Novel supramolecular elastomer films based on linear carboxyl-terminated polydimethylsiloxane oligomers: preparation, characterization, biocompatibility, and application in wound dressings.

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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20

Novel supramolecular elastomer films based on linear carboxyl-terminated polydimethylsiloxane oligomers: preparation, characterization, biocompatibility, and application in wound dressings a

a

b

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Anqiang Zhang , Wenwen Deng , Yaling Lin , Junhui Ye , Yaomin a

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Dong , Yufeng Lei & Hongtao Chen a

Department of Polymer Material Science and Engineering, College of Material Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510641, Guangdong, China b

Department of Pharmaceutical Engineering, College of Resource and Environment, South China Agriculture University, 483 Wushan Road, Guangzhou 510642, Guangdong, China Published online: 24 Jul 2014.

To cite this article: Anqiang Zhang, Wenwen Deng, Yaling Lin, Junhui Ye, Yaomin Dong, Yufeng Lei & Hongtao Chen (2014) Novel supramolecular elastomer films based on linear carboxyl-terminated polydimethylsiloxane oligomers: preparation, characterization, biocompatibility, and application in wound dressings, Journal of Biomaterials Science, Polymer Edition, 25:13, 1346-1361, DOI: 10.1080/09205063.2014.938977 To link to this article: http://dx.doi.org/10.1080/09205063.2014.938977

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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 13, 1346–1361, http://dx.doi.org/10.1080/09205063.2014.938977

Novel supramolecular elastomer films based on linear carboxyl-terminated polydimethylsiloxane oligomers: preparation, characterization, biocompatibility, and application in wound dressings


Anqiang Zhanga*, Wenwen Denga, Yaling Linb*, Junhui Yea, Yaomin Donga, Yufeng Leia and Hongtao Chenb a

Department of Polymer Material Science and Engineering, College of Material Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510641, Guangdong, China; bDepartment of Pharmaceutical Engineering, College of Resource and Environment, South China Agriculture University, 483 Wushan Road, Guangzhou 510642, Guangdong, China (Received 10 April 2014; accepted 23 June 2014) A novel supramolecular elastomer (SESi) based on multiple hydrogen bond associations between low-molecular-weight polydimethylsiloxane chains was obtained through a two-step reaction of linear carboxyl-terminated polydimethylsiloxane oligomers with diethylenetriamine and urea, and the reaction mechanism was characterized. The results of differential scanning calorimetry and X-ray diffraction analyses indicated that the supramolecular network structure is completely amorphous, endowing SESi with rubber-like elastic behavior at room temperature. The transparent SESi film prepared by hot pressing displayed nice viscoelasticity, benign water absorption, water vapor transition rates, and ideal biocompatibility; and did not show cytotoxicity or skin irritation. These properties allow the elastomer to function as an occlusive wound dressing. To demonstrate its potential in wound dressings, a detailed comparison of commercial 3M Tegaderm™ film and the SESi film was conducted. The SESi film exhibited similar effects in wound healing, and the wound bed was covered by the SESi film without the occurrence of significant adverse reactions. Keywords: supramolecular elastomer based on polydimethylsiloxanes; multihydrogen bonds association; biocompatibility; cytotoxicity; wound dressing

1. Introduction Wound healing is a complex and continuous process that involves hemostasis, inflammation, proliferation, and tissue remodeling.[1] Many factors can influence this process, particularly the wound healing environment,[2,3] which can be improved through the use of wound dressings. However, wound dressings were considered to have only a minimal role in the healing process until the theory of wet healing was established in the 1960s. During this period, Winter [4] proposed the concept of the active involvement of a wound dressing in establishing and preserving an optimal environment for wound repair, as occlusive dressings are thought to increase cell proliferation and *Corresponding authors. Email: [email protected] (A. Zhang); [email protected] (Y. Lin) © 2014 Taylor & Francis


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activity by retaining an optimum level of wound exudates. According to studies, occlusive dressings can maintain wound bed moisture, warmth, and hypoxia and also function as a physical barrier, reducing the potential for infection in the injured zone.[5,6] Generally, an ideal wound dressing should maintain a moist environment at the wound surface, allow gaseous exchange, provide a barrier to micro-organisms, absorb wound exudates, reduce wound interface necrosis, stimulate growth factors, and be elastic, non-toxic, non-allergenic, and biocompatible.[7–9] Because polydimethylsiloxane (PDMS)-based materials possess numerous remarkable properties, such as a low glass transition temperature (Tg), high oxygen permeability, good mechanical properties, optical transparency, self-sealing capabilities, convenient processability, chemical stability,[10,11] and excellent biocompatibility, they are widely applied in a variety of medical devices, such as ophthalmologic biomaterials, microfluidic devices, artificial lungs, and finger joints.[12,13] However, these materials could also undoubtedly serve as a desirable wound dressing.[14–16] In fact, silicone wound dressings have been studied for several decades, including silicone foam, which has been used in the healing of wounds caused by diabetes and arteriosclerosis.[17] Because silicone foam is opaque, however, it is extremely difficult to observe the wound condition over time. Although traditional covalent bond cross-linked PDMS films are highly transparent, their poor water absorption would lead to excessive moisture in the wound environment. Therefore, compared with opaque, hydrophobic silicone foam or transparent silicone films, a hydrophilic, transparent silicone film with acceptable water absorption would represent a superior wound dressing. In this study, a novel supramolecular elastomer (SESi) based on linear carboxylterminated polydimethylsiloxane oligomers (PDMS-COOH2) was synthesized by a twostep reaction.[18] The supramolecular network was generated by multiple hydrogen bonds formed by 1,1-dialkylurea and 1,3-dialkylurea groups and imidazolidone derivatives. These highly hydrophilic groups endow the SESi film with satisfactory waterswelling properties and water vapor permeability. The SESi film also shows numerous other properties, such as non-toxicity, high biocompatibility, softness, lack of skin irritation, and elasticity. Moreover, the SESi film adhered strongly to normal tissue while exhibiting low adhesion to the moist wound bed, which would enable easy removal of a wound dressing and reduce patient pain. In the present study, we describe the synthesis and characterization of SESi and evaluate its biocompatibility and effect on wound healing in comparison with commercially available 3M Tegaderm™ film. 2. Experimental section 2.1. Materials Octamethylcyclotetrasiloxane (D4, PMX-0244, 99.9% purity) was provided by Dow Corning Corp UK (Barry, UK). 1,1,3,3-Tetramethyldisiloxane (HMM, 98% purity) was supplied by Kaihua Taicheng Silicone Co., Ltd (Quzhou, China). Tert-butyl methacrylate (98% purity) was supplied by Chemlin Chemical Industry Co., Ltd (Nanjing, China). 3M Tegaderm™ films (transparent film dressing, type 1624W) were supplied by 3M Health Care (St. Paul, MN, USA). The culture medium RPMI 1640 was obtained from Invitrogen (New York, USA). Fetal bovine serum was provided by Hangzhou Sijiqing Biological Engineering Materials Co., Ltd (Hangzhou, China). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was obtained from

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Alfa-Aesar. All other reagents, such as diethylenetriamine (DETA) (98% purity), urea (99% purity), and solvents, were used as received without any further purification.


2.2. Methods Infrared spectra were recorded using a Bruker Vertex 70 Fourier transform infrared spectrometer equipped with a Harrick ATC-024 temperature controller. 1 H-NMR spectra were collected with a Bruker AVANCE III-400 (400 MHz) spectrometer using CDCl3 as the solvent. Gel permeation chromatography (GPC) was performed on an Elite EC2000 GPC apparatus (Dalian, China) equipped with a Shodex K-G guard column and a Shodex K-804L chromatographic column. Detection was achieved using a refractive index detector, and the samples were analyzed at 30 °C using CHCl3 as the eluent at a flow rate of 1 mL/min. The instrument was calibrated using low polydispersity polystyrene standards. Differential scanning calorimetry (DSC) measurements were obtained using a Netzsch DSC 204C apparatus (Selb, Germany). The specimens were first heated and maintained at 150 °C for 5 min and then cooled and held at −150 °C for 5 min. The heating and cooling rates were 10 °C/min. The thermal transitions of these specimens were recorded during the second heating process. X-ray diffraction (XRD) analyses were conducted using a PANalytical X’ Pert-Pro X-ray diffractometer (ALMELO, The Netherlands) with filtered monochromatic Cu Kα radiation in the 2θ range of 5°–90°. Tensile tests were performed on rectangle specimens (50 mm × 10 mm × 1 mm) using a Zwick Z010 tensile testing machine (Zwick GmbH & Co. KG, Ulm, Germany) at a stretching rate of 500 mm/min. Creep tests were also performed on the Z010 tensile testing machine, and the applied load for the creep tests was 0.0125 MPa. 2.3. Synthesis of SESi Linear, low-molecular-weight PDMS-COOH2 (Mn = 0.98 × 104, Mw = 1.59 × 104, Mw/Mn = 1.62, measured by GPC) was synthesized as described previously.[19] The SESi materials were then synthesized according to the two-stage reaction presented in Scheme 1, which was derived from the dimer fatty acid system reported by Leibler [20,21]. The details were as follows. In the first step, PDMS-COOH2 and DETA were added to a 500 mL three-neck round-bottom flask fitted with a reflux condenser, a stirring system, and a nitrogen inlet at room temperature at a [–NH2]/[–COOH] mole ratio of 2.3/1. The mixture was heated to 120 °C at a heating rate of 10 °C/h until a transparent mixture was obtained. The mixture was then heated to 135 °C at a rate of 5 °C/h for 4–6 h. The mixture was cooled to room temperature, dissolved in an adequate volume of chloroform, and washed a minimum of five times with a mixture of water and methanol (5/2 wt/wt). The chloroform was first evaporated using a rotary evaporator and then placed under 200 mL/min nitrogen flow at 80 °C and stirred overnight. FTIR (KBr, cm−1): 3300 (νNH), 3072 (δNH), 2965 (νas CH3), 1651 (νC=O amide), 1606 (νCN imidazoline), 1549 (δNH), 1450 (δas CH3), 1262 (δs Si–CH3), 1020–1100 (νSi–O), 866 (rSi–CH3), 800 (νas Si–CH3), 699(νs Si–CH3). 1 H-NMR δ (CDCl3, 400 MHz, ppm): 0.05–0.15 (m, Si–CH3), 0.75–1.05 (m, Si–CH2–), 1.20 (d, –CH–CH3), 2.38 (m, –CH2–CH–), 2.67 (t, NHCH2CH2NH2),



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2.76 (t, C(O)NHCH2CH2NH), 2.80 (t, NHCH2CH2NH2), 2.86 (t, NH2CH2CH2Nimidazoline), 3.15 (t, NH2CH2CH2N-imidazoline), 3.33 (m, C(O)NHCH2CH2NH), 3.33 (m, C(O)NHCH2CH2N-imidazoline), 3.68 (m, N(C)CH2CH2N-imidazoline). In the second step, urea was added to the reaction apparatus described above. The mixture was heated to 135 °C at a heating rate of 40 °C/h and maintained for 2 h. Then, the mixture was heated to 160 °C in 5 °C increments every 60 min. After further incubation, the mixture became viscous and began to rise up the stirring stem. Once the mixture content had risen for 2–3 h, the reaction was stopped, and the mixture was removed as soon as possible while still hot. As the product cooled to room temperature, it was cut into small fragments and washed in water at 50 °C for 2–3 days. FTIR (KBr, cm−1): 3298 (νNH), 3069 (δNH), 2963 (νas CH3), 1655 (νC=O), 1603 (νNC), 1540 (δNH), 1451 (δas CH3), 1262 (δs Si–CH3), 1020–1100 (νSi–O), 866 (rSi–CH3), 802 (νas Si–CH3), 697(νs Si–CH3). 1 H-NMR δ (CDCl3, 400 MHz, ppm): 0.05–0.15 (m, Si–CH3), 0.75–1.00 (m, Si–CH2–), 1.18 (d, –CH–CH3), 2.39 (m, –CH2–CH–), 3.20–3.50 3.20–3.50 (m, –NCH2CH2NH-urea), 3.20–3.50 (m, CH2–NCH2CH2NHCO), 3.51 (m, C(O)NHCH2CH2N-imidazolidone), 3.85 (t, C(O)NHCH2CH2N-imidazolidone).

Scheme 1.

The synthesis route and structure of the SESi materials.

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2.4. Preparation of the SESi film The SESi material prepared above was first hot pressed at 150 °C for 10 min in a 0.5 or 1 mm-thick mold and then cool pressed at room temperature for 20 min to obtain a 0.5 or 1 mm-thick SESi film that was smooth and transparent.


2.5. Water vapor transition rate (WVTR) The moisture permeability of the SESi film was determined by measuring the WVTR across the material as expressed by ASTM E96.[22] A 0.5 mm-thick SESi film was used to cover the mouth of a conical flask (25 mm diameter) filled with 40 mL of distilled water. The flask was then maintained at 35 °C and 50 ± 2% RH in an incubator. The WVTR was calculated using the following equation: WVTR ¼

G At

(1)

where WVTR is expressed in g/m2 d; G is the weight change in g; A is the area of the cup mouth (m2); and t is time (d). 2.6. Water absorption measurements The water absorption ratio (Aw) was determined by soaking the SESi film samples in distilled water at 37 °C and 80 ± 2% RH until achieving an equilibrium swelling state. The films were then removed and wiped gently with filter paper. The Aw values were calculated using the following formula: Aw ð%Þ ¼

We Wd 100% Wd

(2)

where We and Wd are the film weights at equilibrium and dry state, respectively. 2.7. Evaluation of cytotoxicity (MTT cytotoxicity test) The SESi patches (1 cm × 1 cm × 0.1 cm) were first sterilized for 3 h with ultraviolet light and then soaked in 10 mL of RPMI 1640 culture medium (the film/solution ratio was 1/10 (cm2/mL)) for 24 h to obtain an extract solution of the SESi film. L929 cells (murine aneuploid fibrosarcoma cells) were cultured in RPMI 1640 supplemented with 10% FBS in 96-well plates (100 μL medium/well) at a density of 1.0 × 105 cells/mL. The cells were cultured overnight at 37 °C in a humidified 5% CO2 incubator after inoculation. After culturing the cells for 24, 48, and 72 h, a 5.0 mg/mL MTT solution (in PBS) was added to the wells and incubated for another 4 h at 37 °C. The growth medium was subsequently removed, and 150 μL of DMSO was added, followed by vigorous shaking to dissolve the purple formazan crystals that formed. The absorbance was measured with a Bio-Rad Model 680 MicroplateReader (Bio-Rad, USA) at a wavelength of 570 nm. 2.8. Evaluation of cytotoxicity (direct contact test) An L929 cell (2 mL) suspension was added to the vessels and incubated at 37 °C in a humidified 5% CO2 incubator. Next, each vessel was filled with fresh RPMI 1640 culture medium. The sterilized SESi film patches (1 cm × 1 cm × 0.1 cm) were gently


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placed on the layer of cells in the center of each of the replicate vessels. One-tenth of the cell layer surface was covered by the test sample. Finally, the vessels were incubated in the same environment described above. For comparison, an LDPE film (negative control) and an organotin-stabilized PVC film (positive control) were also prepared.


2.9. Hemolytic activity The hemocompatibility of the SESi film samples was tested by direct and indirect contact methods. Fresh blood was obtained from an albino rabbit of the New Zealand strain (weight: 2.5 kg). The blood was then diluted with saline water (0.9% wt NaCl) to a volume ratio of 1/1.25 (each sample contained 4 mL of fresh blood and 5 mL of saline water). In the direct contact method, 5 g of sterilized SESi film patches (5 mm × 20 mm × 1 mm) and 10 mL of normal saline were added to the dilute blood. For comparison, positive and negative controls were prepared using 10 mL of distilled water and 10 mL of saline water, respectively. After incubation for 30 min at 37 °C, 0.2 mL of diluted blood was added, and the samples were incubated for 1 h. The solution was then centrifuged at 3750 rpm (×800 g) for 5 min, and the optical density (OD) of the clear supernatant fluid was measured at 545 nm. In the indirect contact test, 1.2 g of SESi film patches (5 mm × 25 mm × 1 mm) were equilibrated in 6 mL of normal saline solution at 37 °C for 24, 48, 72, and 96 h. Positive and negative controls were prepared using 0.12 mL diluted blood solution in 6 mL distilled water and 6 mL normal saline, respectively. After incubation in a water bath for 30 min at 37 °C, 0.12 mL of diluted blood was added, and the solutions were incubated for another 60 min at 37 °C. Next, the solutions were centrifuged at 2500 rpm for 5 min, and the OD of the clear supernatant fluid was measured with a Bio-Rad Model 680 MicroplateReader at 545 nm. The hemolysis ratio (HR) was calculated using the following equation: HR ð%Þ ¼

ODtest sample ODðÞcontrol 100% ODðþÞcontrol ODðÞcontrol

(3)

where ODtest sample, OD(−)control, and OD(+)control are the OD values of the SESi film sample, the positive control, and the negative control, respectively. 2.10. Skin irritation Skin irritation was determined according to the ISO 10933.10-2010 method. Before the experiment, the dorsal skin of five albino rabbits of the New Zealand strain was shaved (area of approximately 6 cm × 6 cm). Then, two SESi membranes (2.5 cm × 2.5 cm) and one 3M Tegaderm™ film were applied to the hairless area of each rabbit. The conditions of each application site at 1, 24, 48, and 72 h were recorded and evaluated according to a visual scoring scale immediately after removal of the films. The method for determining the skin irritation score was described previously.[23] 2.11. In vivo wound healing Male Wistar rats (weight range, 220–250 g) were used to evaluate the wound healing characteristics, as shown in Figure S1. All experiments were completed with the


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approval of the Animal Research Ethics Committee of the South China Agriculture University. During the study, all rats had access to chow and water ad libitum. Prior to the test, the rats were anesthetized with chloral hydrate (400 mg/kg of body weight). Next, the dorsal hairs of the rats were shaved with an electric razor, and full-thickness defects of the dermis (diameter of approximately 18 mm) were created on both sides of the back using curved-blade surgical scissors. Then, SESi membranes and Tegaderm™ films were applied to the full-thickness skin defects. The surgery was repeated multiple times to obtain a sample size of five rats per treatment per time point. After the experiment, the treated rats were housed in individual cages, and the wound tissues were observed at different healing times. On days 5, 7, 10, 14, and 21 post-surgery, the rats were anesthetized and sacrificed by cervical dislocation. The new wound area was determined by the diameters of the two wound sites. The wound sites were excised and processed for histological evaluation. The percent wound contraction was calculated using the following formula: Wound contraction ð%Þ ¼

Ao At 100% Ao

(4)

where Ao and At are the original wound area and the area of the wound at the time of biopsy, respectively. The wound area was determined from the images of the wounds. 2.12. Histology For histology, the wound tissues and adjacent normal skin were dissected, fixed with 10% phosphate-buffered formalin, and stained with hematoxylin and eosin (H&E) reagent for histological observations. Digital photos were acquired by a light microscope (Nikon DS-Ri1, Japan) at 40× and 100× magnification. 2.13. Statistics Statistical analyses were performed with at least five samples of each SESi film and 3M Tegaderm™ film. Data are presented as the mean ± SD, and the significance level was set at p < 0.05. Intergroup differences were determined using Microsoft Excel’s statistical function for Student’s t-test for comparisons against a control group. 3. Results and discussion 3.1. Characterization of SESi SESi was prepared via a two-stage reaction, described in Scheme 1, and the process was monitored by acquiring FTIR and 1H-NMR spectra of samples withdrawn during the reaction. At the beginning of the first stage of the reaction, an –NH2 absorption band appeared at 3340 cm−1 due to the addition of DETA. As acylation proceeded, the –COO– absorption bands of PDMS-COOH2 and –NH2 decreased in intensity and transferred to the absorption bands of the C=O amide (1651 cm−1) and –NH– (3298 cm−1), respectively. After the temperature reached 135 °C, an absorption band for the νCN of imidazoline appeared at 1606 cm−1, as shown in Figure S2. For the second step of the reaction, the product of the first step was reacted with urea at 160 °C. As the reaction advanced, the absorption band of νC=O gradually became wider due to the products of the reaction of urea with primary (1,3-dialkylurea)



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and secondary amines (1,1-dialkylurea) as well as cyclization of the –NH–CH2–CH2– NH2 group to form the imidazolidone, as shown in Figure S3(B). Moreover, a distinct increase in molecular weight during the two-step reaction process was observed by GPC, as shown in Figure S4. The reaction mechanism of the two-step reaction was also monitored by acquiring 1 H-NMR spectra of samples withdrawn from the mixture during the reaction (Figure S5). At the end of the first step, a small amount of imidazoline (δ = 3.7 ppm) by-product was generated. In the second step, –NH2 groups were first reacted with partial urea groups to form monoalkylurea derivatives (combined with the disappearance of the signals from –NHCH2CH2NH2 at δ = 2.67 ppm and –NHCH2CH2NH2 at δ = 2.80 ppm), and the –NH– groups then reacted with the residual urea to produce 1,1-dialkylurea derivatives (illustrated by the disappearance of the characteristic signal from –CONHCH2CH2NHCH2CH2– at δ = 2.76 ppm). Moreover, imidazolidone derivatives were formed at longer reaction times (a new signal appeared at δ = 3.85 ppm belonging to C(O)NHCH2CH2 N-imidazolidone, as shown in Figure S5). These results confirm that SESi was obtained by the two-step reaction. The association of hydrogen bonds in the SESi matrix was demonstrated by temperature-dependent infrared spectroscopy. With increasing temperature, the N–H stretching vibration (3100–3500 cm−1) and the C=O stretching vibration (1651–1663 cm−1 from 30 to 160 °C) of SESi shifted to higher wave numbers, whereas the N–H in-plane bending vibration signals shifted to lower wave numbers. When the sample was cooled immediately, the shifting behavior was completely reversed, as shown in Figure S6. Both these behaviors indicate the presence of thermally reversible hydrogen bonding interactions in the SESi matrix. The thermal transitions of the SESi material were evaluated by DSC. As shown in Figure 1, the SESi material exhibited a clear glass transition temperature (Tg) at −126 °C, crystallization (Tc) at −77 °C, and melting (Tm) at −43 °C, similar to unfunctionalized linear PDMS. In addition, no crystalline peaks were observed by XRD, indicating that the SESi is amorphous and could display rubber-like elastic behaviors at room temperature. Indeed, the rubber-like properties of SESi were confirmed by the creep deformation curve at room temperature, shown in Figure 2(A). After releasing the stress, the SESi material could mostly recover its original conformation in a short time. The stress– strain curves of the SESi material were also used to evaluate the association strength of

Figure 1.

Characterization of SESi: (A) DSC and (B) XRD curves.


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Figure 2. Creep deformation curve (A) and stress–strain curve (B) of a SESi sample at room temperature.

these hydrogen bonds. As shown in Figure 2(B), the SESi samples possessed an elongation of approximately 700% and displayed typical rubber-like stress-softening behavior at elongations larger than 100%. 3.2. Water vapor transmission rate (WVTR) and water absorption of the SESi film A suitable WVTR in wound dressings is beneficial to the transmission of O2 and the permeation of CO2 and aqueous vapor. The WVTRs of the SESi film and the 3M Tegaderm™ film were 331 ± 4 and 413 ± 5 g/m2/d, respectively, when measured at 37 °C and 50% RH, as shown in Figure 3(A). Thus, the WVTR of the 3M Tegaderm™ film is slightly superior to that of the SESi film. The water absorption value was determined at 37 °C and 80% RH using distilled water (Figure 3(B)). The equilibrium swelling ratios of the SESi film and the 3M Tegaderm™ film were 19.1 and 9.2%, respectively. Thus, in contrast to the WVTR results, the absorption of the SESi film was superior to that of 3M Tegaderm™ film, which would enable the dressing film to better absorb liquid secretions.

Figure 3. Water vapor loss values (A) and water absorption (B) of the SESi film and 3M Tegaderm™ film.


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3.3. Evaluation of biocompatibility


As biomaterials for a specific end use, wound dressings must meet specific standards in terms of the nature of the physiological environment, the desired functions, and adverse effects in case of failure. A biomaterial must also exhibit good biocompatibility to qualify for biological applications. In this study, biocompatibility was measured by in vitro and in vivo tests, such as cytotoxicity, hemolysis, and skin irritation tests. 3.3.1. Cytotoxicity In this study, the cytotoxicity of SESi toward L929 fibroblast cells was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assays and direct contact tests. For the MTT assays, the original and diluted extracts were prepared and immersed in RPMI 1640 culture medium for 24 h. L929 cells were cultured for 72 h in normal saline, distilled water, or SESi film extract before adding MTT. The morphology of the cells is shown in Figure 4. When cultured in the SESi film extract or normal saline, the L929 cells cultured in the SESi film extract grew well, whereas in distilled

Figure 4. Comparison of cytotoxicity in L929 cells (cultured for 72 h before addition of MTT). (A) Normal saline (negative control), (B) distilled water (positive control), and (C) SESi film extract.

Figure 5. Comparison of the cytotoxicity of a SESi extract, the negative control and the positive control toward L929 cells (n = 6).


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Figure 6. Comparison of cytotoxicity toward L929 cells in direct contact tests. (A) LDPE film (negative control), (B) organotin-stabilized PVC film (positive control), and (C) SESi film.

water, the majority of the cells adopted a bloom form. The cell viability in the SESi extract was similar to that in normal saline, as determined by MTT assay and shown in Figure 5. In addition, the direct contact method was also used to evaluate in vitro cytotoxicity. The morphologies of L929 cells incubated on the SESi film, an LDPE film (negative control), and an organotin-stabilized PVC film (positive control) for 24 h are shown in Figure 6. In agreement with the results of the MTT assay, the cell viability on the SESi film was similar to that on the negative control (LDPE film), and almost all of the cells adopted a bloom form in the positive control. Taken together, these results demonstrate the non-toxicity of SESi toward L929 cells. 3.3.2. Hemocompatibility The hemocompatibility of the SESi film was assessed by measuring hydrolysis, dynamic coagulating time, platelet adhesion, and in vitro cell viability. The hemolytic activity was determined by hemoglobin release under the test conditions. For hemocompatibility, the HR must be less than 5%. The HRs for heparinized blood mixed with extracts of a SESi film patch for 24, 48, 72, and 96 h were 1.17 ± 0.12, 1.04 ± 0.17, 1.43 ± 0.09, and 3.26 ± 0.42%, respectively. These values are within the acceptable limit, indicating the hemocompatibility of the SESi film. 3.3.3. Skin irritation test The results of the skin irritation test are shown in Figure S7. As the blank and 3M Tegaderm™, there was no symptoms of skin irritation were observed. In addition, all the rabbit skin irritation scores for the SESi films were 0, including that for erythema and edema. These results indicate that the SESi film does not cause skin irritation in rabbits. In addition, the rabbits did not display any significant changes in body weight or signs of intoxication. Thus, the SESi film can be safely used on skin. 3.4. Wound healing performance 3.4.1. Gross observation The 3M Tegaderm™ film was compared with the SESi film, as in our previous paper.[23] Figure 7 shows a rough observation of wound healing when covered by a



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Figure 7. Comparison of wound healing state: (A) 3M Tegaderm™ film and (B) SESi wound film dressings.

Figure 8. The percentage of wound contraction for a 3M Tegaderm™ film (n = 5) and SESi film dressings (n = 5). The results (mean ± SD) are for the two experimental groups.

3M Tegaderm™ film or a SESi film. As a quantitative comparison, wound closure (%) is shown in Figure 8. The average wound closure for the two films is approximate, and the difference between them was insignificant (p > 0.05), as analyzed by Student’s t-test. This result indicates that the SESi film dressing and Tegaderm™ similarly promote wound healing and that the SESi film in this work is more effective in the early stages of wound healing than our previous SESi film, especially in the early stages (5–7 days) of wound healing.[23] 3.4.2. Histological analysis The healing patterns of the wounds were analyzed by examining the histology of the wound tissues at days 5, 7, 10, 14, and 21 post-wounding, and the condition of each wound is depicted in Figure 9. However, to quantitatively assess the healing response,


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Figure 9. H&E staining of wound tissues (40×). (A1)–(A5): wound covered by 3M Tegaderm™ film, from days 5, 7, 10, 14, and 21; (B1)–(B5): wound covered by SESi film, from days 5, 7, 10, 14 and 21. Notes: OT, original skin tissue; RT, recovery skin tissue; BS, blood scab; BV, blood vessel; E, reepithelialize.

several images of higher magnification (200×) were also acquired (Figure 10) to observe the details of wound healing, as described below. On the 5th day post-wounding, histology observations revealed that the wounds covered by the 3M Tegaderm™ film and the SESi film were in the inflammatory phase, as shown in Figure 10((A1) and (B1)). In both wounds, the infiltration of lymphocyte cells and fibroblasts as well as vasodilatation could be observed. Moreover, the wound covered by the 3M Tegaderm™ film displayed congestion and hemorrhage. In contrast to the wound covered by the 3M Tegaderm™ film, however, a blood scab and inflammatory granulation tissue were formed in the dermis covered by the SESi film. On the 7th day post-wounding, a blood scab formed on the wound covered by the 3M Tegaderm™ film. At this point, many granulation tissues could be observed under the scabs, and the capillaries were growing perpendicularly. In addition, fibroblasts began to migrate along the fibrin threads, and all wounds began to show increased congestion and hemorrhage, as shown in Figure 10((A2) and (B2)). On the 10th day, the two wounds were in the proliferative phase, and the scabs were reduced. Although the fibroblasts displayed obvious proliferation in all wounds, they were more organized on the wound covered by the SESi film, as shown in Figure 10((A3) and (B3)).



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Figure 10. H&E staining of wound tissues (200×). (A1)–(A5): wound covered by the 3M Tegaderm™ film at days 5, 7, 10, 14, and 21; (B1)–(B5): wound covered by the SESi film at days 5, 7, 10, 14, and 21. Notes: IE, Inflammatory exudates; BS, blood scab; CH, congestion and hemorrhage; GT, granulation tissue; VS, vasodilatation; BV, blood vessel; CF, collagenous fibers; E, reepithelialization; MOCF, more organized fibroblasts; green arrow-heads, lymphocytes; white arrow-heads, fibroblasts; red arrows, skin cuticle. (Please see the online article for the colour version of this figure: http://dx.doi.org/10.1080/09205063.2014.938977.)

After 14 days, all surfaces of the test wounds were nearly completely healed. In this stage, the increased vascularity had diminished in quantity. Fibroblasts were still proliferating, and the collagenous fibers became more organized. Moreover, skin cuticles began to form. However, slightly more skin cuticles could be observed on the wound covered by the 3M Tegaderm™ film, as shown in Figure 10((A4) and (B4)). Twenty-one days after surgery, the two types of wounds were completely healed, with complete reepithelialization across the wounds, and the wound bed had generated more skin cuticles. Beneath the epidermis, the increased vascularity had diminished, and the leukocyte infiltration had nearly vanished. Furthermore, organized collagenous fibers became more obvious in the wound covered by the SESi film, as shown in Figure 10((A5) and (B5)). In summary, the SESi film was able to create a wet wound-healing environment and exhibited similar effectiveness in promoting wound healing as the 3M Tegaderm™ film. 4. Conclusion In this study, we developed a novel supramolecular elastomer based on linear carboxylterminated PDMS oligomers (SESi) in which the chains were cross-linked by multiple hydrogen bonds. The SESi is transparent and soft and exhibits cross-linked rubber-like mechanical properties. In addition, it exhibits excellent biocompatible properties, water absorption abilities, and an acceptable WVTR. We further demonstrated that the SESi

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film could help to create a wet wound-healing environment, showing wound healing efficiency similar to that of 3M Tegaderm™ film. Funding The authors acknowledge financial support from the National Natural Science Foundation of China [grant number 51003032], [grant number 31201552] and the Specialized Research Fund for the Doctoral Program of Higher Education [grant number 20124404120025].

Supplemental data


Supplemental data for this article can be accessed here http://dx.doi.org/10.1080/09205063.2014. 938977.

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