity of FMTMC because of its rigidity structure and steric hindrance. The molecular weight and molecular weight distribution were measured by GPC (Fig. S3). The peaks of P(FMTMC-LA) and P(MTMC-LA) in the GPC spectra are single ones. The number-average molecular weight (Mn) of P(FMTMC-LA) and P(MTMC-LA) is 37,000 and 33,000, respectively. In addition, the molecular weight distribution does not change. The decrease of Mn after the retro Diels-Alder reaction verified that the furan group were removed and P(MTMC-LA) were obtained. For the backbone structure of P (MTMC-LA) is similar to PTMC and PLA, both of which are biodegradable polymers, P(MTMC-LA)
Scheme 1. Synthesis of P(MTMC-LA)-E7.
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Fig. 1. 1H NMR spectrum of P(FMTMC-LA) and P(MTMC-LA).
should show the similar biodegradability of the copolymer of P(TMCLA). 3.2. Synthesis and characterization of E7 modified P(MTMC-LA) It is of great importance to enhance the cell-material interaction and bio-functionalization is an efficient way to fabricate versatile biomaterials with good cell affinity [30]. For example, RGD modified alginate microspheres promoted the attachment and growth of human mesenchymal stem cells (hMSCs) [31]. A BMSCs specific affinity E7 peptide which has been identified to have specific affinity towards BMSCs and promote the adhesion of BMSCs was used to modify P(MTMC-LA)
to enhance its affinity with BMSCs [32]. As depicted in Scheme 1, three kinds of P(MTMC-LA)-E7 with different peptide grafting ratio, i.e. P(MTMC-LA)-E7-15, P(MTMC-LA)-E7-25, and P(MTMC-LA)-E7-50 were prepared. The surface composition of the P(MTMC-LA)-E7 films prepared by spin-coating were investigated by XPS, the spectra of which displayed the signals of nitrogen and carbon of P(MTMC-LA)-E7 films at 397.1 and 282.1 eV (Fig. 2). With the increase of E7 peptide, the signal of N peaks increased, which indicated that the E7 peptide was successfully modified to P(MTMC-LA). Besides, the ratio of nitrogen to carbon of P(MTMC-LA), P(MTMC-LA)-E7-15, P(MTMC-LA)-E7-25 and P(MTMC-LA)-E7-50 is 1:55.5, 1:36.6, 1:25.3 and 1:19.3, based on which the real grafting ratio can be calculated as 5.8%, 14.2%, 23.8%.
Fig. 2. XPS spectra of P(MTMC-LA) and P(MTMC-LA)-E7 films.
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Fig. 3. SEM images of films of (a) P(MTMC-LA), (b) P(MTMC-LA)-E7-15, (c) P(MTMC-LA)-E7-25, (d) P(MTMC-LA)-E7-50.
The surface topography of P(MTMC-LA)-E7 films were characterized by SEM. For the P(MTMC-LA) film (Fig. 3a), there are many pores with size ranging from 200 nm to 2 μm on the surface. And for the P(MTMC-LA)-E7-15 films (Fig. 3b), the surface with inhomogenous roughness was observed. The roughness of the surface of P(MTMCLA)-E7-25 films (Fig. 3c) increased and were relative homogenous. For P(MTMC-LA)-E7-50 films (Fig. 3d), the roughness is the biggest and there are some bulges size ranging from a few microns to dozens of micron on the surface. It is reported that solvent, molecular weight, polymer concentration are closely related to the surface topography of films prepared by spin-coating [33]. In this system, DMF which is slowly evaporating was used as solvent and the solubility of polyester in DMF decreased with the increase of E7 grafting ratio. Therefore, phase separation may occur during the evaporation of solvent resulting in different surface topography. The static water contact angle measurement was performed to test the wettability of P(MTMC-LA)-E7 films (Fig. 4). The results showed that the films became more hydrophilic after the modification of E7 peptide and the water contact angle decreased from 80° to
Fig. 4. Static water contact angle of P(MTMC-LA) and P(MTMC-LA)-E7 films.
57°, which can be attributed to the existence of E7 peptide. It has been proved that cells effectively adhered onto polymer surfaces with water contact angles of 40°–70° [34]. The hydrophilic surface of P(MTMC-LA)-E7 is beneficial for cell adhesion.
3.3. Cell morphology and adhesion force Generally, aliphatic polyesters are hydrophobic resulting in weak interaction between cells and biomaterials [35]. E7 peptide has been proved to promote the attachment of BMSCs [32]. In this study, E7 were immobilized to P(MTMC-LA) through “click reaction” to control the grafting ratio to investigate the affinity effect of E7 on BMSCs. The advantage of chemical conjugation of E7 peptide over physical incorporation mainly represents in the maintenance of the stability of E7 peptide. The morphology of BMSCs after being cultured for 24 h on P(MTMC-LA)-E7 films was observed by F-actin and nucleus staining (Fig. 5). As shown in Fig. 5, BMSCs can adhere on the P(MTMC-LA) and P(MTMC-LA)-E7 films. BMSCs presented contractive shapes on P(MTMC-LA) films. While, on P(MTMC-LA)-E7 films, BMSCs spread well and demonstrated stretching morphology with clear cytoskeleton. The grafting ratio of E7 has no obvious effect on the morphology of BMSCs. The introduction of E7 peptide enhanced the interaction between BMSCs and polymer films. Besides, the hydrophilic surface P(MTMC-LA)-E7 films is also in favor of the adhesion and spreading of BMSCs. Fig. 6 is the cell area and adhesion force results of BMSCs on P(MTMC-LA) and P(MTMC-LA)-E7 films. Compared to P(MTMC-LA) film, BMSCs seeded on P(MTMC-LA)-E7 films showed larger spreading area increasing from 1033 to 1621 μm2 (Fig. 6a). With the increase of E7 grafting ratio, the spreading area increased slightly. The similar trend was found in the adhesion force (Fig. 6b). The adhesion force of P(MTMC-LA)-E7-50 is about twice and 5 times higher than that of P(MTMC-LA)-E7-15 and P(MTMC-LA), respectively. There are no obviously difference between P(MTMC-LA)-E7-50 and P(MTMC-LA)-E725. Adhesion area and force is related to focal adhesion [36]. With the increase of E7 grafting ratio, P(MTMC-LA)-E7 films provided more and more recognition sites for BMSCs resulting in enhanced interaction between BMSCs and P(MTMC-LA)-E7 films through focal adhesion, which accounted for the increasing spreading area and adhesion force.
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Fig. 5. Cell morphology of BMSCs cultured on (a) P(MTMC-LA), (b) P(MTMC-LA)-E7-15, (c) P(MTMC-LA)-E7-25, (d) P(MTMC-LA)-E7-50.
3.4. Cell viability There are evidence indicated that many biomolecules promote the viability and proliferation of BMSCs [35]. In this study, BMSCs were seeded onto the P(MTMC-LA) and P(MTMC-LA)-E7 films for 1 day, 3 days and 5 days for cell viability and proliferation test. The MTT assay was employed to examine the cell viability. As shown in Fig. 7, the increased cell viability was observed in all samples as culture time prolonged, which demonstrated that BMSCs proliferated on all the polymer films. In addition, the BMSCs cultured on P(MTMC-LA)-E7 films had
higher viability compared to P(MTMC-LA) film at each time interval, especially at day 5. Moreover, with the increase of E7 peptide, the viability increased. All the results indicated that P(MTMC-LA)-E7 has good affinity with BMSCs to accelerate the adhesion and proliferation of BMSCs. 4. Conclusions P(MTMC-LA) was successfully synthesized by the copolymerization between FMTMC and LA and a subsequent retro Diels-Alder reaction. The polyester was modified by a BMSCs affinity peptide-E7. The
Fig. 6. (a) Spreading area and (b) adhesion force of BMSCs cultured on P(MTMC-LA) and P(MTMC-LA)-E7 films.
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Fig. 7. Proliferation behaviors of BMSCs cultured on P(MTMC-LA) and P(MTMC-LA)-E7 films.
P(MTMC-LA)-E7 promotes the adhesion and proliferation of BMSCs. Moreover, the adhesion and proliferation is closely related to the peptide grafting ratio. The P(MTMC-LA)-E7 is a promising biomaterial for tissue engineering and regenerative medicine, especially for in situ BMSCs adhesion and capturing. Acknowledgements We acknowledge financial support by the Key Science Technology Innovation Team of Zhejiang Province (2013TD02), the Natural Science Foundation of China (51322302, 51673167, 20934003) and the National Key Research Program of China (2016YFC1101001). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.12.088. References [1] G. Chen, T. Ushida, T. Tateishi, Hybrid biomaterials for tissue engineering: a preparative method for PLA or PLGA–collagen hybrid sponges, Adv. Mater. 12 (2000) 455–457. [2] B. Li, J. Yang, L. Ma, F. Li, Z. Tu, C. Gao, Influence of the molecular weight of poly (lactide-co-glycolide) on the in vivo cartilage repair by a construct of poly (lactide-co-glycolide)/fibrin gel/mesenchymal stem cells/transforming growth factor-β1, Tissue Eng. Part A 20 (2013) 1–11. [3] Y. Zhu, Z. Mao, C. Gao, Control over the gradient differentiation of rat BMSCs on a PCL membrane with surface-immobilized alendronate gradient, Biomacromolecules 14 (2013) 342–349. [4] V. Russo, L. Tammaro, L. Di Marcantonio, A. Sorrentino, M. Ancora, L. Valbonetti, M. Turriani, A. Martelli, C. Cammà, B. Barboni, Amniotic epithelial stem cell biocompatibility for electrospun poly(lactide-co-glycolide), poly(ε-caprolactone), poly(lactic acid) scaffolds, Mater. Sci. Eng. C 69 (2016) 321–329. [5] E.J. Jansen, R.E. Sladek, H. Bahar, A. Yaffe, M.J. Gijbels, R. Kuijer, S.K. Bulstra, N.A. Guldemond, I. Binderman, L.H. Koole, Hydrophobicity as a design criterion for polymer scaffolds in bone tissue engineering, Biomaterials 26 (2005) 4423–4431. [6] X. Liu, J.M. Holzwarth, P.X. Ma, Functionalized synthetic biodegradable polymer scaffolds for tissue engineering, Macromol. Biosci. 12 (2012) 911–919. [7] Y. Ikada, H. Tsuji, Biodegradable polyesters for medical and ecological applications, Macromol. Rapid Commun. 21 (2000) 117–132. [8] G. Bayramoglu, V. Bitirim, Y. Tunali, M.Y. Arica, K.C. Akcali, Poly (hydroxyethyl methacrylate-glycidyl methacrylate) films modified with different functional groups: in vitro interactions with platelets and rat stem cells, Mater. Sci. Eng. C 33 (2013) 801–810. [9] O. Coulembier, P. Degée, J.L. Hedrick, P. Dubois, From controlled ring-opening polymerization to biodegradable aliphatic polyester: especially poly (β-malic acid) derivatives, Prog. Polym. Sci. 31 (2006) 723–747.
[10] X. Bi, Z. You, J. Gao, X. Fan, Y. Wang, A functional polyester carrying free hydroxyl groups promotes the mineralization of osteoblast and human mesenchymal stem cell extracellular matrix, Acta Biomater. 10 (2014) 2814–2823. [11] W. Cai, J. Wu, C. Xi, A.J. Ashe, M.E. Meyerhoff, Carboxyl-ebselen-based layer-by-layer films as potential antithrombotic and antimicrobial coatings, Biomaterials 32 (2011) 7774–7784. [12] A. Philipp, M. Meyer, A. Zintchenko, E. Wagner, Functional modification of amidecrosslinked oligoethylenimine for improved siRNA delivery, React. Funct. Polym. 71 (2011) 288–293. [13] B. Guo, L. Glavas, A.-C. Albertsson, Biodegradable and electrically conducting polymers for biomedical applications, Prog. Polym. Sci. 38 (2013) 1263–1286. [14] S. Hvilsted, Facile design of biomaterials by ‘click’ chemistry, Polym. Int. 61 (2012) 485–494. [15] R. Wang, W. Chen, F. Meng, R. Cheng, C. Deng, J. Feijen, Z. Zhong, Unprecedented access to functional biodegradable polymers and coatings, Macromolecules 44 (2011) 6009–6016. [16] D. Xing, L. Ma, C. Gao, Synthesis of functionalized poly (ester carbonate) with laminin-derived peptide for promoting neurite outgrowth of PC12 cells, Macromol. Biosci. 14 (2014) 1429–1436. [17] K.C. Kemp, J. Hows, C. Donaldson, Bone marrow-derived mesenchymal stem cells, Leuk. Lymphoma 46 (2005) 1531–1544. [18] S. Martino, F. D'Angelo, I. Armentano, J.M. Kenny, A. Orlacchio, Stem cell-biomaterial interactions for regenerative medicine, Biotechnol. Adv. 30 (2012) 338–351. [19] A. Salerno, V. Guarino, O. Oliviero, L. Ambrosio, C. Domingo, Bio-safe processing of polylactic-co-caprolactone and polylactic acid blends to fabricate fibrous porous scaffolds for in vitro mesenchymal stem cells adhesion and proliferation, Mater. Sci. Eng. C 63 (2016) 512–521. [20] Y. Lai, F. Pan, C. Xu, H. Fuchs, L. Chi, In situ surface-modification-induced superhydrophobic patterns with reversible wettability and adhesion, Adv. Mater. 25 (2013) 1682–1686. [21] Y.-x. Sun, K.-f. Ren, J.-l. Wang, G.-x. Chang, J. Ji, Electrochemically controlled stiffness of multilayers for manipulation of cell adhesion, ACS Appl. Mater. Interfaces 5 (2013) 4597–4602. [22] C.H. Seo, H. Jeong, K.S. Furukawa, Y. Suzuki, T. Ushida, The switching of focal adhesion maturation sites and actin filament activation for MSCs by topography of well-defined micropatterned surfaces, Biomaterials 34 (2013) 1764–1771. [23] S.H. Ku, J.S. Lee, C.B. Park, Spatial control of cell adhesion and patterning through mussel-inspired surface modification by polydopamine, Langmuir 26 (2010) 15104–15108. [24] Y. Gustafsson, J. Haag, P. Jungebluth, V. Lundin, M.L. Lim, S. Baiguera, F. Ajalloueian, C. Del Gaudio, A. Bianco, G. Moll, Viability and proliferation of rat MSCs on adhesion protein-modified PET and PU scaffolds, Biomaterials 33 (2012) 8094–8103. [25] H. Zhang, C.-Y. Lin, S.J. Hollister, The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds, Biomaterials 30 (2009) 4063–4069. [26] Z. Shao, X. Zhang, Y. Pi, X. Wang, Z. Jia, J. Zhu, L. Dai, W. Chen, L. Yin, H. Chen, Polycaprolactone electrospun mesh conjugated with an MSC affinity peptide for MSC homing in vivo, Biomaterials 33 (2012) 3375–3387. [27] H. Wang, X. Yan, L. Shen, S. Li, Y. Lin, S. Wang, X. Hou, C. Shi, Y. Yang, J. Dai, Acceleration of wound healing in acute full-thickness skin wounds using a collagen-binding peptide with an affinity for MSCs, Burns Trauma 2 (2014) 181. [28] M. Soleimani, S. Nadri, A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow, Nat. Protoc. 4 (2009) 102–106. [29] C.D. Reyes, A.J. García, A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces, J. Biomed. Mater. Res. A 67 (2003) 328–333. [30] Y.-P. Jiao, F.-Z. Cui, Surface modification of polyester biomaterials for tissue engineering, Biomed. Mater. 2 (2007) R24. [31] J. Yu, K.T. Du, Q. Fang, Y. Gu, S.S. Mihardja, R.E. Sievers, J.C. Wu, R.J. Lee, The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat, Biomaterials 31 (2010) 7012–7020. [32] Z. Man, L. Yin, Z. Shao, X. Zhang, X. Hu, J. Zhu, L. Dai, H. Huang, L. Yuan, C. Zhou, H. Chen, Y. Ao, The effects of co-delivery of BMSC-affinity peptide and rhTGF-β1 from coaxial electrospun scaffolds on chondrogenic differentiation, Biomaterials 35 (2014) 5250–5260. [33] L.L. Spangler, J.M. Torkelson, J.S. Royal, Influence of solvent and molecular weight on thickness and surface topography of spincoated polymer films, Polym. Eng. Sci. 30 (1990) 644–653. [34] Y. Arima, H. Iwata, Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers, Biomaterials 28 (2007) 3074–3082. [35] B. Parrish, R.B. Breitenkamp, T. Emrick, PEG-and peptide-grafted aliphatic polyesters by click chemistry, J. Am. Chem. Soc. 127 (2005) 7404–7410. [36] N.Q. Balaban, U.S. Schwarz, D. Riveline, P. Goichberg, G. Tzur, I. Sabanay, D. Mahalu, S. Safran, A. Bershadsky, L. Addadi, Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates, Nat. Cell Biol. 3 (2001) 466–472.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of E7 peptide-modified biodegradable polyester with the improving affinity to mesenchymal stem cells Qian Li, Dongming Xing, Lie Ma ⁎, Changyou Gao MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e
i n f o
Article history: Received 17 August 2016 Received in revised form 30 November 2016 Accepted 17 December 2016 Available online 22 December 2016 Keywords: Maleimide Biodegradable polyester E7 peptide Affinity BMSCs
a b s t r a c t As the most promising stem cell, bone marrow-derived mesenchymal stem cells (BMSCs) has attracted many attentions and applied widely in regenerative medicine. A biodegradable polyester with tunable affinity to BMSCs plays critical role in determining the properties of the BMSCs-based constructs. In this study, maleimide functionalized biodegradable polyester (P(MTMC-LA)) was synthesized through ring-opening copolymerization between L-lactide (LA) and furan-maleimide functionalized trimethylene carbonate (FMTMC) and a subsequent retro Diels-Alder reaction. P(MTMC-LA) was modified by different amounts of BMSCs specific affinity peptide (EPLQLKM, E7) through click-chemistry to investigate the effect on BMSCs. The E7 peptide modified P(MTMCLA) was casted into films on glass slides and BMSCs were seeded onto the films. In vitro study showed that E7 peptide modified P(MTMC-LA) films supported BMSCs adhesion and proliferation compared to unmodified P(MTMC-LA) film. Besides, the adhesion and proliferation were enhanced by the increasing peptide grafting ratio. These results indicated that the novel biodegradable polyester can serve as a biomaterial with great potential application in tissue engineering and regenerative medicine. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Synthetic biodegradable polyesters such as polylactide (PLA), poly (lactide-co-glycolide) (PLGA) and polycaprolactone (PCL) have widespread applications in biomaterial field [1–4]. However, those conventional polyesters are challenged by their hydrophobicity and the absence of reactive groups, which results in their limited capacity to combine with biomolecules [5]. Recently, the efficient synthesis and post-functionalization of reactive biodegradable polymers have been a powerful strategy to fabricate biomaterials with enhanced bioactivity [6]. In particular, functional polyesters that are biodegradable, biocompatible, and easy-designed have gained increasing attention and hold immense promise in the application of tissue engineering, regenerative medicine, and novel drug delivery system [7,8]. Ring-opening copolymerization is one of the most common method to synthesize functional biodegradable polyesters [9]. Functional biodegradable polyesters that possess hydroxyl, carboxyl, and amine pendant groups have been synthesized through ring-opening copolymerization [10–12]. Versatile polymers based on the bio-functionalization of those functional polyesters have already been used to investigate their effect on cell behaviors [13]. As biomolecules can be easily degraded, the process for the post functionalization of biodegradable polyester should be mild. “Click
⁎ Corresponding author. E-mail address: [email protected] (L. Ma).
http://dx.doi.org/10.1016/j.msec.2016.12.088 0928-4931/© 2016 Elsevier B.V. All rights reserved.
chemistry” is an effective and potential method to modify functional biodegradable polyesters under mild conditions [14]. Wang et al. prepared novel vinyl sulfone-functionalized polyesters which can be synthesized by ring-opening copolymerization and modified by different molecules to control cell adhesion [15]. Poly(ester-carbonate) copolymer with maleimide group has been developed and bio-functionalized through the “click reactions” by Xing et al. [16] The laminin-derived peptide modified biodegradable polyester supported the proliferation and neurite outgrowth of PC12. As one of the key elements in tissue engineering, seed cells paly great role in determining the properties of the repaired tissues. Among them, stem cells especially bone marrow-derived mesenchymal stem cells (BMSCs) have been proved to be promising cells for tissue engineering due to easy isolation, rapid proliferation, self-renew, and multiple differentiation potential [17]. Typically, stem cells should be combined with biomaterial matrix for their application in tissue engineering and regenerative medicine. Thus, the effective adhesion of stem cells on the biomaterial is a vital process to realize its function [18,19]. Cell adhesion is closely related to the surface properties of biomaterial such as wettability, stiffness and topography [20–22]. To enhance the affinity between cells and biomaterial, surface modification is a widely accepted method [23]. A number of studies have fabricated versatile biomaterial system to promote the adhesion of stem cells for tissue regeneration. For example, protein-modified polyethylene terephthalate (PET) and polyurethane (PU) facilitate rats MSC adhesion,
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proliferation and viability [24]. Herein, the functional peptide play the function to establish the ECM mimicking environment for controlling cell behavior because it is more stable, cost-effective, and controllable than functional proteins. For example, functional mimicking peptide has been used to modify materials to establish the ECM mimicking environment for cell behavior because peptide is more stable, cost-effective, and easy to control than functional proteins, such as growth factors. Among these functional peptides, arginine-glycine-aspartic acid (RGD), a peptide derived from fibronectin in extracellular matrix (ECM) have been used widely to promote cell-biomaterial interaction, the functional biomaterials modified by which have been reported by many studies [25]. Recently, a novel peptide with the amino acid sequence of “EPLQLKM” (E7) with highly specific affinity to BMSCs has been identified and applied to specifically enhance the adhesion and proliferation of BMSCs [26]. The E7 modified PCL mesh showed the significant improvement on promoting the attachment and recruitment of BMSCs in vivo. Besides, it is reported that the collagen scaffold modified with E7 accelerated wound healing in acute full-thickness skin wounds [27]. In this study, to design novel biodegradable polyester with enhanced affinity to BMSCs specifically, the maleimide-functionalized poly (carbonate ester) was synthesized through ring-opening copolymerization between furan-maleimide functionalized trimethylene carbonate (FMTMC) and L-lactide. Then, different amounts of E7 peptides were conjugated to the polyester by “click reaction” to enhance the bioactivity. The affinity of BMSCs on the E7-modified polyester was evaluated by in vitro BMSCs culture test. 2. Experimental sections 2.1. Material 2,2-Bis(hydroxymethyl) propionic acid, 2,2-dimethoxypropane, ptoluenesulfonic acid monohydrate, and N,N′-dicyclohexylcarbodiimide (DCC), 4-Dimethylamiopryidine (DMAP) were purchased from Aladdin Industrial Inc. (China). Exo-3, 6-epoxy-1, 2, 3, 6-tetrahydrophthalic anhydride and Dowex H+ 50WX2 were purchased from Alfa Aesar (USA). L-Lactide (LA) was purchased from GLACO Ltd. (China). 1, 8diazabicyclo [5.4.0] undec-7-ene (DBU) was purchased from Sigma-Aldrich (USA). Ethyl chloroformate was purchased from Jinan Dacheng Chemical Co., Ltd. (China). Dichloride methane, tetrahydrofuran (THF), and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd. (SCRC, China) and purified over CaH2 before use. Other agents were purchased from SCRC without purification. E7 peptide was customized using a solid-state peptide synthesis method from GL Biochem Ltd. (China).
2.2. Synthesis of P(MTMC-LA) Maleimide-functionalized poly(carbonate ester) (P(MTMC-LA)) was synthesized through the following steps. Firstly, furan-maleimide functionalized trimethylene carbonate (FMTMC) was synthesized according to previous report [16]. Secondly, furan-maleimide functionalized poly(carbonate ester) (P(FMTMC-LA)) was obtained by copolymerization between FMTMC and LA. Typically, 0.533 g FMTMC (1.5 mmol) and 1.224 g L-lactide (8.5 mmol) were added to a glass vial. After evacuated and charged with nitrogen gas three times, chloroform was added through a syringe under nitrogen atmosphere followed by 21.9 mL (0.15 mmol) of DBU. The vial was sealed and the reaction mixture was stirred at 30 °C for 48 h. The resulting polymer was precipitated and purified in cold methanol. The products were dried under vacuum at room temperature. Finally, maleimide-functionalized polyester (P(MTMC-LA)) was obtained via the retro Diels-Alder reaction of P(FMTMC-LA) at 100 °C in toluene under nitrogen atmosphere for 12 h. 1H NMR, 13C NMR (CDCl3, 99.8 at.% D, Bruker DMX-500
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spectrometer, Switzerland) and Mass spectra (Bruker Esquire 3000plus ion trap mass spectrometer, Germany) were used to characterize FMTMC. The chemical structures of P(FMTMC-LA) and P(MTMC-LA) were characterized by 1H NMR (CDCl3, 99.8 at.% D). Gel permeation chromatography system (GPC, Waters 1515 Isocratic HPLC, USA) was used to determine the molecular weight and molecular weight distribution of P(FMTMC-LA) and P(MTMC-LA). 2.3. Synthesis and characterization of E7-modified P(MTMC-LA) To synthesize the E7-modified P(MTMC-LA), namely P(MTMC-LA)E7, cysteine was linked at the carboxyl terminus of E7 peptide in order to be reacted with P(MTMC-LA). Typically, P(MTMC-LA), E7 peptide, and pyridine were dissolved in DMF. The reaction was carried out under nitrogen atmosphere at room temperature for 24 h. The products was precipitated and purified from cold diethyl ether. Molar ratio of maleimide: -SH: pyridine = 1:0.15:0.15, 1:0.25:0.25, 1:0.5:0.5 were used to synthesize three kinds of P(MTMC-LA)-E7 with different E7 grafting ratio, which were noted as P(MTMC-LA)-E7-15, P(MTMC-LA)E7-25, P(MTMC-LA)-E7-50, respectively. 2.4. Preparation of P(MTMC-LA)-E7 films P(MTMC-LA)-E7 was dissolved in DMF for 24 h to form a solution with 10% mass ratio. P(MTMC-LA)-E7 films were prepared by spincoating on glass slides. The films were washed by ethanol and dried under vacuum at room temperature. X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD, Japan) was used to character the surface element of the P(MTMC-LA)-E7 films. The morphology and wettability of the films were analyzed by scanning electron microscopy (SEM, Hitachi S-4800, Japan) and static water contact angle measurement (Kruss DSA100, Germany). P(MTMC-LA) film was chosen as control. 2.5. Cell culture BMSCs were isolated from Sprague-Dawley male rats (6–8 weeks old) according to the methods reported previously [28]. The procedures were performed in accordance with the “Guidelines for Animal Experimentation” by the Institutional Animal Care and Use Committee, Zhejiang University. Briefly, the marrow was extracted from the femoral shafts using alpha minimum essential medium (α-MEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL streptomycin and 100 U/mL penicillin. The cells from the marrow were cultured in cell dishes in a humidified atmosphere with 5% CO2 at 37 °C. The medium was changed every 3 days. Cells were detached and passaged at 80% confluence. BMSCs at passage 3 were used for further experiments. The films were placed in 24 well plates and sterilized in 75% ethanol for 30 min, followed by three times washes in phosphate buffered saline (PBS, pH 7.2). BMSCs were seeded onto the films with the density of 2 × 104 cells per well. 2.6. Cell morphology Fluorescent staining of F-actin and cell nucleus were carried out to reveal the morphology of BMSCs. Briefly, after being cultured for 1 day, the attached BMSCs were washed with PBS 3 times to remove the suspended BMSCs and then fixed with 4% formaldehyde solution for 30 min at room temperature. The fixed BMSCs were treated with 0.5% Triton/PBS solution at 4 °C for 10 min. After being rinsed with PBS 3 times, the samples were treated with 1% BSA/PBS solution to block nonspecific adsorptions for 2 h. Finally, the BMSCs were stained with DAPI (Sigma, USA) for nucleus and rhodamine phalloidin (Invitrogen, USA) for cytoskeleton at 37 °C for 1 h. After 3 time washes in PBS, the morphology of the BMSCs were observed under a confocal laser scanning microscope (CLSM, LSM510, Carl Zeiss, Germany). The
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BMSCs were stained with fluorescein diacetate (FDA, Sigma, USA) and the images were taken under a fluorescence microscope (IX81, Olympus, Japan) for cell area assay.
ANOVA (SPSS 16.0) using SPSS software (SPSS Inc., Chicago, USA). The significant level was set as p b 0.05. 3. Results and discussion
2.7. Cell adhesion force
3.1. Synthesis and characterization of P(MTMC-LA)
The adhesion force of BMSCs on different P(MTMC-LA)-E7 films were measured according to Reyes's work [29]. Briefly, at day 1, the P(MTMC-LA)-E7 films were washed with PBS to remove the suspended cells, the BMSCs were stained with fluorescein diacetate (FDA) and the number of BMSCs on the films was counted under a fluorescence microscope (Zeiss Axiovert 200). Then the films were placed vertically at the bottom of tubes filled with PBS. The number of BMSCs remained on the films were counted after being centrifuged at 600 and 1000 rpm for 5 min respectively. Then the ratio of BMSCs adhered on the films before and after centrifugation was calculated, based on which the adhesion force can be calculated according to the theory described in Reyes's work. 2.8. Cell viability Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide staining (MTT) assay. Briefly, BMSCs were cultured on the films for 1 day, 3 days and 5 days, respectively. At each time interval, the culture medium was removed and the fresh medium containing 0.5 mg/mL MTT was added, and then BMSCs were incubated for another 4 h at 37 °C. The dark blue formazan crystals generated by mitochondrial dehydrogenase in living cells were dissolved in dimethyl sulfoxide (DMSO). The absorbance at 570 nm was measured by a microplate reader (Biorad Model 550, USA). 2.9. Statistical analysis At least three independent experiments were carried out if not specifically stated. Experimental data were expressed as mean ± standard deviation (SD) and the significant difference were analyzed by one way
FMTMC was synthesized through the cyclization reaction between furan-maleimide diol and ethyl chloroformate at the presence of triethylamine in THF (Scheme S1). The 1H, 13C NMR spectra (Fig. S1) and the Mass spectrum (Fig. S2) results proved that FMTMC was synthesized eventually. As shown in Scheme 1, P(FMTMC-LA) was synthesized through the copolymerization between FMTMC and LA in dry chloroform using DBU as catalyst under N2 atmosphere at 30 °C and P(MTMC-LA) was obtained through retro Diels-Alder reaction of P(FMTMC-LA) at 100 °C in toluene for 12 h. The reason for choosing DBU over Sn(Oct)2 is that the condition of ring-opening copolymerization catalyzed by DBU is mild while the temperature for Sn(Oct)2 is N100 °C which could lead to the crosslink between maleimide groups. All the chemical shift of hydrogen protons of P(FMTMC-LA) can be found in the 1H NMR spectra (Fig. 1), which proved the chemical structure of P(FMTMC-LA). After the retro Diels-Alder reaction, the peaks at δ 5.21 and 2.80 ppm vanished and a new peak at δ 6.67 ppm appeared which is assigned to the vinyl protons of maleimide groups in P(MTMC-LA) (Fig. 1). The change of the characteristic peaks confirmed the successful synthesis of P(MTMC-LA). Besides, through 1H NMR spectra, the actual ratio of MTMC in P(MTMC-LA) was calculated as 14.2% less than theoretical value of 15%, which could be attributed to the lower polymerization activity of FMTMC because of its rigidity structure and steric hindrance. The molecular weight and molecular weight distribution were measured by GPC (Fig. S3). The peaks of P(FMTMC-LA) and P(MTMC-LA) in the GPC spectra are single ones. The number-average molecular weight (Mn) of P(FMTMC-LA) and P(MTMC-LA) is 37,000 and 33,000, respectively. In addition, the molecular weight distribution does not change. The decrease of Mn after the retro Diels-Alder reaction verified that the furan group were removed and P(MTMC-LA) were obtained. For the backbone structure of P (MTMC-LA) is similar to PTMC and PLA, both of which are biodegradable polymers, P(MTMC-LA)
Scheme 1. Synthesis of P(MTMC-LA)-E7.
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Fig. 1. 1H NMR spectrum of P(FMTMC-LA) and P(MTMC-LA).
should show the similar biodegradability of the copolymer of P(TMCLA). 3.2. Synthesis and characterization of E7 modified P(MTMC-LA) It is of great importance to enhance the cell-material interaction and bio-functionalization is an efficient way to fabricate versatile biomaterials with good cell affinity [30]. For example, RGD modified alginate microspheres promoted the attachment and growth of human mesenchymal stem cells (hMSCs) [31]. A BMSCs specific affinity E7 peptide which has been identified to have specific affinity towards BMSCs and promote the adhesion of BMSCs was used to modify P(MTMC-LA)
to enhance its affinity with BMSCs [32]. As depicted in Scheme 1, three kinds of P(MTMC-LA)-E7 with different peptide grafting ratio, i.e. P(MTMC-LA)-E7-15, P(MTMC-LA)-E7-25, and P(MTMC-LA)-E7-50 were prepared. The surface composition of the P(MTMC-LA)-E7 films prepared by spin-coating were investigated by XPS, the spectra of which displayed the signals of nitrogen and carbon of P(MTMC-LA)-E7 films at 397.1 and 282.1 eV (Fig. 2). With the increase of E7 peptide, the signal of N peaks increased, which indicated that the E7 peptide was successfully modified to P(MTMC-LA). Besides, the ratio of nitrogen to carbon of P(MTMC-LA), P(MTMC-LA)-E7-15, P(MTMC-LA)-E7-25 and P(MTMC-LA)-E7-50 is 1:55.5, 1:36.6, 1:25.3 and 1:19.3, based on which the real grafting ratio can be calculated as 5.8%, 14.2%, 23.8%.
Fig. 2. XPS spectra of P(MTMC-LA) and P(MTMC-LA)-E7 films.
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Fig. 3. SEM images of films of (a) P(MTMC-LA), (b) P(MTMC-LA)-E7-15, (c) P(MTMC-LA)-E7-25, (d) P(MTMC-LA)-E7-50.
The surface topography of P(MTMC-LA)-E7 films were characterized by SEM. For the P(MTMC-LA) film (Fig. 3a), there are many pores with size ranging from 200 nm to 2 μm on the surface. And for the P(MTMC-LA)-E7-15 films (Fig. 3b), the surface with inhomogenous roughness was observed. The roughness of the surface of P(MTMCLA)-E7-25 films (Fig. 3c) increased and were relative homogenous. For P(MTMC-LA)-E7-50 films (Fig. 3d), the roughness is the biggest and there are some bulges size ranging from a few microns to dozens of micron on the surface. It is reported that solvent, molecular weight, polymer concentration are closely related to the surface topography of films prepared by spin-coating [33]. In this system, DMF which is slowly evaporating was used as solvent and the solubility of polyester in DMF decreased with the increase of E7 grafting ratio. Therefore, phase separation may occur during the evaporation of solvent resulting in different surface topography. The static water contact angle measurement was performed to test the wettability of P(MTMC-LA)-E7 films (Fig. 4). The results showed that the films became more hydrophilic after the modification of E7 peptide and the water contact angle decreased from 80° to
Fig. 4. Static water contact angle of P(MTMC-LA) and P(MTMC-LA)-E7 films.
57°, which can be attributed to the existence of E7 peptide. It has been proved that cells effectively adhered onto polymer surfaces with water contact angles of 40°–70° [34]. The hydrophilic surface of P(MTMC-LA)-E7 is beneficial for cell adhesion.
3.3. Cell morphology and adhesion force Generally, aliphatic polyesters are hydrophobic resulting in weak interaction between cells and biomaterials [35]. E7 peptide has been proved to promote the attachment of BMSCs [32]. In this study, E7 were immobilized to P(MTMC-LA) through “click reaction” to control the grafting ratio to investigate the affinity effect of E7 on BMSCs. The advantage of chemical conjugation of E7 peptide over physical incorporation mainly represents in the maintenance of the stability of E7 peptide. The morphology of BMSCs after being cultured for 24 h on P(MTMC-LA)-E7 films was observed by F-actin and nucleus staining (Fig. 5). As shown in Fig. 5, BMSCs can adhere on the P(MTMC-LA) and P(MTMC-LA)-E7 films. BMSCs presented contractive shapes on P(MTMC-LA) films. While, on P(MTMC-LA)-E7 films, BMSCs spread well and demonstrated stretching morphology with clear cytoskeleton. The grafting ratio of E7 has no obvious effect on the morphology of BMSCs. The introduction of E7 peptide enhanced the interaction between BMSCs and polymer films. Besides, the hydrophilic surface P(MTMC-LA)-E7 films is also in favor of the adhesion and spreading of BMSCs. Fig. 6 is the cell area and adhesion force results of BMSCs on P(MTMC-LA) and P(MTMC-LA)-E7 films. Compared to P(MTMC-LA) film, BMSCs seeded on P(MTMC-LA)-E7 films showed larger spreading area increasing from 1033 to 1621 μm2 (Fig. 6a). With the increase of E7 grafting ratio, the spreading area increased slightly. The similar trend was found in the adhesion force (Fig. 6b). The adhesion force of P(MTMC-LA)-E7-50 is about twice and 5 times higher than that of P(MTMC-LA)-E7-15 and P(MTMC-LA), respectively. There are no obviously difference between P(MTMC-LA)-E7-50 and P(MTMC-LA)-E725. Adhesion area and force is related to focal adhesion [36]. With the increase of E7 grafting ratio, P(MTMC-LA)-E7 films provided more and more recognition sites for BMSCs resulting in enhanced interaction between BMSCs and P(MTMC-LA)-E7 films through focal adhesion, which accounted for the increasing spreading area and adhesion force.
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Fig. 5. Cell morphology of BMSCs cultured on (a) P(MTMC-LA), (b) P(MTMC-LA)-E7-15, (c) P(MTMC-LA)-E7-25, (d) P(MTMC-LA)-E7-50.
3.4. Cell viability There are evidence indicated that many biomolecules promote the viability and proliferation of BMSCs [35]. In this study, BMSCs were seeded onto the P(MTMC-LA) and P(MTMC-LA)-E7 films for 1 day, 3 days and 5 days for cell viability and proliferation test. The MTT assay was employed to examine the cell viability. As shown in Fig. 7, the increased cell viability was observed in all samples as culture time prolonged, which demonstrated that BMSCs proliferated on all the polymer films. In addition, the BMSCs cultured on P(MTMC-LA)-E7 films had
higher viability compared to P(MTMC-LA) film at each time interval, especially at day 5. Moreover, with the increase of E7 peptide, the viability increased. All the results indicated that P(MTMC-LA)-E7 has good affinity with BMSCs to accelerate the adhesion and proliferation of BMSCs. 4. Conclusions P(MTMC-LA) was successfully synthesized by the copolymerization between FMTMC and LA and a subsequent retro Diels-Alder reaction. The polyester was modified by a BMSCs affinity peptide-E7. The
Fig. 6. (a) Spreading area and (b) adhesion force of BMSCs cultured on P(MTMC-LA) and P(MTMC-LA)-E7 films.
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Fig. 7. Proliferation behaviors of BMSCs cultured on P(MTMC-LA) and P(MTMC-LA)-E7 films.
P(MTMC-LA)-E7 promotes the adhesion and proliferation of BMSCs. Moreover, the adhesion and proliferation is closely related to the peptide grafting ratio. The P(MTMC-LA)-E7 is a promising biomaterial for tissue engineering and regenerative medicine, especially for in situ BMSCs adhesion and capturing. Acknowledgements We acknowledge financial support by the Key Science Technology Innovation Team of Zhejiang Province (2013TD02), the Natural Science Foundation of China (51322302, 51673167, 20934003) and the National Key Research Program of China (2016YFC1101001). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.12.088. References [1] G. Chen, T. Ushida, T. Tateishi, Hybrid biomaterials for tissue engineering: a preparative method for PLA or PLGA–collagen hybrid sponges, Adv. Mater. 12 (2000) 455–457. [2] B. Li, J. Yang, L. Ma, F. Li, Z. Tu, C. Gao, Influence of the molecular weight of poly (lactide-co-glycolide) on the in vivo cartilage repair by a construct of poly (lactide-co-glycolide)/fibrin gel/mesenchymal stem cells/transforming growth factor-β1, Tissue Eng. Part A 20 (2013) 1–11. [3] Y. Zhu, Z. Mao, C. Gao, Control over the gradient differentiation of rat BMSCs on a PCL membrane with surface-immobilized alendronate gradient, Biomacromolecules 14 (2013) 342–349. [4] V. Russo, L. Tammaro, L. Di Marcantonio, A. Sorrentino, M. Ancora, L. Valbonetti, M. Turriani, A. Martelli, C. Cammà, B. Barboni, Amniotic epithelial stem cell biocompatibility for electrospun poly(lactide-co-glycolide), poly(ε-caprolactone), poly(lactic acid) scaffolds, Mater. Sci. Eng. C 69 (2016) 321–329. [5] E.J. Jansen, R.E. Sladek, H. Bahar, A. Yaffe, M.J. Gijbels, R. Kuijer, S.K. Bulstra, N.A. Guldemond, I. Binderman, L.H. Koole, Hydrophobicity as a design criterion for polymer scaffolds in bone tissue engineering, Biomaterials 26 (2005) 4423–4431. [6] X. Liu, J.M. Holzwarth, P.X. Ma, Functionalized synthetic biodegradable polymer scaffolds for tissue engineering, Macromol. Biosci. 12 (2012) 911–919. [7] Y. Ikada, H. Tsuji, Biodegradable polyesters for medical and ecological applications, Macromol. Rapid Commun. 21 (2000) 117–132. [8] G. Bayramoglu, V. Bitirim, Y. Tunali, M.Y. Arica, K.C. Akcali, Poly (hydroxyethyl methacrylate-glycidyl methacrylate) films modified with different functional groups: in vitro interactions with platelets and rat stem cells, Mater. Sci. Eng. C 33 (2013) 801–810. [9] O. Coulembier, P. Degée, J.L. Hedrick, P. Dubois, From controlled ring-opening polymerization to biodegradable aliphatic polyester: especially poly (β-malic acid) derivatives, Prog. Polym. Sci. 31 (2006) 723–747.
[10] X. Bi, Z. You, J. Gao, X. Fan, Y. Wang, A functional polyester carrying free hydroxyl groups promotes the mineralization of osteoblast and human mesenchymal stem cell extracellular matrix, Acta Biomater. 10 (2014) 2814–2823. [11] W. Cai, J. Wu, C. Xi, A.J. Ashe, M.E. Meyerhoff, Carboxyl-ebselen-based layer-by-layer films as potential antithrombotic and antimicrobial coatings, Biomaterials 32 (2011) 7774–7784. [12] A. Philipp, M. Meyer, A. Zintchenko, E. Wagner, Functional modification of amidecrosslinked oligoethylenimine for improved siRNA delivery, React. Funct. Polym. 71 (2011) 288–293. [13] B. Guo, L. Glavas, A.-C. Albertsson, Biodegradable and electrically conducting polymers for biomedical applications, Prog. Polym. Sci. 38 (2013) 1263–1286. [14] S. Hvilsted, Facile design of biomaterials by ‘click’ chemistry, Polym. Int. 61 (2012) 485–494. [15] R. Wang, W. Chen, F. Meng, R. Cheng, C. Deng, J. Feijen, Z. Zhong, Unprecedented access to functional biodegradable polymers and coatings, Macromolecules 44 (2011) 6009–6016. [16] D. Xing, L. Ma, C. Gao, Synthesis of functionalized poly (ester carbonate) with laminin-derived peptide for promoting neurite outgrowth of PC12 cells, Macromol. Biosci. 14 (2014) 1429–1436. [17] K.C. Kemp, J. Hows, C. Donaldson, Bone marrow-derived mesenchymal stem cells, Leuk. Lymphoma 46 (2005) 1531–1544. [18] S. Martino, F. D'Angelo, I. Armentano, J.M. Kenny, A. Orlacchio, Stem cell-biomaterial interactions for regenerative medicine, Biotechnol. Adv. 30 (2012) 338–351. [19] A. Salerno, V. Guarino, O. Oliviero, L. Ambrosio, C. Domingo, Bio-safe processing of polylactic-co-caprolactone and polylactic acid blends to fabricate fibrous porous scaffolds for in vitro mesenchymal stem cells adhesion and proliferation, Mater. Sci. Eng. C 63 (2016) 512–521. [20] Y. Lai, F. Pan, C. Xu, H. Fuchs, L. Chi, In situ surface-modification-induced superhydrophobic patterns with reversible wettability and adhesion, Adv. Mater. 25 (2013) 1682–1686. [21] Y.-x. Sun, K.-f. Ren, J.-l. Wang, G.-x. Chang, J. Ji, Electrochemically controlled stiffness of multilayers for manipulation of cell adhesion, ACS Appl. Mater. Interfaces 5 (2013) 4597–4602. [22] C.H. Seo, H. Jeong, K.S. Furukawa, Y. Suzuki, T. Ushida, The switching of focal adhesion maturation sites and actin filament activation for MSCs by topography of well-defined micropatterned surfaces, Biomaterials 34 (2013) 1764–1771. [23] S.H. Ku, J.S. Lee, C.B. Park, Spatial control of cell adhesion and patterning through mussel-inspired surface modification by polydopamine, Langmuir 26 (2010) 15104–15108. [24] Y. Gustafsson, J. Haag, P. Jungebluth, V. Lundin, M.L. Lim, S. Baiguera, F. Ajalloueian, C. Del Gaudio, A. Bianco, G. Moll, Viability and proliferation of rat MSCs on adhesion protein-modified PET and PU scaffolds, Biomaterials 33 (2012) 8094–8103. [25] H. Zhang, C.-Y. Lin, S.J. Hollister, The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds, Biomaterials 30 (2009) 4063–4069. [26] Z. Shao, X. Zhang, Y. Pi, X. Wang, Z. Jia, J. Zhu, L. Dai, W. Chen, L. Yin, H. Chen, Polycaprolactone electrospun mesh conjugated with an MSC affinity peptide for MSC homing in vivo, Biomaterials 33 (2012) 3375–3387. [27] H. Wang, X. Yan, L. Shen, S. Li, Y. Lin, S. Wang, X. Hou, C. Shi, Y. Yang, J. Dai, Acceleration of wound healing in acute full-thickness skin wounds using a collagen-binding peptide with an affinity for MSCs, Burns Trauma 2 (2014) 181. [28] M. Soleimani, S. Nadri, A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow, Nat. Protoc. 4 (2009) 102–106. [29] C.D. Reyes, A.J. García, A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces, J. Biomed. Mater. Res. A 67 (2003) 328–333. [30] Y.-P. Jiao, F.-Z. Cui, Surface modification of polyester biomaterials for tissue engineering, Biomed. Mater. 2 (2007) R24. [31] J. Yu, K.T. Du, Q. Fang, Y. Gu, S.S. Mihardja, R.E. Sievers, J.C. Wu, R.J. Lee, The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat, Biomaterials 31 (2010) 7012–7020. [32] Z. Man, L. Yin, Z. Shao, X. Zhang, X. Hu, J. Zhu, L. Dai, H. Huang, L. Yuan, C. Zhou, H. Chen, Y. Ao, The effects of co-delivery of BMSC-affinity peptide and rhTGF-β1 from coaxial electrospun scaffolds on chondrogenic differentiation, Biomaterials 35 (2014) 5250–5260. [33] L.L. Spangler, J.M. Torkelson, J.S. Royal, Influence of solvent and molecular weight on thickness and surface topography of spincoated polymer films, Polym. Eng. Sci. 30 (1990) 644–653. [34] Y. Arima, H. Iwata, Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers, Biomaterials 28 (2007) 3074–3082. [35] B. Parrish, R.B. Breitenkamp, T. Emrick, PEG-and peptide-grafted aliphatic polyesters by click chemistry, J. Am. Chem. Soc. 127 (2005) 7404–7410. [36] N.Q. Balaban, U.S. Schwarz, D. Riveline, P. Goichberg, G. Tzur, I. Sabanay, D. Mahalu, S. Safran, A. Bershadsky, L. Addadi, Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates, Nat. Cell Biol. 3 (2001) 466–472.