J Mater Sci: Mater Med (2014) 25:273–282 DOI 10.1007/s10856-013-5068-1
Synthesis, characterization and cytocompatibility of a degradable polymer using ferric catalyst for esophageal tissue engineering Yu-Na Lei • Ya-Bin Zhu • Chang-Feng Gong Jing-Jing Lv • Chen Kang • Lin-Xi Hou
•
Received: 5 June 2013 / Accepted: 9 October 2013 / Published online: 23 October 2013 Ó Springer Science+Business Media New York 2013
Abstract This study focused on the synthesis, characterization and cytocompatibility of a biodegradable polymer by the cross-linking from poly(ethylene glycol-colactide) dimethacrylate (PLEGDMA), polyethylene glycol diacrylate (PEGDA) and N-isopropylacrylamide, where PLEGDMA was synthesized by ring-opening oligomerization of poly(ethylene glycol) with different molecular weights (Mn = 400, 600, 1000, 2000 Da) and L-lactide using low toxic iron(III) acetylacetonate (Fe(acac)3) as the catalyst and subsequently being terminated with dimethacrylate. The product, PLEGDMA, was analyzed to confirm its chemistry using FTIR spectroscopy, 1H NMR spectra and gel permeation chromatography etc. The thermodynamic properties, mechanical behaviors, surface hydrophilicity, degradability and cytotoxicity of the crosslinked product were evaluated bydifferential scanning calorimetry, tensile tests, contact angle measurements and cell cultures. The effects of reaction variables such as PEGDA content and reactants ratio were optimized to achieve a material with low glass transition temperature (Tg), high wettability and preferable mechanical characteristics. Using a tubular mould which has been patented in our group, a tubular scaffold with predetermined dimension and pattern was fabricated, which aims at guiding the growth and phenotype regulation of esophageal primary
Y.-N. Lei L.-X. Hou (&) School of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, China e-mail: [email protected] Y.-B. Zhu (&) C.-F. Gong J.-J. Lv C. Kang School of Medicine, Ningbo University, Ningbo 315211, China e-mail: [email protected]
cells like fibroblast and smooth muscle cell towards fabricating tissue engineered esophagus in future.
1 Introduction The biomaterials based on poly(L-lactide) (PLLA) have been widely used in pharmaceutical and other medical applications such as sutures, implants for bone fixation, carriers in drug delivery and temporary matrices or scaffolds in tissue engineering due to their low immunogenicity, good biocompatibility and be able to acceptance by living organism etc. [1, 2] However, PLLA is a stiff polymer with high glass transition temperature (Tg) of 65 °C, which can’t meet the requirements of soft tissue engineering like esophagus [3–6]. Blending with or covalently linking a low glass transition temperature component is usually an effective way to improve its elongation and elasticity. Poly(ethylene glycol) (PEG) is known as one kind of materials with outstanding biological and physicochemical properties like hydrophilicity, little toxicity, absence of antigenicity and immunogenicity. It has been much used in biomedical and pharmaceutical applications [7–14]. Herein, together with hydrophilic N-isopropylacrylamide (NIPAAm), it was used as cross-linking component to improve the hydrophilicity and decrease the glass transition temperature of the ultimate PLLA-based products. The other issue in PLLA-based polymers is that the polymerization of lactide is usually carried out using stannum compounds as catalysts [15–18]. This kind of catalysts covalently links to the molecular chain of the ultimate product after polymerization. When they were applied for in vivo transplant, stannum element will accumulate in body as the material gradually degraded, resulting in poisoning the human body.
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Our group has studied the availability of polymerization for aliphatic cyclic ester monomers using low toxic iron compounds as catalysts in stead of stannum compounds. Products with monomer conversion (*90 %) and number average molecular weight (1.2 9 105) had been obtained under our reaction protocol using iron(III) acetylacetonate (Fe(acac)3) as the catalyst. It is believed to be beneficial for aliphatic polyesters to be applied safely as biomaterials in life science [19]. On the basis of these experimental results, the poly(ethylene glycol-co-lactide) dimethacrylate (PLEGDMA) was synthesized firstly via ring opening polymerization of L-LA and PEG under Fe(acac)3 initiation, and subsequently terminated with methacryloyl chloride. Oligomer PLEGDMA, PEG diacrylate and NIPAAm were input into a film or tube mould to let cross-linking occur under ultraviolet (UV) photoinitiation to obtain expected polymer sheet or tubular scaffold with predetermined patterns and dimensions. These products were characterized by Fourier transform infrared spectroscopy (FTIR) spectroscopy, Hydrogen-1 nuclear magnetic resonance (1H NMR) spectra and gel permeation chromatography (GPC) etc. The qualities of its mechanical property, hydrophilicity, cytocompatibility and degradability, etc. were also evaluated. It is believed that scaffolds fabricated from this cross-linked polymer will be a promising candidate for esophagus regeneration.
2 Materials and methods 2.1 Materials PEG (Mn = 400, 600, 1000, 2000, Aladdin reagents Co., Shanghai, China) were used after being dried at 80 °C for 4 h under vacuum. L-lactide (L-LA, Sinopharm Chemical Reagent Co., China) was purified by re-crystallization from ethyl acetate. Iron(III) acetylacetonatenate (Fe(acac)3), methacryloyl chloride, triethylamine(Et3N), PEGDA, NIPAAm, 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) supplied by Aladdin reagents (Shanghai) Co. were used as received. Dichloromethane (CH2Cl2) was dried by refluxing over CaH2 and distilled. All other chemical reagents were analytically pure and purchased from Sinopharm Chemical Reagent without further purification. Adult rabbits were offered by the Experimental Animal Center of Ningbo University under Animal Protection Act. Anti-a-Smooth Muscle Actin (a-SMA) is purchased from Wuhan Boster Biological Engineering Co. All cell culture reagents were obtained from HyClone Co. unless otherwise specified.
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extended by L-LA, and finally terminated with methacryloyl chloride (Scheme 1). Detailedly, predetermined amount of dry PEG (Mn 0.4, 0.6, 1.0 or 2.0k) and L-LA (molar ratio of lactate repeat unit to ethylene oxide (LLA/EO), 6/1) were firstly introduced into a polymerization tube. Fe(acac)3/ CH2Cl2 solution was then added. After degassing for 3 h, the tube was sealed with vacuum and placed entirely into an oil bath. The polymerizing reaction was allowed to proceed at 130 °C under stirring. 32 h later, the first product (named PLEG) was obtained via dissolution in chloroform and precipitation in ethyl alcohol. It was then washed with ethyl alcohol and dried under vacuum, yielding output of *90 %. For the sake of simplicity, the triblock PLEG were expressed as P-ak where -ak means PEG type; for example, P-2k for the element type of PEG with molecular weight 2,000. PLEGs were redissolved separately in anhydrous dichloromethane in a three-neck round-bottomed flask and refrigerated to 0 °C in an ice bath. Methacryloyl chloride and triethylamine, both dissolved in anhydrous dichloromethane in advance, were added dropwise into the flask. After reaction at 0 °C for 8 h and then at room temperature for 14 h under stirring, the produced polymer (PLEGDMA) was then filtered to remove the precipitate and rinsed sequentially with dilute HCl (1 M), saturated NaHCO3 and deionized water. Finally, PLEGDMA was dried with MgSO4 for 30 min, and filtered it away. The solvent (dichloromethane) was removed by rotary evaporation at 40 °C, producing a clear light yellow solid with yields of *85 % (Scheme 1). (For the sake of simplicity, the PLEGDMA oligomers were expressed as PD-ak. For example, PD-2k was referred to the original PEG molecular weight of 2,000.) 2.3 Cross-linking occurrence PLEGDMA oligomers, PEGDA and NIPAAm (molar ratio was listed in Table 1) were dissolved in 1,4-dioxane containing photoinitiator (Irgacure 651, 0.5 wt%) (Scheme 2). This mixture was cast onto a glass mold, covered with a plastic film to isolate the mixture from air and cured for 15 min under UV irradiation. After the solvent was evaporated at room temperature, a sheet with a thickness of *200 lm was taken off the mold. It was washed with acetone to extract residues of unreacted reagents or/and solvent. The film legends were listed in Table 1. Using this cross-linking protocol, a tube with predetermined wall patterns can be fabricated. 2.4 Characterizations
2.2 Synthesis of oligomer PLEGDMA
2.4.1 Oligomer PLEGDMAs
The PLEGDMA were prepared by a two-step addition process, in which PEG was used as a central block, both ends
Fourier transform infrared (FTIR) spectra were recorded over the wave number range 4,000–400 cm-1 using an
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Scheme 1 Synthesis diagram of oligomer PLEGDMA using Fe(acac)3 as the catalyst Table 1 The component feeding ratios of the cross-linked polymer sheets Cross-linked films
PLEGDMA
PEGDA (mmol)
PLEGDMA/PEGDA/ NIPAAm
F-0.4k F-0.6k
PD-0.4k PD-0.6k
0.39 0.39
1:85:60 1:85:60
F-1k
PD-1k
0.39
1:85:60
F-2k
PD-2k
0.39
1:85:60
F-2k-1
PD-2k
0.10
1:22:60
F-2k-2
PD-2k
0.20
1:43:60
F-2k-3
PD-2k
0.59
1:128:60
F-2k-4
PD-2k
0.78
1:170:60
The static contact angle of each cross-linked film was surveyed on Surface Tension-Contact Angle Meter (DIGIDROP, GBX, France) at ambient humidity and temperature. Drops of deionized water about 1.0 lL in volume were applied to test the contact angle of the sample surface. All data were averaged from three different locations and expressed as mean ± SD. Mechanical testing was performed on dumbbells with a 15 mm gauge length and *0.2 9 3 mm cross-section using a tensile tester (Instron 5566, USA) at the deformation rate of 10 mm per minute. Three repeats were performed for each sample.
Oligomer PLEGDMA was set as 0.0046 mmol and NIPAAm as 0.27 mmol. Cross-linking took place under UV irradiation with 0.5 wt% initiator (Irgacure 651)
2.5 Degradability measurement of the cross-linked films
FTIR instrument (Digilab FTS 3100, USA). Samples were dried to remove all moisture before FTIR analysis. For 1H NMR measurement, samples were dissolved in CDCl3 and measured on a nuclear magnetic resonance (Bruker Avance, 400 MHz, Switzerland). Molecular weight (Mn and Mw) and molecular weight distribution (Mw/Mn) were determined by gel permeation chromatography (GPC, Polymer Laboratories PL-GPC 50 plus, England) using polystyrene as the standard. Samples were analyzed at 40 °C using tetrahydrofuran (THF) as eluent at a flow rate of 1.0 mL/min.
Oligomer PLEGDMAs were cross-linked with PEGDA and NIPAAm, shaped to plane sheet with *200 lm thick and stated as F-0.4k, F-0.6k, F-1k and F-2k with corresponding PLEG component PD-0.4k, PD-0.6k, PD-1k and PD-2k, respectively. Three specimens for each kind sample (W0) were placed in a sealed tube filled with sterilized phosphate buffer saline (PBS, pH 7.4) at 37 °C. After some time, the specimens were taken out, washed with distilled water, dried under vacuum at room temperature to constant weight and weighed the mass (Wl). The degradability of samples was related to the weight loss and expressed as.
2.4.2 Cross-linked polymer
Weight loss ð%Þ ¼
Glass transition temperature (Tg) of the cross-linked films was tested with differential scanning calorimetry (DSC, Pyris Diamond DSC, USA) under nitrogen atmosphere. The first heating ranged from 25 to 100 °C at a speed of 20 °C/min with 1 min station to clear the thermal history, then cooled down to -50 °C at a speed of 10 °C/min. The second heating started from -50 to 100 °C at 20 °C/min. Tg values of samples were taken from the second heating round.
2.6 Cell culture
W0 Wl 100 % W0
Human fibroblasts were harvested from human esophageal mucousa (sacrificed by Lihuili Hospital in Ningbo, China, under agreement of an esophageal carcinoma sufferer) using tissue extending method due to fibroblast’s strong extending and attachment capability. Mucosa and submucosa tissue was rinsed well in sterile PBS containing antibiotics (1,000 U/mL penicillin and 1,000 lg/mL
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Scheme 2 Diagram of crosslinking reaction among PLEGDMA, PEG and NIPAAm under UV initiation
streptomycin sulfate), sterilized in 75 % ethanol for 30 s and then 1,000 ppm NaClO solution with PBS washing in each performance. After then, the tissue was cut to cubes with *1 9 1 9 1 mm using a fine scissor and forceps, attached to a culture dish containing a small amount of culture medium made of Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS, 10 %), penicillin (50 IU/mL) and streptomycin (50 IU/mL). The culture medium was amended to top up the tissue 1 day later. After several days, fibroblasts extended from the cubes and attached to the culture plate. These primary fibroblasts were collected and subcultured for the following application. Smooth muscle cell (SMC) explanted from rabbit esophagus tissue was cultured using the same protocol. Due to shortage of human smooth muscle, cells from rabbit esophagus tissue were used instead. Cells were used from 3rd to 5th passages to test the cytocompatibility of cross-linked F-2k film. After some days’ culture, cells were characterized by cell number counting, morphology observation and immunohistochemistry staining. Before harvesting the adhered cells by trypsinization, three gentle washes with PBS were performed. The cells were counted using a haemocytometer. For the morphology observation, cells were washed with PBS for three times and fixed with 2.5 wt% glutaraldehyde solution for 30 min, and then washed with water followed by overnight desiccation in a freeze-dryer (Freeze Zone 2.5, LABCONCO, USA). After gold metallization with Ion Sputter (Hitachi E-1010, Japan), the cells were observed under a scanning electron microscope (SEM, Hitachi S-3400N, Japan). For SMCs, cells were cultured for 7 days followed by immunohistochemistry staining using anti a-smooth muscle actin (Boster, China) as primary antibody and processed as Strept Avidin–Biotin Complex kit (SABC, Boster, China) introduced.
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3 Results and discussions 3.1 Synthesis of oligomer PLEGDMA The synthesis of PLEGDMA was confirmed by FTIR spectra (Fig. 1), which exhibited spectrum of PEG (a), PLLA (b), P-0.4k (c) and PD-0.4k (d). Curve c displayed the peak summary of curve a and b verifying the reaction of PEG and L-LA under Fe(acac)3 initiation. The peak at 3,449 cm-1 due to the hydroxyl group in the end group of PEG molecule (a) disappeared completely after the acrylation reaction (d), suggesting complete or nearly complete conversion from the diol group of P-0.4k to both dimethacrylate ends of oligomer PLEGDMA. The C=CH2 group gave two absorptions at 810 cm-1 (C=C bond twisting) and 1,637 cm-1 (C=C bond stretching), confirming that the acrylation occurred successfully. The peak at 1,758 cm-1 appeared in curve b, c and d, which was attributed to ester stretching absorption existing in all L-LA, P-0.4k and PD0.4k compounds. Four dimethacrylate oligomers, i.e. PD-0.4k, PD-0.6k, PD-1.0k and PD-2.0k, were characterized by 1H NMR measurement. Particularly, the total proton assignment of PD-0.4k in 1H-NMR spectrum was specified in Fig. 2; CH tertiary carbon attached to hydroxy and carbonyl groups at 5.14–5.19 ppm from L-LA moiety exhibited a broad peak (b). Lateral methyl CH3 groups at 1.57–1.59 ppm (a), CH2CH2 segment attached to two oxygen atoms from EO moiety at 3.63–3.64 ppm (c), CH2= ends at 5.63 and 6.2 ppm (d), CH3 groups attached to CH2=C vinyl groups at 1.96 ppm (f) were all indicated in the 1H NMR spectrum (Fig. 2). The LLA/EO molar ratios in the four dimethacrylate oligomers were also determined by 1H NMR (Table 2). These results confirmed, on one hand, the occurrence of the polymerization of PEG and L-LA under
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Fe(acac)3 initiator and further acrylation at both ends of oligomer molecules. On the other hand, as shown in Table 2, the LLA/EO molar ratios in products almost remain unchanged as the feeding’s. Molecular weights and molecular weight distribution of oligomers PLEGDMA with various PEG components, i.e. Mn 0.4, 0.6, 1 and 2k, respectively, were measured on GPC instrument (Table 2). The results presented a narrow molecular weight distribution (1.40–1.72) with a step-up increasing of Mn and Mw (46–11 and 80–150 kDa). Higher molecular weight of PEG component produced higher molecular weight and narrower molecular weight distribution
Fig. 1 FTIR spectrum of PEG (a), PLLA (b), P-0.4k (c) and PD-0.4k (d). PEG (Mn = 400) was used as reference after being dried at 80 °C for 4 h under vacuum. PLLA and P-0.4k were prepared by ringopening polymerization of L-LA and the copolymerization of L-LA and PEG, respectively, Both were catalyzed by Fe(acac)3 with [n(catalyst)/n(L-LA)] 1.0 9 10-3 for 32 h at 130 °C. PD-0.4k was prepared from P-0.4k endcapped with methacryloyl chloride
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of the product PLEGDMA. This finding can be assigned to the fact that the terminal hydroxyl groups of PEG can be readily activated by Fe(acac)3 to further function as co-initiator for the ring opening polymerization of lactide towards forming triblock copolymers (PLEG). PEG with the higher molecular weight got less space resistance for LLA homopolymeric block to join the molecular chain more easily to finally produce the triblock PLEG. 3.2 Properties of the cross-linked polymer PLEGDMA, PEGDA and NIPAAm were mixed and photoinitiated to achieve crosslinked polymer sheet and tubular scaffold in the corresponding moulds. For the plane sheet, four types of PEG with various molecular weights were employed and thus four kinds of films were produced, named F-0.4k, F-0.6k, F-1k and F-2k (Table 1). Using PD2k as the substrate component, different proportions of reactants were explored to produce the ultimate crosslinked films with *200 lm thick, which were marked as F-2k-1, F-2k-2, F-2k-3 and F-2k-4. The glass transition temperature (Tg) of these polymeric films was separately examined on DSC instrument and summarized in Table 3. All of the cross-linked films (from F-0.4k to F-2k) had the glass transition temperatures (5.60–16.24 °C) lower than the body temperature, and far lower than that of PLLA (61.9 °C) [19]. Particularly, Tg of film F-2k is low down to 5.6 °C. Besides, their wettabilities were measured in static contact angle model and listed in the last column of Table 3. Hydrophilic material’s surface is usually more biocompatible than the hydrophobic surface does when the materials contact with cells or/and tissue. The surface wettability is often determined by
Fig. 2 1H NMR spectrum of oligomer PD-0.4k
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Table 2 Data summary of 1H NMR and GPC measurements of oligomer PLEGDMA Copolymer
LLA/EO in feeding (mol)
LLA/EO in product (mol)
Mn (Da)
Mw (Da)
PD-0.4k
6:1
7.10:1
46,485
PD-0.6k
6:1
7.22:1
PD-1k
6:1
7.61:1
PD-2k
6:1
6.46:1
Mw/ Mn
80,166
1.72
55,648
91,349
1.64
85,633
121,863
1.42
109,292
153,155
1.40
measuring the contact angle of the material [20]. The data of these four films were slightly altered around 78° regardless of the different molecular weight of PEG component. All these values are slightly lower than the contact angle of pure PLLA homopolymer, i.e. 80.2 ± 2.1 [21]. That is, the hydrophilicity of this crosslinked polymer was improved after the substrate PLLA was copolymerized with hydrophilic PEG and NIPAAm. Therefore, we were convinced that these polymers would exhibit good elasticity and good hydrophilicity. Good cytocompatibility to esophageal cells of this crosslinked polymer due to the better wattability than PLLA is expected [22, 23]. The mechanical properties of these polymeric films were tested. The tensile stress–strain tendencies of these four films with four kinds of PEG lengths were displayed in Fig. 3A
and the average values of ultimate stress (o´), maximum strain (e) and Young’s modulus (E) were summarized in Table 3. F-0.4k(a), F-0.6k(b) and F-1k(c) exhibited low tensile stress and strain whilst F-2k(d) showed much higher tensile stress and ultimate strain (Fig. 3 and Table 3). Correspondingly, F-2k manifested the lowest Young’s modulus (E, 8.37 ± 0.75 MPa). Among all these four samples, F-2k has the longest PEG component. We induced that it has the longest molecular chain and the highest crosslinking degree, resulting in the highest tensile strength. Because the samples were manufactured from the random crosslinking of three different components, higher crosslinking degree led to the lower regularity or crystallization degree in molecular structure. That is why product F-2k obtained the highest strain property. As a reference, homopolymer PLLA was synthesized under Fe(acac)3 initiator. Its mechanical test results were listed in Table 3, the last row. PLLA has got the highest modulus, much higher than that of all crosslinked samples. Therefore, we concluded that crosslinking reaction among oligomer PLEGDMA, PEGDA and NIPAAm had substantially taken place. The crosslinked products demonstrated the improved tensile strength and elasticity, which is believed to be beneficiary for these polymers to be applied as scaffold substrate in esophageal tissue engineering research. The films’ degradation properties were detected during 20-week span (Fig. 3B). The results demonstrated that
Table 3 Quantitative results of DSC, mechanical properties and contact angle measurements of the cross-linked films Cross-linked films
Tg (°C)
´ (MPa) O
e (mm/mm)
E (MPa)
Contact angle (°)
F-0.4k
16.2
4.80 ± 0.15
0.79 ± 0.09
12.10 ± 1.06
79.5 ± 2.4
F-0.6k
14.6
4.73 ± 0.43
0.98 ± 0.10
9.37 ± 0.51
78.7 ± 2.0
6.2
6.74 ± 0.24
0.55 ± 0.03
33.80 ± 6.20
74.7 ± 2.8
5.6
10.50 ± 0.97
3.11 ± 0.04
8.37 ± 0.75
4.21 ± 0.26
0.29 ± 0.07
54.65 ± 4.58
F-1k F-2k PLLA
61.9 [19]
Tg and contact angle of PLLA were cited from literatures
Fig. 3 Stress–strain curves (A) and weight loss (B) of cross-linked film F-0.4k (a), F-0.6k (b), F-1k (c) and F-2k (d)
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78.6 ± 0.7 80.2 ± 2.1 [20]
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Table 4 Properties of cross-linked films (F-2k) as functions of various PEGDA amounts with the fixed components of PLEGDMA (PD2k) and NIPAAm ´ (MPa) O
e (mm/mm)
Cross-linked films
Tg (°C)
Contact angle (°)
F-2k-1
26.42
72.4 ± 1.58
F-2k-2
15.53
78.1 ± 1.45
8.53 ± 1.02
3.47 ± 0.55
5.60
78.6 ± 0.65
10.50 ± 0.97
3.11 ± 0.04
F-2k
5.88 ± 0.32
3.18 ± 0.31
F-2k-3
-5.05
85.3 ± 1.53
5.90 ± 0.57
1.50 ± 0.05
F-2k-4
-6.53
87.1 ± 0.40
5.68 ± 0.47
1.04 ± 0.08
The detailed amount and ratio of PEGDA were listed in Table 1
F-0.4k and F-0.6k showed higher weight loss than F-1k and F-2k did. For example, F-0.4k F-0.6k, F-1k and F-2k lost 17.8, 14.9, 10.8 and 11.9 % after 11 weeks and lost 24.4, 24.5, 19.2 and 17.6 % of their initial weight after 19 weeks, respectively. It seems to imply that molecular weight of the crosslinked polymers has an inverse relationship with their weight loss. It is reasonable because polymer with higher molecular weight was obtained from higher crosslinking density under the same reaction components, thus induced the higher tensile strength of the
polymer. In our case, the crosslinked product with higher molecular weight which was resulted from higher PEG component length leaded to decreasing degradation rate (decreasing weight loss) due to its high strength. Thus, F-2k sheet displayed the slowest degradation rate, 20 % mass loss in 5 months. PLLA is well-known biodegradable polyester used in biomedical applications. Its degradating behavior has been much investigated in vivo and ex vivo. Due to its hydrophobicity and crystallinity, PLLA exhibited considerably slow degradation kinetics. Ho¨glund and co-workers found that it started to hydrolyze the ester bonds and formed water soluble oligomers after 28 days at 60 °C and after 133 days at 37 °C [24]. Bergsma considered that the complete absorption of PLLA in rats should occur after 3.5 years of implantation [25]. Tune et al. implanted poly-L-lactide rods in the medullary cavity of rabbits. The PLLA weight reduced by 5 % in 25 weeks [26]. Comparably, our PLLA-based films displayed degradation with much higher rate after they were cross-linked with hydrophilic components PEG and NIPAAm. Improved hydrophilicity of these polymers accounts for the relatively fast degradation behaviors. The in vivo degradation test is being designed to perform in SD rats in our group.
Fig. 4 a A tubular mould in which dimension-predetermined PDMS templates with 200 lm long, 30 lm deep and 30 lm wall thick were embedded in both lumens but their direction was vertical. Overview (b) and micrograph (c) of tubular scaffold were observed and photographed with camera and SEM equipment, respectively. The crosslinking was conducted using the same protocol of F-2k
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Fig. 5 SEM image with different magnification (a, b) at day 4 and growth curves during 12 days culture span (c) of esophageal fibroblasts on F-2k film. The seeding density was 5 9 104 cells/mL
In order to detect the impact of PEGDA quantity on the properties of the ultimate cross-linked materials, measurements of Tg, contact angles, ultimate stress (o´) and maximum strain (e) were conducted and summarized in Table 4. It shows that the Tg of the product became much lower with the increase of the amount of component PEGDA. The introduction of PEGDA segments contributed to reduce the film’s Tg. Correspondingly, the contact angles of the films’ surfaces increased greatly, which resulted from the more ether groups introduced into the molecular chain with the more PEGDA components. Poor hydrophilicity is believed to be unfavorable for these materials to support cells’ growth. In the mechanical property cases, F-2k exhibited the largest ultimate stress (o´) and the comparative maximum strain (e) (Table 4). This result could be explained from the relative amount of PLEGDMA and the ultimate crosslink density. The long chain oligomer, PLEGDMA, mainly contributed to the good strength of the crosslinked product. However, correspondingly, the relative amount of PLEGDMA gradually reduced with the increasing of PEGDA amount, which is disadvantageous for the product’s strength. On the other hand, PEGDA is one of the crosslinking components with high reactivity. The final crosslinking density must obviously increase with the increase of PEGDA amount, which
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conversely reduced the product’s elasticity. Therefore, we got a conclusion that F-2k with mediate component ratio was a good candidate to be a scaffold matrix. Its interaction to fibroblast and smooth muscle cell was evaluated as following experiments. With this crosslinking protocol, a tubular scaffold with predetermined dimension and patterns were fabricated (Fig. 4) using a tubular mould designed in our group (China patent ZL 2009 2 0121779.7). Solution mixture of PD-2k, PEGDA and NIPAAm with 0.5 wt% initiator was input inside the mould from the solution entrance, cured for 30 min under UV irradiation, a flexible tubular scaffold with circular micro-channel on lumen and vertical micro-channel on outer face was produced. The channel characters were predetermined from silica wafer. Soft polydimethylsiloxane (PDMS) was used to be a daughter mould and embedded in the lumens of the designed mould (Fig. 4a). The tube sizes and the channel patterns are changeable according to the application requirements. In this experiment, for example, the channel is 200 lm wide and 30 lm deep with an interval wall of 30 lm width and height. The structure parameters of the tube were verified under SEM observation, except for a little contraction of the scaffold during freeze-drying process before SEM test (Fig. 4c).
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Fig. 6 SEM image (a) and immunohistochemical staining (b) of esophageal SMC after it was seeded on F-2k film for 7 days. c, d SMC morphology observed under SEM after it was seeded on micro-channels of tubular scaffold lumen. Anti-a-smooth muscle
actin was used as the primary antibody and processed as SABC kit introduced. Cytoplasm displayed brown from a-SMA staining and nucleus displayed dark blue from DAPI staining. The seeding density was 5 9 104 cells/mL
The muscle tissue of esophagus consists of internalcircular and outer-longitudinal muscle cell bilayers and serves as peristalsis motion for food and water transport from mouth to stomach. Some literatures demonstrated that micro-channels and micro-walls could direct smooth muscle cells’orientation and switch their shape from extensive synthetic phenotype to quiescent and contractile phenotype [27, 28], which is favorable for artificial muscle tissue to keep its mechanical properties and to remodel peristalsis function. Cell alignment and phenotype regulation of porcine esophageal SMC and skeletal muscle cell cultured on 3D micro-patterned polyurethane membrane were discovered in our previous cell culture experiment (not published). Herein, the tubular scaffold with circular and longitudinal bi-structure obtained under the present protocol will be supplied to our group towards systemically study on SMC morphology and phenotype and further peristalsis remodeling of esophageal smooth muscle and skeletal muscle tissue in future.
film to evaluate the film’s cytocompatibility. Figure 5 shows the long spindle phenotype and the growth curves of fibroblasts. Cells spread and attached well on F-2k film after it was cultured for 4 days. The fibroblast’s growth curves during 12-day span displayed a step-up increase of cell number (Fig. 5c). Though the absolute number of fibroblast on the film is lower than that on TCPS at the earlier culture stage, it is tending to the same at day 12 when the cells reached confluence on both culture surfaces. SMCs were seeded both on F-2k film and on the microchannels of tubular scaffold lumen. On F-2k film, SMC showed a confluent monolayer after they were cultured for 7 days (Fig. 6a). Using anti-a-smooth muscle actin (aSMA) as the primary antibody, cells were immunohistochemically stained and exhibited a-SMA positive phenotype to confirm their smooth muscle origin (Fig. 6b). After SMCs were cultured on the lumen micro-channels of the tubular scaffold for 7 days, cells displayed spread and adhered very well onto the tube matrix (Fig. 6c, d, arrows). Moreover, SMCs exhibited aligning along the channel direction. This result convinced us that this micro-patterned scaffold was a good protocol to remodel SMC’s phenotype. We will conduct systemically study on the regeneration of esophageal muscle tissue with biological constitution and function in the next work.
3.3 Cytocompatibility to primary fibroblasts and SMCs Because both fibroblast and SMC are the main cell components of esophagus tissue, primary fibroblasts and SMCs from esophagus tissue were separately seeded on crosslinked F-2k
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4 Conclusions Using iron compound Fe(acac)3 as the catalyst, a series of oligomer poly(ethylene glycol-co-lactide) dimethacrylate (PLEGDMA) with different molecular weights was synthesized and analyzed via FTIR spectroscopy, 1H NMR spectra and GPC etc. Upon UV irradiation, these oligomers reacted with PEGDA and NIPAAm to form highly cross-linked networks and thus the crosslinked film and tubular scaffold were respectively obtained after optimization of reactant contents and amount ratios. The properties like thermodynamics, mechanical behavior, surface hydrophilicity and degradability etc. of four films, i.e. F-0.4k F-0.6k, F-1k and F-2k, were detailedly detected and discussed. F-2k was determined to be the optimal substrate with a lower Tg, higher mechanical strength, better hydrophilicity and degradability than those of all other samples. Its in vitro cytocompatibility to esophageal fibroblasts and SMCs was quantitatively tested and immunohistochemically evaluated. On this basis, a tubular scaffold with predetermined dimension and pattern was successfully fabricated using our patented tubular mould and the crosslinking protocol, which is believed to be favorable to guide the growth and phenotype regulation of esophageal cells like fibroblast and smooth muscle cell upon our cell culture results. The constitution of biofunctional esophageal prosthesis and in vivo regeneration are under way in our laboratory. Acknowledgments Financial supports by the National Science Foundation (81171476), Natural Science Funds and NSF for Distinguished Youth team of Zhejiang Province (LY12B01005, R2101166) and Scientific Innovation Team Project of Ningbo (No. 2011B82014), China, are gratefully acknowledged. This work was also sponsored by K.C. Wang Magna Fund of Ningbo University.
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Synthesis, characterization and cytocompatibility of a degradable polymer using ferric catalyst for esophageal tissue engineering Yu-Na Lei • Ya-Bin Zhu • Chang-Feng Gong Jing-Jing Lv • Chen Kang • Lin-Xi Hou
•
Received: 5 June 2013 / Accepted: 9 October 2013 / Published online: 23 October 2013 Ó Springer Science+Business Media New York 2013
Abstract This study focused on the synthesis, characterization and cytocompatibility of a biodegradable polymer by the cross-linking from poly(ethylene glycol-colactide) dimethacrylate (PLEGDMA), polyethylene glycol diacrylate (PEGDA) and N-isopropylacrylamide, where PLEGDMA was synthesized by ring-opening oligomerization of poly(ethylene glycol) with different molecular weights (Mn = 400, 600, 1000, 2000 Da) and L-lactide using low toxic iron(III) acetylacetonate (Fe(acac)3) as the catalyst and subsequently being terminated with dimethacrylate. The product, PLEGDMA, was analyzed to confirm its chemistry using FTIR spectroscopy, 1H NMR spectra and gel permeation chromatography etc. The thermodynamic properties, mechanical behaviors, surface hydrophilicity, degradability and cytotoxicity of the crosslinked product were evaluated bydifferential scanning calorimetry, tensile tests, contact angle measurements and cell cultures. The effects of reaction variables such as PEGDA content and reactants ratio were optimized to achieve a material with low glass transition temperature (Tg), high wettability and preferable mechanical characteristics. Using a tubular mould which has been patented in our group, a tubular scaffold with predetermined dimension and pattern was fabricated, which aims at guiding the growth and phenotype regulation of esophageal primary
Y.-N. Lei L.-X. Hou (&) School of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, China e-mail: [email protected] Y.-B. Zhu (&) C.-F. Gong J.-J. Lv C. Kang School of Medicine, Ningbo University, Ningbo 315211, China e-mail: [email protected]
cells like fibroblast and smooth muscle cell towards fabricating tissue engineered esophagus in future.
1 Introduction The biomaterials based on poly(L-lactide) (PLLA) have been widely used in pharmaceutical and other medical applications such as sutures, implants for bone fixation, carriers in drug delivery and temporary matrices or scaffolds in tissue engineering due to their low immunogenicity, good biocompatibility and be able to acceptance by living organism etc. [1, 2] However, PLLA is a stiff polymer with high glass transition temperature (Tg) of 65 °C, which can’t meet the requirements of soft tissue engineering like esophagus [3–6]. Blending with or covalently linking a low glass transition temperature component is usually an effective way to improve its elongation and elasticity. Poly(ethylene glycol) (PEG) is known as one kind of materials with outstanding biological and physicochemical properties like hydrophilicity, little toxicity, absence of antigenicity and immunogenicity. It has been much used in biomedical and pharmaceutical applications [7–14]. Herein, together with hydrophilic N-isopropylacrylamide (NIPAAm), it was used as cross-linking component to improve the hydrophilicity and decrease the glass transition temperature of the ultimate PLLA-based products. The other issue in PLLA-based polymers is that the polymerization of lactide is usually carried out using stannum compounds as catalysts [15–18]. This kind of catalysts covalently links to the molecular chain of the ultimate product after polymerization. When they were applied for in vivo transplant, stannum element will accumulate in body as the material gradually degraded, resulting in poisoning the human body.
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Our group has studied the availability of polymerization for aliphatic cyclic ester monomers using low toxic iron compounds as catalysts in stead of stannum compounds. Products with monomer conversion (*90 %) and number average molecular weight (1.2 9 105) had been obtained under our reaction protocol using iron(III) acetylacetonate (Fe(acac)3) as the catalyst. It is believed to be beneficial for aliphatic polyesters to be applied safely as biomaterials in life science [19]. On the basis of these experimental results, the poly(ethylene glycol-co-lactide) dimethacrylate (PLEGDMA) was synthesized firstly via ring opening polymerization of L-LA and PEG under Fe(acac)3 initiation, and subsequently terminated with methacryloyl chloride. Oligomer PLEGDMA, PEG diacrylate and NIPAAm were input into a film or tube mould to let cross-linking occur under ultraviolet (UV) photoinitiation to obtain expected polymer sheet or tubular scaffold with predetermined patterns and dimensions. These products were characterized by Fourier transform infrared spectroscopy (FTIR) spectroscopy, Hydrogen-1 nuclear magnetic resonance (1H NMR) spectra and gel permeation chromatography (GPC) etc. The qualities of its mechanical property, hydrophilicity, cytocompatibility and degradability, etc. were also evaluated. It is believed that scaffolds fabricated from this cross-linked polymer will be a promising candidate for esophagus regeneration.
2 Materials and methods 2.1 Materials PEG (Mn = 400, 600, 1000, 2000, Aladdin reagents Co., Shanghai, China) were used after being dried at 80 °C for 4 h under vacuum. L-lactide (L-LA, Sinopharm Chemical Reagent Co., China) was purified by re-crystallization from ethyl acetate. Iron(III) acetylacetonatenate (Fe(acac)3), methacryloyl chloride, triethylamine(Et3N), PEGDA, NIPAAm, 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) supplied by Aladdin reagents (Shanghai) Co. were used as received. Dichloromethane (CH2Cl2) was dried by refluxing over CaH2 and distilled. All other chemical reagents were analytically pure and purchased from Sinopharm Chemical Reagent without further purification. Adult rabbits were offered by the Experimental Animal Center of Ningbo University under Animal Protection Act. Anti-a-Smooth Muscle Actin (a-SMA) is purchased from Wuhan Boster Biological Engineering Co. All cell culture reagents were obtained from HyClone Co. unless otherwise specified.
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extended by L-LA, and finally terminated with methacryloyl chloride (Scheme 1). Detailedly, predetermined amount of dry PEG (Mn 0.4, 0.6, 1.0 or 2.0k) and L-LA (molar ratio of lactate repeat unit to ethylene oxide (LLA/EO), 6/1) were firstly introduced into a polymerization tube. Fe(acac)3/ CH2Cl2 solution was then added. After degassing for 3 h, the tube was sealed with vacuum and placed entirely into an oil bath. The polymerizing reaction was allowed to proceed at 130 °C under stirring. 32 h later, the first product (named PLEG) was obtained via dissolution in chloroform and precipitation in ethyl alcohol. It was then washed with ethyl alcohol and dried under vacuum, yielding output of *90 %. For the sake of simplicity, the triblock PLEG were expressed as P-ak where -ak means PEG type; for example, P-2k for the element type of PEG with molecular weight 2,000. PLEGs were redissolved separately in anhydrous dichloromethane in a three-neck round-bottomed flask and refrigerated to 0 °C in an ice bath. Methacryloyl chloride and triethylamine, both dissolved in anhydrous dichloromethane in advance, were added dropwise into the flask. After reaction at 0 °C for 8 h and then at room temperature for 14 h under stirring, the produced polymer (PLEGDMA) was then filtered to remove the precipitate and rinsed sequentially with dilute HCl (1 M), saturated NaHCO3 and deionized water. Finally, PLEGDMA was dried with MgSO4 for 30 min, and filtered it away. The solvent (dichloromethane) was removed by rotary evaporation at 40 °C, producing a clear light yellow solid with yields of *85 % (Scheme 1). (For the sake of simplicity, the PLEGDMA oligomers were expressed as PD-ak. For example, PD-2k was referred to the original PEG molecular weight of 2,000.) 2.3 Cross-linking occurrence PLEGDMA oligomers, PEGDA and NIPAAm (molar ratio was listed in Table 1) were dissolved in 1,4-dioxane containing photoinitiator (Irgacure 651, 0.5 wt%) (Scheme 2). This mixture was cast onto a glass mold, covered with a plastic film to isolate the mixture from air and cured for 15 min under UV irradiation. After the solvent was evaporated at room temperature, a sheet with a thickness of *200 lm was taken off the mold. It was washed with acetone to extract residues of unreacted reagents or/and solvent. The film legends were listed in Table 1. Using this cross-linking protocol, a tube with predetermined wall patterns can be fabricated. 2.4 Characterizations
2.2 Synthesis of oligomer PLEGDMA
2.4.1 Oligomer PLEGDMAs
The PLEGDMA were prepared by a two-step addition process, in which PEG was used as a central block, both ends
Fourier transform infrared (FTIR) spectra were recorded over the wave number range 4,000–400 cm-1 using an
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Scheme 1 Synthesis diagram of oligomer PLEGDMA using Fe(acac)3 as the catalyst Table 1 The component feeding ratios of the cross-linked polymer sheets Cross-linked films
PLEGDMA
PEGDA (mmol)
PLEGDMA/PEGDA/ NIPAAm
F-0.4k F-0.6k
PD-0.4k PD-0.6k
0.39 0.39
1:85:60 1:85:60
F-1k
PD-1k
0.39
1:85:60
F-2k
PD-2k
0.39
1:85:60
F-2k-1
PD-2k
0.10
1:22:60
F-2k-2
PD-2k
0.20
1:43:60
F-2k-3
PD-2k
0.59
1:128:60
F-2k-4
PD-2k
0.78
1:170:60
The static contact angle of each cross-linked film was surveyed on Surface Tension-Contact Angle Meter (DIGIDROP, GBX, France) at ambient humidity and temperature. Drops of deionized water about 1.0 lL in volume were applied to test the contact angle of the sample surface. All data were averaged from three different locations and expressed as mean ± SD. Mechanical testing was performed on dumbbells with a 15 mm gauge length and *0.2 9 3 mm cross-section using a tensile tester (Instron 5566, USA) at the deformation rate of 10 mm per minute. Three repeats were performed for each sample.
Oligomer PLEGDMA was set as 0.0046 mmol and NIPAAm as 0.27 mmol. Cross-linking took place under UV irradiation with 0.5 wt% initiator (Irgacure 651)
2.5 Degradability measurement of the cross-linked films
FTIR instrument (Digilab FTS 3100, USA). Samples were dried to remove all moisture before FTIR analysis. For 1H NMR measurement, samples were dissolved in CDCl3 and measured on a nuclear magnetic resonance (Bruker Avance, 400 MHz, Switzerland). Molecular weight (Mn and Mw) and molecular weight distribution (Mw/Mn) were determined by gel permeation chromatography (GPC, Polymer Laboratories PL-GPC 50 plus, England) using polystyrene as the standard. Samples were analyzed at 40 °C using tetrahydrofuran (THF) as eluent at a flow rate of 1.0 mL/min.
Oligomer PLEGDMAs were cross-linked with PEGDA and NIPAAm, shaped to plane sheet with *200 lm thick and stated as F-0.4k, F-0.6k, F-1k and F-2k with corresponding PLEG component PD-0.4k, PD-0.6k, PD-1k and PD-2k, respectively. Three specimens for each kind sample (W0) were placed in a sealed tube filled with sterilized phosphate buffer saline (PBS, pH 7.4) at 37 °C. After some time, the specimens were taken out, washed with distilled water, dried under vacuum at room temperature to constant weight and weighed the mass (Wl). The degradability of samples was related to the weight loss and expressed as.
2.4.2 Cross-linked polymer
Weight loss ð%Þ ¼
Glass transition temperature (Tg) of the cross-linked films was tested with differential scanning calorimetry (DSC, Pyris Diamond DSC, USA) under nitrogen atmosphere. The first heating ranged from 25 to 100 °C at a speed of 20 °C/min with 1 min station to clear the thermal history, then cooled down to -50 °C at a speed of 10 °C/min. The second heating started from -50 to 100 °C at 20 °C/min. Tg values of samples were taken from the second heating round.
2.6 Cell culture
W0 Wl 100 % W0
Human fibroblasts were harvested from human esophageal mucousa (sacrificed by Lihuili Hospital in Ningbo, China, under agreement of an esophageal carcinoma sufferer) using tissue extending method due to fibroblast’s strong extending and attachment capability. Mucosa and submucosa tissue was rinsed well in sterile PBS containing antibiotics (1,000 U/mL penicillin and 1,000 lg/mL
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Scheme 2 Diagram of crosslinking reaction among PLEGDMA, PEG and NIPAAm under UV initiation
streptomycin sulfate), sterilized in 75 % ethanol for 30 s and then 1,000 ppm NaClO solution with PBS washing in each performance. After then, the tissue was cut to cubes with *1 9 1 9 1 mm using a fine scissor and forceps, attached to a culture dish containing a small amount of culture medium made of Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS, 10 %), penicillin (50 IU/mL) and streptomycin (50 IU/mL). The culture medium was amended to top up the tissue 1 day later. After several days, fibroblasts extended from the cubes and attached to the culture plate. These primary fibroblasts were collected and subcultured for the following application. Smooth muscle cell (SMC) explanted from rabbit esophagus tissue was cultured using the same protocol. Due to shortage of human smooth muscle, cells from rabbit esophagus tissue were used instead. Cells were used from 3rd to 5th passages to test the cytocompatibility of cross-linked F-2k film. After some days’ culture, cells were characterized by cell number counting, morphology observation and immunohistochemistry staining. Before harvesting the adhered cells by trypsinization, three gentle washes with PBS were performed. The cells were counted using a haemocytometer. For the morphology observation, cells were washed with PBS for three times and fixed with 2.5 wt% glutaraldehyde solution for 30 min, and then washed with water followed by overnight desiccation in a freeze-dryer (Freeze Zone 2.5, LABCONCO, USA). After gold metallization with Ion Sputter (Hitachi E-1010, Japan), the cells were observed under a scanning electron microscope (SEM, Hitachi S-3400N, Japan). For SMCs, cells were cultured for 7 days followed by immunohistochemistry staining using anti a-smooth muscle actin (Boster, China) as primary antibody and processed as Strept Avidin–Biotin Complex kit (SABC, Boster, China) introduced.
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3 Results and discussions 3.1 Synthesis of oligomer PLEGDMA The synthesis of PLEGDMA was confirmed by FTIR spectra (Fig. 1), which exhibited spectrum of PEG (a), PLLA (b), P-0.4k (c) and PD-0.4k (d). Curve c displayed the peak summary of curve a and b verifying the reaction of PEG and L-LA under Fe(acac)3 initiation. The peak at 3,449 cm-1 due to the hydroxyl group in the end group of PEG molecule (a) disappeared completely after the acrylation reaction (d), suggesting complete or nearly complete conversion from the diol group of P-0.4k to both dimethacrylate ends of oligomer PLEGDMA. The C=CH2 group gave two absorptions at 810 cm-1 (C=C bond twisting) and 1,637 cm-1 (C=C bond stretching), confirming that the acrylation occurred successfully. The peak at 1,758 cm-1 appeared in curve b, c and d, which was attributed to ester stretching absorption existing in all L-LA, P-0.4k and PD0.4k compounds. Four dimethacrylate oligomers, i.e. PD-0.4k, PD-0.6k, PD-1.0k and PD-2.0k, were characterized by 1H NMR measurement. Particularly, the total proton assignment of PD-0.4k in 1H-NMR spectrum was specified in Fig. 2; CH tertiary carbon attached to hydroxy and carbonyl groups at 5.14–5.19 ppm from L-LA moiety exhibited a broad peak (b). Lateral methyl CH3 groups at 1.57–1.59 ppm (a), CH2CH2 segment attached to two oxygen atoms from EO moiety at 3.63–3.64 ppm (c), CH2= ends at 5.63 and 6.2 ppm (d), CH3 groups attached to CH2=C vinyl groups at 1.96 ppm (f) were all indicated in the 1H NMR spectrum (Fig. 2). The LLA/EO molar ratios in the four dimethacrylate oligomers were also determined by 1H NMR (Table 2). These results confirmed, on one hand, the occurrence of the polymerization of PEG and L-LA under
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Fe(acac)3 initiator and further acrylation at both ends of oligomer molecules. On the other hand, as shown in Table 2, the LLA/EO molar ratios in products almost remain unchanged as the feeding’s. Molecular weights and molecular weight distribution of oligomers PLEGDMA with various PEG components, i.e. Mn 0.4, 0.6, 1 and 2k, respectively, were measured on GPC instrument (Table 2). The results presented a narrow molecular weight distribution (1.40–1.72) with a step-up increasing of Mn and Mw (46–11 and 80–150 kDa). Higher molecular weight of PEG component produced higher molecular weight and narrower molecular weight distribution
Fig. 1 FTIR spectrum of PEG (a), PLLA (b), P-0.4k (c) and PD-0.4k (d). PEG (Mn = 400) was used as reference after being dried at 80 °C for 4 h under vacuum. PLLA and P-0.4k were prepared by ringopening polymerization of L-LA and the copolymerization of L-LA and PEG, respectively, Both were catalyzed by Fe(acac)3 with [n(catalyst)/n(L-LA)] 1.0 9 10-3 for 32 h at 130 °C. PD-0.4k was prepared from P-0.4k endcapped with methacryloyl chloride
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of the product PLEGDMA. This finding can be assigned to the fact that the terminal hydroxyl groups of PEG can be readily activated by Fe(acac)3 to further function as co-initiator for the ring opening polymerization of lactide towards forming triblock copolymers (PLEG). PEG with the higher molecular weight got less space resistance for LLA homopolymeric block to join the molecular chain more easily to finally produce the triblock PLEG. 3.2 Properties of the cross-linked polymer PLEGDMA, PEGDA and NIPAAm were mixed and photoinitiated to achieve crosslinked polymer sheet and tubular scaffold in the corresponding moulds. For the plane sheet, four types of PEG with various molecular weights were employed and thus four kinds of films were produced, named F-0.4k, F-0.6k, F-1k and F-2k (Table 1). Using PD2k as the substrate component, different proportions of reactants were explored to produce the ultimate crosslinked films with *200 lm thick, which were marked as F-2k-1, F-2k-2, F-2k-3 and F-2k-4. The glass transition temperature (Tg) of these polymeric films was separately examined on DSC instrument and summarized in Table 3. All of the cross-linked films (from F-0.4k to F-2k) had the glass transition temperatures (5.60–16.24 °C) lower than the body temperature, and far lower than that of PLLA (61.9 °C) [19]. Particularly, Tg of film F-2k is low down to 5.6 °C. Besides, their wettabilities were measured in static contact angle model and listed in the last column of Table 3. Hydrophilic material’s surface is usually more biocompatible than the hydrophobic surface does when the materials contact with cells or/and tissue. The surface wettability is often determined by
Fig. 2 1H NMR spectrum of oligomer PD-0.4k
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Table 2 Data summary of 1H NMR and GPC measurements of oligomer PLEGDMA Copolymer
LLA/EO in feeding (mol)
LLA/EO in product (mol)
Mn (Da)
Mw (Da)
PD-0.4k
6:1
7.10:1
46,485
PD-0.6k
6:1
7.22:1
PD-1k
6:1
7.61:1
PD-2k
6:1
6.46:1
Mw/ Mn
80,166
1.72
55,648
91,349
1.64
85,633
121,863
1.42
109,292
153,155
1.40
measuring the contact angle of the material [20]. The data of these four films were slightly altered around 78° regardless of the different molecular weight of PEG component. All these values are slightly lower than the contact angle of pure PLLA homopolymer, i.e. 80.2 ± 2.1 [21]. That is, the hydrophilicity of this crosslinked polymer was improved after the substrate PLLA was copolymerized with hydrophilic PEG and NIPAAm. Therefore, we were convinced that these polymers would exhibit good elasticity and good hydrophilicity. Good cytocompatibility to esophageal cells of this crosslinked polymer due to the better wattability than PLLA is expected [22, 23]. The mechanical properties of these polymeric films were tested. The tensile stress–strain tendencies of these four films with four kinds of PEG lengths were displayed in Fig. 3A
and the average values of ultimate stress (o´), maximum strain (e) and Young’s modulus (E) were summarized in Table 3. F-0.4k(a), F-0.6k(b) and F-1k(c) exhibited low tensile stress and strain whilst F-2k(d) showed much higher tensile stress and ultimate strain (Fig. 3 and Table 3). Correspondingly, F-2k manifested the lowest Young’s modulus (E, 8.37 ± 0.75 MPa). Among all these four samples, F-2k has the longest PEG component. We induced that it has the longest molecular chain and the highest crosslinking degree, resulting in the highest tensile strength. Because the samples were manufactured from the random crosslinking of three different components, higher crosslinking degree led to the lower regularity or crystallization degree in molecular structure. That is why product F-2k obtained the highest strain property. As a reference, homopolymer PLLA was synthesized under Fe(acac)3 initiator. Its mechanical test results were listed in Table 3, the last row. PLLA has got the highest modulus, much higher than that of all crosslinked samples. Therefore, we concluded that crosslinking reaction among oligomer PLEGDMA, PEGDA and NIPAAm had substantially taken place. The crosslinked products demonstrated the improved tensile strength and elasticity, which is believed to be beneficiary for these polymers to be applied as scaffold substrate in esophageal tissue engineering research. The films’ degradation properties were detected during 20-week span (Fig. 3B). The results demonstrated that
Table 3 Quantitative results of DSC, mechanical properties and contact angle measurements of the cross-linked films Cross-linked films
Tg (°C)
´ (MPa) O
e (mm/mm)
E (MPa)
Contact angle (°)
F-0.4k
16.2
4.80 ± 0.15
0.79 ± 0.09
12.10 ± 1.06
79.5 ± 2.4
F-0.6k
14.6
4.73 ± 0.43
0.98 ± 0.10
9.37 ± 0.51
78.7 ± 2.0
6.2
6.74 ± 0.24
0.55 ± 0.03
33.80 ± 6.20
74.7 ± 2.8
5.6
10.50 ± 0.97
3.11 ± 0.04
8.37 ± 0.75
4.21 ± 0.26
0.29 ± 0.07
54.65 ± 4.58
F-1k F-2k PLLA
61.9 [19]
Tg and contact angle of PLLA were cited from literatures
Fig. 3 Stress–strain curves (A) and weight loss (B) of cross-linked film F-0.4k (a), F-0.6k (b), F-1k (c) and F-2k (d)
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78.6 ± 0.7 80.2 ± 2.1 [20]
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Table 4 Properties of cross-linked films (F-2k) as functions of various PEGDA amounts with the fixed components of PLEGDMA (PD2k) and NIPAAm ´ (MPa) O
e (mm/mm)
Cross-linked films
Tg (°C)
Contact angle (°)
F-2k-1
26.42
72.4 ± 1.58
F-2k-2
15.53
78.1 ± 1.45
8.53 ± 1.02
3.47 ± 0.55
5.60
78.6 ± 0.65
10.50 ± 0.97
3.11 ± 0.04
F-2k
5.88 ± 0.32
3.18 ± 0.31
F-2k-3
-5.05
85.3 ± 1.53
5.90 ± 0.57
1.50 ± 0.05
F-2k-4
-6.53
87.1 ± 0.40
5.68 ± 0.47
1.04 ± 0.08
The detailed amount and ratio of PEGDA were listed in Table 1
F-0.4k and F-0.6k showed higher weight loss than F-1k and F-2k did. For example, F-0.4k F-0.6k, F-1k and F-2k lost 17.8, 14.9, 10.8 and 11.9 % after 11 weeks and lost 24.4, 24.5, 19.2 and 17.6 % of their initial weight after 19 weeks, respectively. It seems to imply that molecular weight of the crosslinked polymers has an inverse relationship with their weight loss. It is reasonable because polymer with higher molecular weight was obtained from higher crosslinking density under the same reaction components, thus induced the higher tensile strength of the
polymer. In our case, the crosslinked product with higher molecular weight which was resulted from higher PEG component length leaded to decreasing degradation rate (decreasing weight loss) due to its high strength. Thus, F-2k sheet displayed the slowest degradation rate, 20 % mass loss in 5 months. PLLA is well-known biodegradable polyester used in biomedical applications. Its degradating behavior has been much investigated in vivo and ex vivo. Due to its hydrophobicity and crystallinity, PLLA exhibited considerably slow degradation kinetics. Ho¨glund and co-workers found that it started to hydrolyze the ester bonds and formed water soluble oligomers after 28 days at 60 °C and after 133 days at 37 °C [24]. Bergsma considered that the complete absorption of PLLA in rats should occur after 3.5 years of implantation [25]. Tune et al. implanted poly-L-lactide rods in the medullary cavity of rabbits. The PLLA weight reduced by 5 % in 25 weeks [26]. Comparably, our PLLA-based films displayed degradation with much higher rate after they were cross-linked with hydrophilic components PEG and NIPAAm. Improved hydrophilicity of these polymers accounts for the relatively fast degradation behaviors. The in vivo degradation test is being designed to perform in SD rats in our group.
Fig. 4 a A tubular mould in which dimension-predetermined PDMS templates with 200 lm long, 30 lm deep and 30 lm wall thick were embedded in both lumens but their direction was vertical. Overview (b) and micrograph (c) of tubular scaffold were observed and photographed with camera and SEM equipment, respectively. The crosslinking was conducted using the same protocol of F-2k
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Fig. 5 SEM image with different magnification (a, b) at day 4 and growth curves during 12 days culture span (c) of esophageal fibroblasts on F-2k film. The seeding density was 5 9 104 cells/mL
In order to detect the impact of PEGDA quantity on the properties of the ultimate cross-linked materials, measurements of Tg, contact angles, ultimate stress (o´) and maximum strain (e) were conducted and summarized in Table 4. It shows that the Tg of the product became much lower with the increase of the amount of component PEGDA. The introduction of PEGDA segments contributed to reduce the film’s Tg. Correspondingly, the contact angles of the films’ surfaces increased greatly, which resulted from the more ether groups introduced into the molecular chain with the more PEGDA components. Poor hydrophilicity is believed to be unfavorable for these materials to support cells’ growth. In the mechanical property cases, F-2k exhibited the largest ultimate stress (o´) and the comparative maximum strain (e) (Table 4). This result could be explained from the relative amount of PLEGDMA and the ultimate crosslink density. The long chain oligomer, PLEGDMA, mainly contributed to the good strength of the crosslinked product. However, correspondingly, the relative amount of PLEGDMA gradually reduced with the increasing of PEGDA amount, which is disadvantageous for the product’s strength. On the other hand, PEGDA is one of the crosslinking components with high reactivity. The final crosslinking density must obviously increase with the increase of PEGDA amount, which
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conversely reduced the product’s elasticity. Therefore, we got a conclusion that F-2k with mediate component ratio was a good candidate to be a scaffold matrix. Its interaction to fibroblast and smooth muscle cell was evaluated as following experiments. With this crosslinking protocol, a tubular scaffold with predetermined dimension and patterns were fabricated (Fig. 4) using a tubular mould designed in our group (China patent ZL 2009 2 0121779.7). Solution mixture of PD-2k, PEGDA and NIPAAm with 0.5 wt% initiator was input inside the mould from the solution entrance, cured for 30 min under UV irradiation, a flexible tubular scaffold with circular micro-channel on lumen and vertical micro-channel on outer face was produced. The channel characters were predetermined from silica wafer. Soft polydimethylsiloxane (PDMS) was used to be a daughter mould and embedded in the lumens of the designed mould (Fig. 4a). The tube sizes and the channel patterns are changeable according to the application requirements. In this experiment, for example, the channel is 200 lm wide and 30 lm deep with an interval wall of 30 lm width and height. The structure parameters of the tube were verified under SEM observation, except for a little contraction of the scaffold during freeze-drying process before SEM test (Fig. 4c).
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Fig. 6 SEM image (a) and immunohistochemical staining (b) of esophageal SMC after it was seeded on F-2k film for 7 days. c, d SMC morphology observed under SEM after it was seeded on micro-channels of tubular scaffold lumen. Anti-a-smooth muscle
actin was used as the primary antibody and processed as SABC kit introduced. Cytoplasm displayed brown from a-SMA staining and nucleus displayed dark blue from DAPI staining. The seeding density was 5 9 104 cells/mL
The muscle tissue of esophagus consists of internalcircular and outer-longitudinal muscle cell bilayers and serves as peristalsis motion for food and water transport from mouth to stomach. Some literatures demonstrated that micro-channels and micro-walls could direct smooth muscle cells’orientation and switch their shape from extensive synthetic phenotype to quiescent and contractile phenotype [27, 28], which is favorable for artificial muscle tissue to keep its mechanical properties and to remodel peristalsis function. Cell alignment and phenotype regulation of porcine esophageal SMC and skeletal muscle cell cultured on 3D micro-patterned polyurethane membrane were discovered in our previous cell culture experiment (not published). Herein, the tubular scaffold with circular and longitudinal bi-structure obtained under the present protocol will be supplied to our group towards systemically study on SMC morphology and phenotype and further peristalsis remodeling of esophageal smooth muscle and skeletal muscle tissue in future.
film to evaluate the film’s cytocompatibility. Figure 5 shows the long spindle phenotype and the growth curves of fibroblasts. Cells spread and attached well on F-2k film after it was cultured for 4 days. The fibroblast’s growth curves during 12-day span displayed a step-up increase of cell number (Fig. 5c). Though the absolute number of fibroblast on the film is lower than that on TCPS at the earlier culture stage, it is tending to the same at day 12 when the cells reached confluence on both culture surfaces. SMCs were seeded both on F-2k film and on the microchannels of tubular scaffold lumen. On F-2k film, SMC showed a confluent monolayer after they were cultured for 7 days (Fig. 6a). Using anti-a-smooth muscle actin (aSMA) as the primary antibody, cells were immunohistochemically stained and exhibited a-SMA positive phenotype to confirm their smooth muscle origin (Fig. 6b). After SMCs were cultured on the lumen micro-channels of the tubular scaffold for 7 days, cells displayed spread and adhered very well onto the tube matrix (Fig. 6c, d, arrows). Moreover, SMCs exhibited aligning along the channel direction. This result convinced us that this micro-patterned scaffold was a good protocol to remodel SMC’s phenotype. We will conduct systemically study on the regeneration of esophageal muscle tissue with biological constitution and function in the next work.
3.3 Cytocompatibility to primary fibroblasts and SMCs Because both fibroblast and SMC are the main cell components of esophagus tissue, primary fibroblasts and SMCs from esophagus tissue were separately seeded on crosslinked F-2k
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4 Conclusions Using iron compound Fe(acac)3 as the catalyst, a series of oligomer poly(ethylene glycol-co-lactide) dimethacrylate (PLEGDMA) with different molecular weights was synthesized and analyzed via FTIR spectroscopy, 1H NMR spectra and GPC etc. Upon UV irradiation, these oligomers reacted with PEGDA and NIPAAm to form highly cross-linked networks and thus the crosslinked film and tubular scaffold were respectively obtained after optimization of reactant contents and amount ratios. The properties like thermodynamics, mechanical behavior, surface hydrophilicity and degradability etc. of four films, i.e. F-0.4k F-0.6k, F-1k and F-2k, were detailedly detected and discussed. F-2k was determined to be the optimal substrate with a lower Tg, higher mechanical strength, better hydrophilicity and degradability than those of all other samples. Its in vitro cytocompatibility to esophageal fibroblasts and SMCs was quantitatively tested and immunohistochemically evaluated. On this basis, a tubular scaffold with predetermined dimension and pattern was successfully fabricated using our patented tubular mould and the crosslinking protocol, which is believed to be favorable to guide the growth and phenotype regulation of esophageal cells like fibroblast and smooth muscle cell upon our cell culture results. The constitution of biofunctional esophageal prosthesis and in vivo regeneration are under way in our laboratory. Acknowledgments Financial supports by the National Science Foundation (81171476), Natural Science Funds and NSF for Distinguished Youth team of Zhejiang Province (LY12B01005, R2101166) and Scientific Innovation Team Project of Ningbo (No. 2011B82014), China, are gratefully acknowledged. This work was also sponsored by K.C. Wang Magna Fund of Ningbo University.
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