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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Electrospun polyurethane-dextran nanofiber mats loaded with Estradiol for post-menopausal wound dressing
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Afeesh Rajan Unnithan a,1 , Arathyram Ramachandra Kurup Sasikala a,1 , Priya Murugesan b , Malarvizhi Gurusamy b , Dongmei Wu b , Chan Hee Park c,∗ , Cheol Sang Kim a,c,∗ a
Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju 561-756, Republic of Korea Department of BIN Fusion Technology, Chonbuk National University, Jeonju 561-756, Republic of Korea c Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea
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Article history: Received 11 November 2014 Received in revised form 15 January 2015 Accepted 15 February 2015 Available online xxx
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Keywords: Wound dressing Estradiol Dextran
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1. Introduction
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Post-menopausal wound care management is a substantial burden on health services, since there are an increased number of elderly populations linked with age-related delayed wound healing. The controlled estrogen replacement can accelerate healing of acute cutaneous wounds, linked to its potent anti-inflammatory activity. The electrospinning technique can be used to introduce the desired therapeutic agents to the nanofiber matrix. So here we introduce a new material for wound tissue dressing, in which a polyurethane–dextran composite nanofibrous wound dressing material loaded with -estradiol was obtained through electrospinning. Dextran can promote neovascularization and skin regeneration in chronic wounds. This study involves the characterization of these nanofibers and analysis of cell growth and proliferation to determine the efficiency of tissue regeneration on these biocomposite polymer nanofibrous scaffolds and to study the possibility of using it as a potential wound dressing material in the in vivo models. © 2015 Published by Elsevier B.V.
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There has been a rapid growth in the magnitude of elderly population mainly due to the recent advancements in the healthcare management and due to the various epidemiological factors. Along with this scenario, there has an increase in morbidity associated with age-related delayed wound healing. Generally the wounds heal through a multifaceted series of overlapping tightly controlled stages. As we became older, these complex processes were disturbed and repair efficiency was reduced. According to the previous reports, in elderly women the estrogen hormone level in the body quickly reduced after the menopause and this might contributed towards to delay in wound healing. Estrogens possess a considerable role in wound repair that can reverse the reduced wound healing process. Those who were at the post-menopausal condition
∗ Corresponding authors. Tel.: +82 63 270 4284; fax: +82 63 270 2460. E-mail addresses: [email protected] (A.R. Unnithan), [email protected] (C.H. Park), [email protected] (C.S. Kim). 1 The two authors equally contributed to this work.
possess an increased risk of developing an excessive inflammation condition, mainly due to a number of deteriorating pathological properties [1,2]. During the menopause condition, considerable changes to the normal female skin have been occurred. The profound changes were the decrease in dermal collagen and reduced skin thickness and both of which can be overturned by topical estrogen application [3,4]. According to the studies, there exist a strong correlation between the systemic hormone levels and wound healing. It has been reported that the women at the post-menopausal stage taking systemic hormone replacement therapy heal wounds more effectively than the control ones [5]. The cutaneous wound healing is a complicated process linked with an initial inflammatory response, restoration of the epithelial barrier and matrix deposition. The improved deposition of cell matrix, prompt re-epithelialization process, and the potential anti-inflammatory activity can be achieved by the systemic estrogen replacement and which can contribute to accelerate the healing of acute cutaneous wounds [6,7]. In this concern, hormone replacement therapy (HRT) can be a good substitute to prevent the development of chronic wounds in post-menopausal women [8,9]. According to the studies, the exogenous treatment of estrogen can
http://dx.doi.org/10.1016/j.ijbiomac.2015.02.044 0141-8130/© 2015 Published by Elsevier B.V.
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reverses the delayed cutaneous wound healing by inhibiting unnecessary neutrophil recruitment, endorsing re-epithelialization and increasing collagen deposition [10,11]. Dextran is a biocompatible, biodegradable, non-immunogenic and non-antigenic biopolymer and hence it has been widely applied in various biomedical applications, such as drug delivery applications [12], as scaffolds for tissue engineering applications [13,14] and as molecular arms [15], etc. Scaffold materials made up of dextran were soft and flexible and hence it improves the handling efficiency for the management of wound treatment [16]. Moreover the dextran-based hydrogel materials have been used as scaffolds for neovascularization and re-epithelialization in wound tissue engineering. In the preparation aspect, dextran is soluble in both water and organic solvents make it as a fine material for bioapplications. So the dextran can be directly blended with suitable polymers such as PU to prepare composite nanofibrous membranes by electrospinning [17]. The physical and biological properties of dextran can be thus manipulated according to the application and hence better biocompatibility can be achieved [18]. The precise adherence to the wound location and good exudate absorbance should be the promising factors of a perfect wound dressing material. They should also retain appropriate moisture along with easy practice and removal [19,20]. Electrospun nanofibers were found to be very effective to be used as the wound dressing material mainly due to its architectural superiority. The electrospun nanofiber can mimic the extra cellular matrix (ECM) environment which will help the host cells to grow and create a new natural cellular matrix [21,22]. Most importantly the preferred therapeutic materials can be introduced to the nanofiber matrix using the electrospinning process [17,23,24], that can seriously affect the wound healing. We introduce a new PU-dextran based nanofibrous material for potential wound dressing utilizing -estradiol, the most bioactive endogenous estrogen. In this preliminary work, a composite nanofibrous wound dressing material loaded with -estradiol was fabricated through electrospinning. The continuous systemic estrogen release from the Estradiol loaded nanofibrous mat at the wound area can accelerate healing of acute cutaneous wounds, linked to its potent anti-inflammatory activity. This preliminary study involves the fabrication, characterization of these composite nanofibers and analysis of cell growth and proliferation to determine the efficiency of tissue regeneration on these biocomposite polymer nanofibrous scaffolds and to study the possibility of using it as an effective wound dressing material in the in vivo models.
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2. Experimental procedure
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2.1. Materials and methods
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2.2. Characterizations The morphology of the electrospun composite mats was observed by using field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan). The bonding configurations of the samples were characterized by means of Fourier-transform infrared (FT-IR) spectroscopy. Contact angle (wettability) was measured by using the deionized water contact angle measurement system, using contact angle meter (Digidrop, GBX, France). Deionized water was automatically dropped (drop diameter 6 m) onto the mat. 2.3. Platelet activation study and whole blood clotting assay The blood clotting studies were done based on reported literature [25]. Blood was mixed with anticoagulant agent acid citrate dextrose at a ratio of 9:1. Later blood was added to each composite nanofiber mats and placed in a 25-mL plastic Petri dish, which was followed by the addition of 10 L of 0.2 M CaCl2 solutions to initiate blood clotting and PU mat was used as negative control. These mats were then incubated at 37 ◦ C for 15 min. 1 mL of distilled water was then added drop wise without disturbing the clot. Subsequently, 1 mL of solution was taken from the dishes and was centrifuged at 1000 rpm for 1 min. The supernatant was collected for each sample and kept at 37 ◦ C for 1 h. Two hundred microliters (200 L) of this solution was transferred to a 96-well plate. The optical density was measured at 530 nm using a plate reader (Dynex Technologies, USA). For platelet activation studies blood mixed with anticoagulant was centrifuged at 2500 rpm for 10 min. The supernatant rich in platelet plasma was collected and added directly on to the nanofibers. Samples were fixed with glutaraldehyde and then washed with PBS. Later these samples were dehydrated using gradient alcohol treatment and then viewed under SEM. 2.4. In vitro drug release study Estradiol loaded composite nanofibers (2 × 2 cm) were immersed into eppendorf tubes containing 3 mL of phosphate buffer solution (pH 7.4) at 37 ◦ C with continuous shaking at 120 rpm. A fixed volume of the release medium was withdrawn at continuous intervals and replenished with the same volume of the fresh PBS solution. All samples were prepared in triplicate and analyzed using UV–vis. 2.5. Cytocompatibility study
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Polyurethane, 10 wt% (PU, Estane® Skythane® X595A-11, Lubrizol) was prepared by dissolving in solvent mixture of Dimethyl sulfoxide (DMSO, Sigma Aldrich, Korea) and Tetrahydrofuran (THF, Sigma Aldrich, Korea) (50/50, wt:wt%). 5 wt% The Dextran (from Leconostoc mesenteroides, average Mw = 8500–11500, Sigma Aldrich) has been added to those respective solutions along with 2 wt% -estradiol (Sigma Aldrich, Germany). The obtained solutions were placed in a plastic syringe tube and fed through a metal capillary (nozzle) with a diameter di = 0.21 mm (21 G) attached to a 1-D robot-system that moves laterally and is controlled by the LabVIEW 9.0 software program (National Instrument). The feeding rate was maintained at 0.5 mL/h via a controllable syringe pump. Electrospinning was carried out at a voltage of 18 kV and working distance of 15 cm at room temperature. After electrospinning the mats were carefully removed and kept at overnight vacuum drying to remove the residual solvents.
The viability of cultured 3T3-L1 fibroblasts (preadipocytes, Korean Cell Line Bank, Korea) was monitored on the third and sixth day of culture using the colorimetric MTT assay (Sigma, USA). The nanofiber scaffolds were washed twice with PBS and were then treated with approximately 50 L of the MTT solution (DMEM); the scaffolds, after mixing of the contents by side-tapping, were incubated at 37 ◦ C for 2 h. The nanofiber scaffolds containing MTT-cell mixtures were gently rocked to deposit the cells. The supernatant of the MTT solution was pipette out and then acid–isopropanol (95 mL isopropanol with 5 mL 3 N HCl) was added to the colored cell deposit. After gently mixing the acid–alcohol-treated scaffolds, it was then allowed to react for 5 min. 100 L of the purple–blue colored supernatant that contained the solubilized formazan in each sample was added to a well in a 96-well plate for analysis at 580 nm in an ELISA reader. The cell viability was obtained by comparing the absorbance of cells cultured on the nanofibers scaffold to that of control well containing cells. The results were expressed as the mean ± standard error of the mean. The data were analyzed via the Student’s t test and repeated measures of analyses of variance
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Fig. 1. A SEM images of electrospun (A) PU(10 wt%):dextran (10 wt%) mat, (B) PU(10 wt%):dextran (15 wt%) mat, (C) PU(10 wt%):dextran (2 wt%) mat and (D) PU(10 wt%):dextran (5 wt%) nanofiber mat. (B) FESEM images of electrospun (A) PU mat, (B) PU-Dextran mat and (C) Estradiol loaded PU-dextran mat.
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(ANOVA) test. A probability of less than 0.01 was considered to be statistically significant.
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2.6. In vivo wound dressing study
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In vivo animal study was approved by the Institutional Animal Ethical Committee, IACUC certification number CBU 2014-00062 at Chonbuk National University, Jeonju, South Korea. Wistar rats, weighing 200–250 g and 4–6 weeks of age, were used in this study.
The rats were divided into two groups and each group contains three rats (n = 3); rats were allowed to take normal rat feed and water without restriction. On the day of wounding, the rats were anaesthetized by intraperitoneal injection of 80 mg/kg ketamine and 8 mg/kg xylazine. The dorsal area of the rats depilated and the operative area of skin cleaned with alcohol. A partial thickness skin wound of 1.5 cm square was prepared by excising the dorsum of the rat using surgical scissors and forceps. The prepared wounds were then covered with the Estradiol loaded PU-dextran nanofibers
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Fig. 2. FTIR spectra of electrospun (A) -Estradiol, (B) pristine PU and (C) Estradiol loaded PU-dextran nanofibrous mat.
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and Rats with bare wound were kept covered using cotton gauze. After applying the dressing materials, the rats were housed individually in cages under normal room temperature. The dressing materials were changed at weeks 1, 2, and 3. During the changing of dressings the animals hairs cropped and photographs were taken and the wound area was measured using a transparent polyethylene sheet. The sheet was kept on top of the wound and area was marked using a marker pen. After Week 3, the skin wound tissue of the rat was excised, fixed with 10% formalin, and stained with a hematoxylin–eosin (H&E) reagent for histological observations.
3. Results and discussion
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3.1. Physical characterizations of composite nanofibers
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Fig. 1A (A–D) shows the SEM images of electrospun composite nanofibers with different concentrations of PU and dextran. As shown in Fig. 1A ((A)–(C)) PU (10 wt%):dextran (10 wt%) mat, PU(10 wt%):dextran (15 wt%) mat and PU(10 wt%):dextran (2 wt%) mat, respectively, showed beaded morphology. The formation of beaded fibers in may be possibly due to the change in viscosity of PU in blend polymer solution with dextran, then causing different stretching time from droplet to fiber during e-spinning. But Fig. 1A (D) showed promising fiber morphology with PU (10 wt%):Dextran (5 wt%). From these data it is clear that the concentration of PU and dextran in the final solution having a direct effect on the fiber morphology. Fig. 1B (A)–(C) shows the FESEM images of final study material, electrospun PU, PU-dextran and PU-dextran-Estradiol loaded composite nanofibers, respectively. It can be observed that these randomly oriented as-spun nanofibers exhibited bead-free, smooth surface with almost uniform diameters along their lengths. The diameters of these composite nanofibers were determined to be in the range of 500–600 nm. For PU electrospun mat (Fig. 1A), the fibers appear well-defined without any interconnection among the fibers. But the composite mats containing Estradiol and dextran showed some changes in fibrous morphology (Fig. 1C). It may be due to the combined effect of Estradiol and dextran with the PU solution. The addition of Estradiol above 2 wt% caused the drastic decrease in solution viscosity and hence the final concentration of Estradiol was set as 2 wt%. The addition of Estradiol can significantly decrease solution viscosity; however, it did not affect the electrospinning of the composite fibers. According to the reports, solution viscosity seriously influences the morphological structure and average size of resulting fibers [26]. Different concentrations of PU and dextran were electrospun and then decided (Fig. 1A) the final concentration of PU as 10 wt% and dextran as 5 wt%. The
Fig. 3. Water contact angles of electrospun (A) PU nanofibrous mat, (B) PU-dextran nanofibrous mat and (C) Estradiol loaded PU-dextran nanofibrous mat.
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Fig. 4. (A) Blood clotting efficiency of nanofibrous mats using OD values, SEM images of platelet activation of (B) Estradiol loaded PU-dextran composite nanofibrous mat (white dots indicate the platelet adhesion on composite mat) respectively.
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Estradiol and dextran can affect the viscosity of the polymeric solution and some studies reported that some materials can be confined between polymeric chains, and thus they can as a plasticizer [27]. The composite electrospun nanofiber mat produced in this study was desirably smooth and flexible and were successfully used for the desired studies. The changes of the functional groups that occur during the blending of Estradiol and dextran with PU were examined by FTIR spectroscopy. Fig. 2 explains the FTIR spectra of PU, PU-dextran and Estradiol loaded PU-dextran nanofibers. The FT-IR spectra of electrospun PU has characteristic absorption band at 3320, 2960, 1710, 1530, 1220, 1110 and 777 cm−1 , which represents (N–H) , (C–H) , (C–O) , (C–C) ,(C–C) , (C–O) , (C–H) on substituted benzene, respectively (Jiang, Yuan, Li & Chow, 2006). Also the characteristic bands of dextran were located at 759 and 849 cm−1 (CH bend); 1272 cm−1 (C–O stretch); 1435 cm−1 (CH3 bend); 2922 cm−1 (CH stretch); 3117 cm−1 (CH3 stretch); 3335 cm−1 (OH stretch, end group), 1140, 1122 and 1033 cm−1 (saccharide structure) [28]. It was observed that all the characteristic peaks of PU and dextran were visible in PU-dextran nanofibers and some peaks were being overlapped. The characteristic absorption bands of Estradiol were confirmed and are explained as follows [29]. The FT-IR spectrum of Estradiol indicates two broad intense O–H bands at 3446 and 3240 cm−1 . In the IR spectrum of -estradiol, the band at 1359 cm−1 is assigned to deformation vibration of the phenol OH group. The IR band at 1283 cm−1 in the absorption spectrum of -estradiol is assigned to the ıOH vibration. The presence of the polar C–O bond in phenols leads to occurrence of the intense absorption band at 1230–1140 cm−1 . The symmetric deformations of methyl group were observed in a narrow range of 1385–1370 cm−1 . Our studies showed them around 1381–1379 cm−1 in Estradiol. The absorption band at 1218 cm−1 observed in the Estradiol IR spectrum have been assigned to vibrations of the CH2 -groups. Most of the peaks of PU, dextran and Estradiol have been overlapped in the Estradiol loaded PU-dextran nanofibers due to the relative similarity and shifting. So the blending of Estradiol and dextran with PU has been confirmed with FTIR. The contact angle measurement details of composite nanofibers were shown in Fig. 3. As expected pristine PU nanofiber showed a hydrophobic nature (Fig. 3A) with contact angle around 126.1 ± 4◦ .
Due to the addition of dextran the contact angle has been modified to 80.9 ± 2◦ , which shows the hydrophilic behaviour of composite nanofibers. The dextran played a crucial role in converting the hydrophobic mat to a hydrophilic mat (Fig. 3B). The Estradiol loaded PU-dextran mat showed a contact angle of 82.9 ± 2◦ , attributed to the changed manner of the composite nanofiber towards hydrophilic nature (Fig. 3A). This result demonstrates that addition of dextran to the composite mat resulted in a hydrophilic nature and the less amount of Estradiol did not affect much towards the wettability of the electrospun nanofibers. A more hydrophilic nanofiber surface will be helpful for the cell attachment which is very essential in wound healing. An optimized amount of hydrophilic bioactive material such as dextran is very crucial in improving hydrophilicity of the nanofibrous mat. The hydrophilicity of the composite mat can enhance the cytocompatibility and so it is important and should be taken in order to keep good surface wettability and fiber stability.
Fig. 5. In vitro release profile from Estradiol loaded PU-dextran composite nanofibrous mat.
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Fig. 6. MTT cell growth measurement assay. The viability of control cells was set at 100%, and viability relative to the control was expressed.
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3.2. Blood clotting ability of composite nanofibers In order to evaluate the hemostatic potential of nanofiber scaffolds, whole-blood clotting study was conducted. After the addition of blood, the nanofiber mat was fully covered with the blood. The composite mat showed a perfect blood clotting ability after 15 min incubation. Absorbance value of the resulted hemoglobin solution showed a higher absorbance value, which indicated a slower
clotting rate [25]. The measurements showed that Estradiol loaded PU-dextran composite nanoscaffold showed enhanced blood clotting ability in comparison with pristine PU as given in Fig. 4A. The result was further confirmed by the platelet activation analysis by SEM. Pristine PU did not activate the platelets while Estradiol loaded PU-dextran composite nanoscaffold promoted platelet activation as seen in Fig. 4B. The SEM images showed that the platelets were spread all over the Estradiol loaded PU-dextran composite
Fig. 7. Photographs of the in vivo wound healing study. Note the extent of wound closure in the wounds treated with Estradiol loaded PU-dextran composite mat compared to bare wound.
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observed for first 24 h due to the diffusion controlled release of the drug and the higher water adsorption of the electrospun nanofibers. The higher surface area of the nanofibers may also cause more drug molecules to be diffused from the nanofibers to the surrounding medium. Therefore the drug molecule showed a more quick diffusion from the fiber into the aqueous medium. After 24 h, the profile reached the equilibrium and the rates became slower. The constant release rate and longer drug release sustainability be crucial for proper healing. 3.4. Invitro cytocompatibility study
Fig. 8. Evaluation of the wound area closure.
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nanofibrous mat surface. More platelet activation was observed in Estradiol loaded PU-dextran composite nanoscaffold (Fig. 4B). The uniform distribution of platelets along the composite mats indicate the perfect platelet adhesion and hence the enhanced clotting ability.
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3.3. Drug release study
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In vitro release profiles of Estradiol from the Estradiol loaded PU-dextran composite nanofibrous mats was shown in Fig. 5. The release rate of PTX in phosphate buffered saline (PBS) solutions was examined by immersing Estradiol loaded PU-dextran composite fiber mats into solutions with pH of 7.4. A burst release was
Cytocompatibility is a most important parameter for a wound dressing material. The cell viability of fibroblasts on neat PU, Estradiol loaded PU and Estradiol loaded PU-dextran composite nanofibrous mats were evaluated by MTT assay on days 2, 4 and 6 (Fig. 6). Estradiol loaded PU-dextran composite nanofibrous scaffolds were more viable for the growth of fibroblasts as compared to PU scaffolds, attaining a significant level of increase in cell proliferation after 4 days and 6 days of culture. The presence of dextran and estradiol enhanced the bioactivity and cell affinity of the composite scaffolds as seen in Fig. 6. The biocompatibility of dextran has been well studied. Moreover dextran is subject to enzymatic degradation by dextranase, which exists in human tissues. Higher number of cell attachment and growth were observed on the Estradiol loaded PU-dextran nanofibers as the composite nanofibers become more hydrophilic and bioactive. Results clearly indicate that the presence of dextran and estradiol highly accelerates the proliferation of fibroblasts on composite scaffolds. The fibroblasts must bind to the Estradiol loaded PU-dextran nanofibers scaffold, so the quality of extracellular matrix is also crucial for re-epithelialization, which is satisfied by the morphology of nanofiber matrix. Moreover the hormone estrogen (-estradiol) has a key role in cell
Fig. 9. Photomicrographs of hematoxilin and eosin (H&E)-stained (A and B) bare wound, (C and D) Estradiol loaded PU-dextran composite mat treated wounds.
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proliferation and differentiation [30] and hence the cell growth is more in Estradiol loaded PU nanofibers compared to pristine PU. But the Estradiol loaded PU-dextran nanofibers showed much enhanced cell growth due to the combined effect of dextran and estradiol. An ECM mimicking nanofiber structure along with the rough surface of the nanofibrous membrane [31] and enhanced cytocompatibility of dextran supported the proliferation and enhanced regeneration of cells, which is very essential in wound healing.
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3.5. Evaluation of in vivo wound healing
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In vivo study conducted in wistar rats proved the enhanced wound healing ability of the prepared Estradiol loaded PU-dextran composite mat. Fig. 7 shows photographs of the in vivo wound healing study. Estradiol loaded PU-dextran composite mat showed excellent healing after one and two weeks, compared to the bare wound. The extent of wound closure was evaluated and observed that after three weeks, the wounds treated with the Estradiol loaded PU-dextran composite mat achieved significant closure to ∼95%, compared to the bare wounds, which showed ∼70% wound closure (Fig. 8). According to the literature, the delayed healing can be reversed by topical application of -estradiol treatment [32]. The -Estradiol is a keratinocyte mitogen, promoting re-epithelialization after in vivo wounding [2,33] and stimulating keratinocyte proliferation in vitro [34]. It directly modulates endothelial cells, increasing adhesion to a range of extracellular matrix substrates and promoting endothelial cell migration in vitro [35]. The controlled release of Estradiol from the nanofibers and the presence of dextran enhanced the healing rate compared to the bare wound, which resulted in altered wound inflammation, re-epithelialization, and contraction. Furthermore, qualitative histomorphology in routinely stained hematoxilin and eosin sections indicated densely packed keratinocytes in the epidermis on the wounds treated with Estradiol loaded PU-dextran composite mat, compared to the bare wound (Fig. 9). Furthermore, the presence of Estradiol enhanced the rate of healing in wounds treated with composite mat, which was evident from the H&E-stained images. Estradiol receptors are known to exist in cells involved in the healing process such as macrophages, fibroblasts, endothelium, and in the cytoplasm and/or nucleus of various cells in the dermis [36], and are reported to indirectly influence the proliferative phase of the wound healing by increasing the production of growth factors [37]. All these studies indicated that this advanced Estradiol loaded PU-dextran composite mats can be successfully applied as the potential wound dressing material for post-menopausal stage.
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4. Conclusions
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In this preliminary study, we clearly demonstrated the importance of -Estradiol in cutaneous wound healing. Continuous uniform nanofibers of Estradiol loaded PU-dextran was electrospun and successfully applied as a wound dressing material. Using a validated in vivo model, we have proven that incisional wounds take far longer time to heal in the absence of Estradiol loaded PUdextran mat. However, knowledge about the effects of -Estradiol on wound cell-specific function is very limited, and most importantly, there exist a scarcity of literature addressing the issue. From our study, we witnessed the significance of -Estradiol in wound healing, and this understanding should be translated in the near future into improved treatments for post-menopausal wound dressing.
Acknowledgement This research was supported by grants from the Basic Science Research Program through the National Research Foundation of Q3 Korea (NRF) funded by the Ministry of Education, Science and Technology (Project no. 2013-012911 and 2013R1A2A2A04015484). References [1] G.S. Ashcroft, K.J. Lei, W.W. Jin, G. Longenecker, A.B. Kulkarni, T. GreenwellWild, H. Hale-Donze, G. McGrady, X.Y. Song, S.M. Wahl, Nat. Med. 6 (2000) 1147–1153. [2] G.S. Ashcroft, J. Dodsworth, E. vanBoxtel, R.W. Tarnuzzer, M.A. Horan, G.S. Schultz, M.W.J. Ferguson, Nat. Med. 3 (1997) 1209–1215. [3] J.B. Schmidt, M. Binder, W. Macheiner, C. Kainz, G. Gitsch, C. Bieglmayer, Maturitas 20 (1994) 25–30. [4] T. Mogi, N. Ohtake, M. Yoshida, R. Chimura, Y. Kamaga, S. Ando, T. Tsukamoto, T. Nakajima, H. Uenodan, M. Otsuka, Y. Matsuda, H. Ohshima, K. Makino, Colloid Surf. B 17 (2000) 153–165. [5] G.S. Roth, S.M. Harman, S.I. Lamberg, P Soc Exp Biol Med 166 (1981) 17–23. [6] E. Pirila, M. Parikka, N.S. Ramamurthy, P. Maisi, S. McClain, A. Kucine, T. Tervahartiala, K. Prikk, L.M. Golub, T. Salo, V. Sorsa, Wound Repair Regen 10 (2002) 38–51. [7] L.C. Chang, C.J. Cheng, T.H. Tsai, C.W. Liu, T.R. Tsai, J Biomed Nanotechnol 9 (2013) 1724–1735. [8] J. Knauss, D.J. Margolis, W. Bilker, Lancet 359 (2002) 675–677. [9] A. Berard, S.R. Kahn, L. Abenhaim, Pharmacoepidem Drug Safety 10 (2001) 245–251. [10] G.S. Ashcroft, T. Greenwell-Wild, M.A. Horan, S.M. Wahl, M.W.J. Ferguson, Am J Pathol 155 (1999) 1137–1146. [11] E.D. Son, J.Y. Lee, S. Lee, M.S. Kim, B.G. Lee, I.S. Chang, J.H. Chung, J Invest Dermatol 124 (2005) 1149–1161. [12] A. Anitha, V.G. Deepagan, V.V.D. Rani, D. Menon, S.V. Nair, R. Jayakumar, Carbohyd Polym 84 (2011) 1158–1164. [13] W. Ritcharoen, Y. Thaiying, Y. Saejeng, I. Jangchud, R. Rangkupan, C. Meechaisue, P. Supaphol, Cellulose 15 (2008) 435–444. [14] M.F.A. Cutiongco, M.H. Tan, M.Y.K. Ng, C. Le Visage, E.K.F. Yim, Acta Biomaterialia 10 (2014) 4410–4418. [15] Z.Y. Li, H.W. Yang, N.Y. He, W.B. Liang, C. Ma, M.A.A. Shah, Y.J. Tang, S. Li, H.N. Liu, H.S. Jiang, Y.F. Guo, J Biomed Nanotechnol 9 (2013) 1945–1949. [16] G.M. Sun, X.J. Zhang, Y.I. Shen, R. Sebastian, L.E. Dickinson, K. Fox-Talbot, M. Reinblatt, C. Steenbergen, J.W. Harmon, S. Gerecht, Proc Natl Acad Sci USA, 108 (2011) 20976–20981. [17] A.R. Unnithan, N.A.M. Barakat, P.B.T. Pichiah, G. Gnanasekaran, R. Nirmala, Y.S. Cha, C.H. Jung, M. El-Newehy, H.Y. Kim, Carbohyd Polym 90 (2012) 1786–1793. [18] H.L. Jiang, D.F. Fang, B.S. Hsiao, B. Chu, W.L. Chen, Biomacromolecules 5 (2004) 326–333. [19] P.T.S. Kumar, V.K. Lakshmanan, R. Biswas, S.V. Nair, R. Jayakumar, J Biomed Nanotechnol 8 (2012) 891–900. [20] J.J. Elsner, I. Berdicevsky, M. Zilberman, Acta Biomaterialia 7 (2011) 325–336. [21] M. Jaiswal, A. Gupta, A.K. Agrawal, M. Jassal, A.K. Dinda, V. Koul, J Biomed Nanotechnol 9 (2013) 1495–1508. [22] A. Hadjizadeh, A. Ajji, M. Jolicoeur, B. Liberelle, G. De Crescenzo, J Biomed Nanotechnol 9 (2013) 1195–1209. [23] O.J. Lee, H.W. Ju, J.H. Kim, J.M. Lee, C.S. Ki, J.H. Kim, B.M. Moon, H.J. Park, F.A. Sheikh, C.H. Park, J Biomed Nanotechnol 10 (2014) 1294–1303. [24] M. Mohiti-Asli, B. Pourdeyhimi, E.G. Loboa, Acta Biomaterialia 10 (2014) 2096–2104. [25] S.Y. Ong, J. Wu, S.M. Moochhala, M.H. Tan, J. Lu, Biomaterials 29 (2008) 4323–4332. [26] C.J. Thompson, G.G. Chase, A.L. Yarin, D.H. Reneker, Polymer 48 (2007) 6913–6922. [27] M. Zamani, M. Morshed, J. Varshosaz, M. Jannesari, Eur J Pharm Biopharm 75 (2010) 179–185. [28] J. Zhi, K.J. Yuan, S.F. Li, W.K. Chow, Spectrosc Spect Anal 26 (2006) 624–628. [29] G. Cakmak, I. Togan, F. Severcan, Aquat Toxicol 77 (2006) 53–63. [30] I. Meneses-Morales, A.C. Tecalco-Cruz, T. Barrios-Garcia, V. Gomez-Romero, I. Trujillo-Gonzalez, S. Reyes-Carmona, E. Garcia-Zepeda, E. Mendez-Enriquez, R. Cervantes-Roldan, V. Perez-Sanchez, F. Recillas-Targa, A. Mohar-Betancourt, A. Leon-Del-Rio, Nucleic Acids Res 42 (2014) 6885–6900. [31] T. Lou, M. Leung, X.J. Wang, J.Y.F. Chang, C.T. Tsao, J.G.C. Sham, D. Edmondson, M.Q. Zhang, J Biomed Nanotechnol 10 (2014) 1105–1113. [32] K. Tomoda, A. Watanabe, K. Suzuki, T. Inagi, H. Terada, K. Makino, Colloid Surf B 97 (2012) 84–89. [33] M.J. Hardman, A. Waite, L. Zeef, M. Burow, T. Nakayama, G.S. Ashcroft, Am J Pathol 167 (2005) 1561–1574. [34] N. Kanda, S. Watanabe, J Invest Dermatol 123 (2004) 319–328. [35] D.E. Morales, K.A. Mcgowan, D.S. Grant, S. Maheshwari, D. Bhartiya, M.C. Cid, H.K. Kleinman, H.W. Schnaper, Circulation 91 (1995) 755–763. [36] M. Calvin, Maturitas 34 (2000) 195–210. [37] D.M. Oh, T.J. Phillips, Wounds 18 (2006) 8–18.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Electrospun polyurethane-dextran nanofiber mats loaded with Estradiol for post-menopausal wound dressing
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Afeesh Rajan Unnithan a,1 , Arathyram Ramachandra Kurup Sasikala a,1 , Priya Murugesan b , Malarvizhi Gurusamy b , Dongmei Wu b , Chan Hee Park c,∗ , Cheol Sang Kim a,c,∗ a
Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju 561-756, Republic of Korea Department of BIN Fusion Technology, Chonbuk National University, Jeonju 561-756, Republic of Korea c Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea
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Article history: Received 11 November 2014 Received in revised form 15 January 2015 Accepted 15 February 2015 Available online xxx
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Keywords: Wound dressing Estradiol Dextran
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1. Introduction
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Post-menopausal wound care management is a substantial burden on health services, since there are an increased number of elderly populations linked with age-related delayed wound healing. The controlled estrogen replacement can accelerate healing of acute cutaneous wounds, linked to its potent anti-inflammatory activity. The electrospinning technique can be used to introduce the desired therapeutic agents to the nanofiber matrix. So here we introduce a new material for wound tissue dressing, in which a polyurethane–dextran composite nanofibrous wound dressing material loaded with -estradiol was obtained through electrospinning. Dextran can promote neovascularization and skin regeneration in chronic wounds. This study involves the characterization of these nanofibers and analysis of cell growth and proliferation to determine the efficiency of tissue regeneration on these biocomposite polymer nanofibrous scaffolds and to study the possibility of using it as a potential wound dressing material in the in vivo models. © 2015 Published by Elsevier B.V.
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There has been a rapid growth in the magnitude of elderly population mainly due to the recent advancements in the healthcare management and due to the various epidemiological factors. Along with this scenario, there has an increase in morbidity associated with age-related delayed wound healing. Generally the wounds heal through a multifaceted series of overlapping tightly controlled stages. As we became older, these complex processes were disturbed and repair efficiency was reduced. According to the previous reports, in elderly women the estrogen hormone level in the body quickly reduced after the menopause and this might contributed towards to delay in wound healing. Estrogens possess a considerable role in wound repair that can reverse the reduced wound healing process. Those who were at the post-menopausal condition
∗ Corresponding authors. Tel.: +82 63 270 4284; fax: +82 63 270 2460. E-mail addresses: [email protected] (A.R. Unnithan), [email protected] (C.H. Park), [email protected] (C.S. Kim). 1 The two authors equally contributed to this work.
possess an increased risk of developing an excessive inflammation condition, mainly due to a number of deteriorating pathological properties [1,2]. During the menopause condition, considerable changes to the normal female skin have been occurred. The profound changes were the decrease in dermal collagen and reduced skin thickness and both of which can be overturned by topical estrogen application [3,4]. According to the studies, there exist a strong correlation between the systemic hormone levels and wound healing. It has been reported that the women at the post-menopausal stage taking systemic hormone replacement therapy heal wounds more effectively than the control ones [5]. The cutaneous wound healing is a complicated process linked with an initial inflammatory response, restoration of the epithelial barrier and matrix deposition. The improved deposition of cell matrix, prompt re-epithelialization process, and the potential anti-inflammatory activity can be achieved by the systemic estrogen replacement and which can contribute to accelerate the healing of acute cutaneous wounds [6,7]. In this concern, hormone replacement therapy (HRT) can be a good substitute to prevent the development of chronic wounds in post-menopausal women [8,9]. According to the studies, the exogenous treatment of estrogen can
http://dx.doi.org/10.1016/j.ijbiomac.2015.02.044 0141-8130/© 2015 Published by Elsevier B.V.
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reverses the delayed cutaneous wound healing by inhibiting unnecessary neutrophil recruitment, endorsing re-epithelialization and increasing collagen deposition [10,11]. Dextran is a biocompatible, biodegradable, non-immunogenic and non-antigenic biopolymer and hence it has been widely applied in various biomedical applications, such as drug delivery applications [12], as scaffolds for tissue engineering applications [13,14] and as molecular arms [15], etc. Scaffold materials made up of dextran were soft and flexible and hence it improves the handling efficiency for the management of wound treatment [16]. Moreover the dextran-based hydrogel materials have been used as scaffolds for neovascularization and re-epithelialization in wound tissue engineering. In the preparation aspect, dextran is soluble in both water and organic solvents make it as a fine material for bioapplications. So the dextran can be directly blended with suitable polymers such as PU to prepare composite nanofibrous membranes by electrospinning [17]. The physical and biological properties of dextran can be thus manipulated according to the application and hence better biocompatibility can be achieved [18]. The precise adherence to the wound location and good exudate absorbance should be the promising factors of a perfect wound dressing material. They should also retain appropriate moisture along with easy practice and removal [19,20]. Electrospun nanofibers were found to be very effective to be used as the wound dressing material mainly due to its architectural superiority. The electrospun nanofiber can mimic the extra cellular matrix (ECM) environment which will help the host cells to grow and create a new natural cellular matrix [21,22]. Most importantly the preferred therapeutic materials can be introduced to the nanofiber matrix using the electrospinning process [17,23,24], that can seriously affect the wound healing. We introduce a new PU-dextran based nanofibrous material for potential wound dressing utilizing -estradiol, the most bioactive endogenous estrogen. In this preliminary work, a composite nanofibrous wound dressing material loaded with -estradiol was fabricated through electrospinning. The continuous systemic estrogen release from the Estradiol loaded nanofibrous mat at the wound area can accelerate healing of acute cutaneous wounds, linked to its potent anti-inflammatory activity. This preliminary study involves the fabrication, characterization of these composite nanofibers and analysis of cell growth and proliferation to determine the efficiency of tissue regeneration on these biocomposite polymer nanofibrous scaffolds and to study the possibility of using it as an effective wound dressing material in the in vivo models.
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2. Experimental procedure
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2.1. Materials and methods
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2.2. Characterizations The morphology of the electrospun composite mats was observed by using field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan). The bonding configurations of the samples were characterized by means of Fourier-transform infrared (FT-IR) spectroscopy. Contact angle (wettability) was measured by using the deionized water contact angle measurement system, using contact angle meter (Digidrop, GBX, France). Deionized water was automatically dropped (drop diameter 6 m) onto the mat. 2.3. Platelet activation study and whole blood clotting assay The blood clotting studies were done based on reported literature [25]. Blood was mixed with anticoagulant agent acid citrate dextrose at a ratio of 9:1. Later blood was added to each composite nanofiber mats and placed in a 25-mL plastic Petri dish, which was followed by the addition of 10 L of 0.2 M CaCl2 solutions to initiate blood clotting and PU mat was used as negative control. These mats were then incubated at 37 ◦ C for 15 min. 1 mL of distilled water was then added drop wise without disturbing the clot. Subsequently, 1 mL of solution was taken from the dishes and was centrifuged at 1000 rpm for 1 min. The supernatant was collected for each sample and kept at 37 ◦ C for 1 h. Two hundred microliters (200 L) of this solution was transferred to a 96-well plate. The optical density was measured at 530 nm using a plate reader (Dynex Technologies, USA). For platelet activation studies blood mixed with anticoagulant was centrifuged at 2500 rpm for 10 min. The supernatant rich in platelet plasma was collected and added directly on to the nanofibers. Samples were fixed with glutaraldehyde and then washed with PBS. Later these samples were dehydrated using gradient alcohol treatment and then viewed under SEM. 2.4. In vitro drug release study Estradiol loaded composite nanofibers (2 × 2 cm) were immersed into eppendorf tubes containing 3 mL of phosphate buffer solution (pH 7.4) at 37 ◦ C with continuous shaking at 120 rpm. A fixed volume of the release medium was withdrawn at continuous intervals and replenished with the same volume of the fresh PBS solution. All samples were prepared in triplicate and analyzed using UV–vis. 2.5. Cytocompatibility study
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Polyurethane, 10 wt% (PU, Estane® Skythane® X595A-11, Lubrizol) was prepared by dissolving in solvent mixture of Dimethyl sulfoxide (DMSO, Sigma Aldrich, Korea) and Tetrahydrofuran (THF, Sigma Aldrich, Korea) (50/50, wt:wt%). 5 wt% The Dextran (from Leconostoc mesenteroides, average Mw = 8500–11500, Sigma Aldrich) has been added to those respective solutions along with 2 wt% -estradiol (Sigma Aldrich, Germany). The obtained solutions were placed in a plastic syringe tube and fed through a metal capillary (nozzle) with a diameter di = 0.21 mm (21 G) attached to a 1-D robot-system that moves laterally and is controlled by the LabVIEW 9.0 software program (National Instrument). The feeding rate was maintained at 0.5 mL/h via a controllable syringe pump. Electrospinning was carried out at a voltage of 18 kV and working distance of 15 cm at room temperature. After electrospinning the mats were carefully removed and kept at overnight vacuum drying to remove the residual solvents.
The viability of cultured 3T3-L1 fibroblasts (preadipocytes, Korean Cell Line Bank, Korea) was monitored on the third and sixth day of culture using the colorimetric MTT assay (Sigma, USA). The nanofiber scaffolds were washed twice with PBS and were then treated with approximately 50 L of the MTT solution (DMEM); the scaffolds, after mixing of the contents by side-tapping, were incubated at 37 ◦ C for 2 h. The nanofiber scaffolds containing MTT-cell mixtures were gently rocked to deposit the cells. The supernatant of the MTT solution was pipette out and then acid–isopropanol (95 mL isopropanol with 5 mL 3 N HCl) was added to the colored cell deposit. After gently mixing the acid–alcohol-treated scaffolds, it was then allowed to react for 5 min. 100 L of the purple–blue colored supernatant that contained the solubilized formazan in each sample was added to a well in a 96-well plate for analysis at 580 nm in an ELISA reader. The cell viability was obtained by comparing the absorbance of cells cultured on the nanofibers scaffold to that of control well containing cells. The results were expressed as the mean ± standard error of the mean. The data were analyzed via the Student’s t test and repeated measures of analyses of variance
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Fig. 1. A SEM images of electrospun (A) PU(10 wt%):dextran (10 wt%) mat, (B) PU(10 wt%):dextran (15 wt%) mat, (C) PU(10 wt%):dextran (2 wt%) mat and (D) PU(10 wt%):dextran (5 wt%) nanofiber mat. (B) FESEM images of electrospun (A) PU mat, (B) PU-Dextran mat and (C) Estradiol loaded PU-dextran mat.
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(ANOVA) test. A probability of less than 0.01 was considered to be statistically significant.
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In vivo animal study was approved by the Institutional Animal Ethical Committee, IACUC certification number CBU 2014-00062 at Chonbuk National University, Jeonju, South Korea. Wistar rats, weighing 200–250 g and 4–6 weeks of age, were used in this study.
The rats were divided into two groups and each group contains three rats (n = 3); rats were allowed to take normal rat feed and water without restriction. On the day of wounding, the rats were anaesthetized by intraperitoneal injection of 80 mg/kg ketamine and 8 mg/kg xylazine. The dorsal area of the rats depilated and the operative area of skin cleaned with alcohol. A partial thickness skin wound of 1.5 cm square was prepared by excising the dorsum of the rat using surgical scissors and forceps. The prepared wounds were then covered with the Estradiol loaded PU-dextran nanofibers
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Fig. 2. FTIR spectra of electrospun (A) -Estradiol, (B) pristine PU and (C) Estradiol loaded PU-dextran nanofibrous mat.
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and Rats with bare wound were kept covered using cotton gauze. After applying the dressing materials, the rats were housed individually in cages under normal room temperature. The dressing materials were changed at weeks 1, 2, and 3. During the changing of dressings the animals hairs cropped and photographs were taken and the wound area was measured using a transparent polyethylene sheet. The sheet was kept on top of the wound and area was marked using a marker pen. After Week 3, the skin wound tissue of the rat was excised, fixed with 10% formalin, and stained with a hematoxylin–eosin (H&E) reagent for histological observations.
3. Results and discussion
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3.1. Physical characterizations of composite nanofibers
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Fig. 1A (A–D) shows the SEM images of electrospun composite nanofibers with different concentrations of PU and dextran. As shown in Fig. 1A ((A)–(C)) PU (10 wt%):dextran (10 wt%) mat, PU(10 wt%):dextran (15 wt%) mat and PU(10 wt%):dextran (2 wt%) mat, respectively, showed beaded morphology. The formation of beaded fibers in may be possibly due to the change in viscosity of PU in blend polymer solution with dextran, then causing different stretching time from droplet to fiber during e-spinning. But Fig. 1A (D) showed promising fiber morphology with PU (10 wt%):Dextran (5 wt%). From these data it is clear that the concentration of PU and dextran in the final solution having a direct effect on the fiber morphology. Fig. 1B (A)–(C) shows the FESEM images of final study material, electrospun PU, PU-dextran and PU-dextran-Estradiol loaded composite nanofibers, respectively. It can be observed that these randomly oriented as-spun nanofibers exhibited bead-free, smooth surface with almost uniform diameters along their lengths. The diameters of these composite nanofibers were determined to be in the range of 500–600 nm. For PU electrospun mat (Fig. 1A), the fibers appear well-defined without any interconnection among the fibers. But the composite mats containing Estradiol and dextran showed some changes in fibrous morphology (Fig. 1C). It may be due to the combined effect of Estradiol and dextran with the PU solution. The addition of Estradiol above 2 wt% caused the drastic decrease in solution viscosity and hence the final concentration of Estradiol was set as 2 wt%. The addition of Estradiol can significantly decrease solution viscosity; however, it did not affect the electrospinning of the composite fibers. According to the reports, solution viscosity seriously influences the morphological structure and average size of resulting fibers [26]. Different concentrations of PU and dextran were electrospun and then decided (Fig. 1A) the final concentration of PU as 10 wt% and dextran as 5 wt%. The
Fig. 3. Water contact angles of electrospun (A) PU nanofibrous mat, (B) PU-dextran nanofibrous mat and (C) Estradiol loaded PU-dextran nanofibrous mat.
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Fig. 4. (A) Blood clotting efficiency of nanofibrous mats using OD values, SEM images of platelet activation of (B) Estradiol loaded PU-dextran composite nanofibrous mat (white dots indicate the platelet adhesion on composite mat) respectively.
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Estradiol and dextran can affect the viscosity of the polymeric solution and some studies reported that some materials can be confined between polymeric chains, and thus they can as a plasticizer [27]. The composite electrospun nanofiber mat produced in this study was desirably smooth and flexible and were successfully used for the desired studies. The changes of the functional groups that occur during the blending of Estradiol and dextran with PU were examined by FTIR spectroscopy. Fig. 2 explains the FTIR spectra of PU, PU-dextran and Estradiol loaded PU-dextran nanofibers. The FT-IR spectra of electrospun PU has characteristic absorption band at 3320, 2960, 1710, 1530, 1220, 1110 and 777 cm−1 , which represents (N–H) , (C–H) , (C–O) , (C–C) ,(C–C) , (C–O) , (C–H) on substituted benzene, respectively (Jiang, Yuan, Li & Chow, 2006). Also the characteristic bands of dextran were located at 759 and 849 cm−1 (CH bend); 1272 cm−1 (C–O stretch); 1435 cm−1 (CH3 bend); 2922 cm−1 (CH stretch); 3117 cm−1 (CH3 stretch); 3335 cm−1 (OH stretch, end group), 1140, 1122 and 1033 cm−1 (saccharide structure) [28]. It was observed that all the characteristic peaks of PU and dextran were visible in PU-dextran nanofibers and some peaks were being overlapped. The characteristic absorption bands of Estradiol were confirmed and are explained as follows [29]. The FT-IR spectrum of Estradiol indicates two broad intense O–H bands at 3446 and 3240 cm−1 . In the IR spectrum of -estradiol, the band at 1359 cm−1 is assigned to deformation vibration of the phenol OH group. The IR band at 1283 cm−1 in the absorption spectrum of -estradiol is assigned to the ıOH vibration. The presence of the polar C–O bond in phenols leads to occurrence of the intense absorption band at 1230–1140 cm−1 . The symmetric deformations of methyl group were observed in a narrow range of 1385–1370 cm−1 . Our studies showed them around 1381–1379 cm−1 in Estradiol. The absorption band at 1218 cm−1 observed in the Estradiol IR spectrum have been assigned to vibrations of the CH2 -groups. Most of the peaks of PU, dextran and Estradiol have been overlapped in the Estradiol loaded PU-dextran nanofibers due to the relative similarity and shifting. So the blending of Estradiol and dextran with PU has been confirmed with FTIR. The contact angle measurement details of composite nanofibers were shown in Fig. 3. As expected pristine PU nanofiber showed a hydrophobic nature (Fig. 3A) with contact angle around 126.1 ± 4◦ .
Due to the addition of dextran the contact angle has been modified to 80.9 ± 2◦ , which shows the hydrophilic behaviour of composite nanofibers. The dextran played a crucial role in converting the hydrophobic mat to a hydrophilic mat (Fig. 3B). The Estradiol loaded PU-dextran mat showed a contact angle of 82.9 ± 2◦ , attributed to the changed manner of the composite nanofiber towards hydrophilic nature (Fig. 3A). This result demonstrates that addition of dextran to the composite mat resulted in a hydrophilic nature and the less amount of Estradiol did not affect much towards the wettability of the electrospun nanofibers. A more hydrophilic nanofiber surface will be helpful for the cell attachment which is very essential in wound healing. An optimized amount of hydrophilic bioactive material such as dextran is very crucial in improving hydrophilicity of the nanofibrous mat. The hydrophilicity of the composite mat can enhance the cytocompatibility and so it is important and should be taken in order to keep good surface wettability and fiber stability.
Fig. 5. In vitro release profile from Estradiol loaded PU-dextran composite nanofibrous mat.
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Fig. 6. MTT cell growth measurement assay. The viability of control cells was set at 100%, and viability relative to the control was expressed.
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3.2. Blood clotting ability of composite nanofibers In order to evaluate the hemostatic potential of nanofiber scaffolds, whole-blood clotting study was conducted. After the addition of blood, the nanofiber mat was fully covered with the blood. The composite mat showed a perfect blood clotting ability after 15 min incubation. Absorbance value of the resulted hemoglobin solution showed a higher absorbance value, which indicated a slower
clotting rate [25]. The measurements showed that Estradiol loaded PU-dextran composite nanoscaffold showed enhanced blood clotting ability in comparison with pristine PU as given in Fig. 4A. The result was further confirmed by the platelet activation analysis by SEM. Pristine PU did not activate the platelets while Estradiol loaded PU-dextran composite nanoscaffold promoted platelet activation as seen in Fig. 4B. The SEM images showed that the platelets were spread all over the Estradiol loaded PU-dextran composite
Fig. 7. Photographs of the in vivo wound healing study. Note the extent of wound closure in the wounds treated with Estradiol loaded PU-dextran composite mat compared to bare wound.
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observed for first 24 h due to the diffusion controlled release of the drug and the higher water adsorption of the electrospun nanofibers. The higher surface area of the nanofibers may also cause more drug molecules to be diffused from the nanofibers to the surrounding medium. Therefore the drug molecule showed a more quick diffusion from the fiber into the aqueous medium. After 24 h, the profile reached the equilibrium and the rates became slower. The constant release rate and longer drug release sustainability be crucial for proper healing. 3.4. Invitro cytocompatibility study
Fig. 8. Evaluation of the wound area closure.
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nanofibrous mat surface. More platelet activation was observed in Estradiol loaded PU-dextran composite nanoscaffold (Fig. 4B). The uniform distribution of platelets along the composite mats indicate the perfect platelet adhesion and hence the enhanced clotting ability.
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In vitro release profiles of Estradiol from the Estradiol loaded PU-dextran composite nanofibrous mats was shown in Fig. 5. The release rate of PTX in phosphate buffered saline (PBS) solutions was examined by immersing Estradiol loaded PU-dextran composite fiber mats into solutions with pH of 7.4. A burst release was
Cytocompatibility is a most important parameter for a wound dressing material. The cell viability of fibroblasts on neat PU, Estradiol loaded PU and Estradiol loaded PU-dextran composite nanofibrous mats were evaluated by MTT assay on days 2, 4 and 6 (Fig. 6). Estradiol loaded PU-dextran composite nanofibrous scaffolds were more viable for the growth of fibroblasts as compared to PU scaffolds, attaining a significant level of increase in cell proliferation after 4 days and 6 days of culture. The presence of dextran and estradiol enhanced the bioactivity and cell affinity of the composite scaffolds as seen in Fig. 6. The biocompatibility of dextran has been well studied. Moreover dextran is subject to enzymatic degradation by dextranase, which exists in human tissues. Higher number of cell attachment and growth were observed on the Estradiol loaded PU-dextran nanofibers as the composite nanofibers become more hydrophilic and bioactive. Results clearly indicate that the presence of dextran and estradiol highly accelerates the proliferation of fibroblasts on composite scaffolds. The fibroblasts must bind to the Estradiol loaded PU-dextran nanofibers scaffold, so the quality of extracellular matrix is also crucial for re-epithelialization, which is satisfied by the morphology of nanofiber matrix. Moreover the hormone estrogen (-estradiol) has a key role in cell
Fig. 9. Photomicrographs of hematoxilin and eosin (H&E)-stained (A and B) bare wound, (C and D) Estradiol loaded PU-dextran composite mat treated wounds.
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proliferation and differentiation [30] and hence the cell growth is more in Estradiol loaded PU nanofibers compared to pristine PU. But the Estradiol loaded PU-dextran nanofibers showed much enhanced cell growth due to the combined effect of dextran and estradiol. An ECM mimicking nanofiber structure along with the rough surface of the nanofibrous membrane [31] and enhanced cytocompatibility of dextran supported the proliferation and enhanced regeneration of cells, which is very essential in wound healing.
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In vivo study conducted in wistar rats proved the enhanced wound healing ability of the prepared Estradiol loaded PU-dextran composite mat. Fig. 7 shows photographs of the in vivo wound healing study. Estradiol loaded PU-dextran composite mat showed excellent healing after one and two weeks, compared to the bare wound. The extent of wound closure was evaluated and observed that after three weeks, the wounds treated with the Estradiol loaded PU-dextran composite mat achieved significant closure to ∼95%, compared to the bare wounds, which showed ∼70% wound closure (Fig. 8). According to the literature, the delayed healing can be reversed by topical application of -estradiol treatment [32]. The -Estradiol is a keratinocyte mitogen, promoting re-epithelialization after in vivo wounding [2,33] and stimulating keratinocyte proliferation in vitro [34]. It directly modulates endothelial cells, increasing adhesion to a range of extracellular matrix substrates and promoting endothelial cell migration in vitro [35]. The controlled release of Estradiol from the nanofibers and the presence of dextran enhanced the healing rate compared to the bare wound, which resulted in altered wound inflammation, re-epithelialization, and contraction. Furthermore, qualitative histomorphology in routinely stained hematoxilin and eosin sections indicated densely packed keratinocytes in the epidermis on the wounds treated with Estradiol loaded PU-dextran composite mat, compared to the bare wound (Fig. 9). Furthermore, the presence of Estradiol enhanced the rate of healing in wounds treated with composite mat, which was evident from the H&E-stained images. Estradiol receptors are known to exist in cells involved in the healing process such as macrophages, fibroblasts, endothelium, and in the cytoplasm and/or nucleus of various cells in the dermis [36], and are reported to indirectly influence the proliferative phase of the wound healing by increasing the production of growth factors [37]. All these studies indicated that this advanced Estradiol loaded PU-dextran composite mats can be successfully applied as the potential wound dressing material for post-menopausal stage.
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4. Conclusions
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In this preliminary study, we clearly demonstrated the importance of -Estradiol in cutaneous wound healing. Continuous uniform nanofibers of Estradiol loaded PU-dextran was electrospun and successfully applied as a wound dressing material. Using a validated in vivo model, we have proven that incisional wounds take far longer time to heal in the absence of Estradiol loaded PUdextran mat. However, knowledge about the effects of -Estradiol on wound cell-specific function is very limited, and most importantly, there exist a scarcity of literature addressing the issue. From our study, we witnessed the significance of -Estradiol in wound healing, and this understanding should be translated in the near future into improved treatments for post-menopausal wound dressing.
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