Acta Biomaterialia xxx (2014) xxx–xxx

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Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes Shih-Hsien Chen a,1, Chih-Hao Chen a,b,1, Yi Teng Fong a, Jyh-Ping Chen a,c,⇑ a

Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 333, Taiwan, ROC Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kwei-San, Taoyuan 333, Taiwan, ROC c Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kwei-San, Taoyuan 333, Taiwan, ROC b

a r t i c l e

i n f o

Article history: Received 25 January 2014 Received in revised form 30 June 2014 Accepted 25 August 2014 Available online xxxx Keywords: Anti-adhesion Nanofibrous membranes Polycaprolactone Chitosan Surface grafting

a b s t r a c t As one of the common complications after tendon injury and subsequent surgery, peritendinous adhesions could be minimized by directly placing a physical barrier between the injured site and the surrounding tissue. With the aim of solving the shortcomings of current biodegradable anti-adhesion barrier membranes, we propose the use of an electrospun chitosan-grafted polycaprolactone (PCL-g-CS) nanofibrous membrane (NFM) to prevent peritendinous adhesions. After introducing carboxyl groups on the surface by oxygen plasma treatment, the polycaprolactone (PCL) NFM was covalently grafted with chitosan (CS) molecules, with carbodiimide as the coupling agent. Compared with PCL NFM, PCL-g-CS NFM showed a similar fiber diameter, permeation coefficient for bovine serum albumin, ultimate tensile strain, reduced pore diameter, lower water contact angle, increased water sorption and tensile strength. With its submicrometer pore diameter (0.6–0.9 lm), both NFMs could allow the diffusion of nutrients and waste while blocking fibroblast penetration to prevent adhesion formation after tendon surgery. Cell culture experiments verified that PCL-g-CS NFM can reduce fibroblast attachment while maintaining the biocompatibility of PCL NFM, implicating a synergistic anti-adhesion effect to raise the anti-adhesion efficacy. In vivo studies with a rabbit flexor digitorum profundus tendon surgery model confirmed that PCL-g-CS NFM effectively reduced peritendinous adhesion from gross observation, histology, joint flexion angle, gliding excursion and biomechanical evaluation. An injured tendon wrapped with PCL-g-CS NFM showed the same tensile strength as the naturally healed tendon, indicating that the anti-adhesion NFM will not compromise tendon healing. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The formation of adhesions usually accompanies tissue injury, cerebral ischemia, foreign body reaction, infection and bleeding [1]. Although adhesions is an important component of a physically inevitable wound healing process, poor post-operative adhesions can cause serious complications, including pain, functional obstruction and the demand for a second surgical intervention [2]. The repair of injuries to the flexor tendon is also complicated by fibrotic adhesions that compromise post-operative gliding and limit the range of joint flexion [3]. Adhesions are especially exacerbated in injuries involving the flexor digitorum profundus and flexor digitorum superficialis tendons in zone II of the hand [4]. ⇑ Corresponding author at: Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 333, Taiwan, ROC. Tel.: +886 3 2118800; fax: +886 3 2118668. E-mail address: [email protected] (J.-P. Chen). 1 These authors contributed equally to this work.

The biological mechanisms of adhesion formation following tendon repair or reconstruction are still poorly understood, but are thought to arise through intrinsic or extrinsic fibrosis, among other factors, including gap length and postoperative protocols [5]. U.S. Food and Drug Administration-approved anti-adhesion barriers such as Seprafilm™ and SurgiWrap™ are rarely applied to prevent peritendinous adhesions. During the mid-term healing phase of abdominal surgery, Seprafilm™, a dense film of sodium hyaluronate and carboxymethylcellulose, can be applied to the surface of the wound to keep it mechanically separated from the surrounding tissue [6]. Since the healing time after tendon surgery is relatively long (more than 6 weeks) compared with that after abdominal surgery, Seprafilm™ could not be an effective anti-adhesion barrier after tendon surgery considering its fast degradation time (within 1 week) in vivo. SurgiWrap™ is a dense membrane of polylactides [7]. However, the hydrophobic nature of SurgiWrap™ may lead to the hindered exchange of nutrients and waste at the surgical site, and could cause complications

http://dx.doi.org/10.1016/j.actbio.2014.08.030 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Chen S-H et al. Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.08.030

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during the healing phase. Besides, since both anti-adhesion barriers are dense membranes, which leads to difficulty in exchange of nutrients and waste, their applications at surgical sites with limited blood circulation (such as tendons) is questionable. The tendon sheath is a protective layer of membrane around a tendon. The functions of tendon sheath are (i) to secrete synovial fluid for tendon nutrition and lubrication; (ii) to keep synovial fluid around the tendon; and (iii) to maintain the alignment of flexor tendons. The membrane-like sheath structure consists of an outer fibrotic layer and an inner synovial layer. The fibrotic layer acts as an effective biological barrier, while the synovial layer secretes synovial fluid, an important source for tendon nutrition and a lubricant for tendon gliding [8,9]. Therefore, a biomimetic tendon sheath that could effectively reduce peritendinous adhesion formation should have dual functions, serving as a cell barrier layer without impeding the diffusion of nutrients and waste, allowing tendon gliding at the membrane–tendon interface. Polycaprolactone (PCL) offers several advantages when used in an anti-adhesion polymer membrane, such as stability under ambient conditions, low costs, ready availability in large quantities and good mechanical properties. However, the application of PCL in anti-adhesion products has been limited because of its stiffness and hydrophobicity [10,11]. Chitosan (CS) is a natural amino polysaccharide with many important biological characteristics; for example, it is biocompatible, biodegradable, hemostatic, non-toxic and antibacterial, and reduces fibroblast adhesions [12]. Many reports have shown poor cell adhesion on CS membranes [13,14]. This observation has prompted the investigation of this biomaterial with regard to preventing post-surgical adhesion formation. In vivo studies have demonstrated that N,O-carboxymethylchitosan is safe and efficacious as an anti-adhesion barrier [15]. A modified chitosan–dextran gel was used to prevent peritoneal adhesions in a rat model and significantly reduced the formation of intra-abdominal adhesions without adversely affecting wound healing [16]. Chitosan–alginate mixtures have also been patented for post-surgical adhesion barrier use [17]. Electrospinning (ES) is a versatile method for manufacturing nanofibrous membranes (NFMs) from natural and synthetic materials for biomedical applications [18]. An NFM is expected to offer effective tissue separation, due to its high porosity, good permeability and submicrometer pore size, thus preventing fibroblast penetration. Previously, anti-adhesion barrier properties have been reported for NFMs made from PCL, poly(lactide-co-glycolide), polylactide-poly(ethylene glycol) tri-block copolymer and a chitosan/ alginate blend [19–22]. However, the anti-adhesion barrier effects of these NFMs were only tested in abdominal anti-adhesion animal models and were not investigated with regard to preventing peritendinous adhesion. Recently, NFMs have been used as barrier films to prevent peritendinous adhesion. One study developed a bilayer NFM with a hyaluronic acid-loaded PCL inner membrane layer and a PCL outer membrane layer [23]. A second study used an ibuprofen-loaded poly(L-lactic acid)-polyethylene glycol diblock copolymer NFM [24]. An NFM made from a polymer blend or a synthesized copolymer is expected to augment the anti-adhesion function of an NFM made from a single component homopolymer. However, this approach will inevitably demand well-controlled ES conditions and complex copolymer synthesis steps. Post-modification, an NFM may be preferred since it offers the flexibility of choosing the most suitable molecule to modify a single-component NFM based on the membrane’s intended use, rather than searching for sophisticated ES conditions to prepare a composite NFM. Considering the different healing times after tendon or abdominal surgery, a facile preparation of peritendinous anti-adhesion barrier by ES is warranted. Given the good mechanical properties of PCL NFM and its intrinsic anti-adhesion property, we envision a PCL NFM membrane surface grafted with CS would improve its

peritendinous anti-adhesion performance. Thus, the first objective of this work is to prepare and characterize electrospun PCL and CSgrafted PCL NFMs. Secondly, with the combined advantages of a nanofibrous membrane vs. a dense film structure and CS grafting, we want to demonstrate the improved efficacy of PCL-g-CS over PCL NFM and Seprafilm™ in preventing peritendinous adhesions in vivo (Scheme 1). 2. Materials and methods 2.1. Materials PCL (molecular weight = 80,000 Da), CS (molecular weight = 1  105 Da, degree of deacetylation = 98%), 2-(N-morpholino) ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), antibiotics and trypsin–EDTA were purchased from Sigma-Aldrich. CellTiter 96Ò AQueous one solution was purchased from Promega. Dulbecco’s modified Eagle’s medium (DMEM; Sigma) and fetal bovine serum (FBS; HyClone) were used for cell culture. The actin cytoskeleton and focal adhesion staining kit (FAK100) was purchased from Millipore. 2.2. Preparation of NFM by electrospinning A 12% (w/v) PCL polymer solution was prepared in a methylene chloride and N,N0 -dimethylformamide (weight ratio = 4:1) mixed solvent system. To prepare the NFM by ES, we used a 23-gauge stainless-steel needle fitted to a glass syringe and a syringe pump (KD Scientific). A high-voltage power supply (Glassman) provided a 20 kV voltage difference between the needle tip and a grounded collector (aluminum foil). The electrostatic force drew the PCL solution horizontally from the needle tip to reach a collector that was placed 15 cm from the needle tip. The flow rate of the polymer solution was controlled at 1.0 ml h1. 2.3. Surface modification of electrospun NFM The fabricated PCL NFM was dried overnight in a vacuum oven. The modification of the membrane was carried out by a DC-pulsed plasma system equipped with a pulse controller delivering a square-wave 28 ms/7 ms output on/off time [25]. Alumina alloy electrodes coupled to a bipolar device could modify the NFM sample simultaneously. The NFM (9 cm  5 cm) was placed in the reactor 5 cm apart from each electrode. After being evacuated to 4 Pa, the reactor was purged with high-purity oxygen (99.9%) and maintained at a gas pressure of 26.7 Pa. The NFM was treated at a working voltage of 600 V for approximately 60 s. After plasma treatment, the NFM (8 cm  4 cm) was reacted with 10 mg ml1 CS in 200 ml of 0.1 M MES buffer (pH 5) containing 10 mg ml1 EDC and 10 mg ml1 NHS for 24 h to produce PCL-g-CS NFM. The PCLg-CS NFM was washed with copious distilled deionized (DDI) water, dried for 24 h in a vacuum oven and stored in a desiccator. The amount of CS grafted onto the PCL-g-CS NFM was determined directly using a dye-binding assay by first reacting a 1 cm  1 cm PCL-g-CS NFM with 10 ml of 50 g ml1 Remazol Brilliant Red 3BS (Everlight Chemical) solution at 80 °C for 30 min, then measuring the absorbance of the dye solution at 541 nm from six samples [26]. 2.4. Characterization of electrospun NFM The morphology of an NFM was observed with a scanning electron microscope (SEM; Hitachi S3000N). The diameters were calculated by measuring at least 100 random fibers from 10 images

Please cite this article in press as: Chen S-H et al. Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.08.030

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Scheme 1. Schematic illustration of preparation of PCL-g-CS NFM by surface grafting of CS to PCL and the use of an NFM as a biomimetic tendon sheath to prevent peritendinous adhesion.

using Image J software. The pore size of the NFM was measured by capillary flow porometry (PMI CFP-1100-AI, Porous Materials, Inc.) with a 21 dynes cm1 surface tension wetting agent to 30 psi for six samples [27]. Chemical analysis was carried out with Fourier transform infrared (FTIR) spectroscopy using a Horiba FT-730 spectrometer over a wave number range between 600 and 4000 cm1 with a resolution of 2 cm1 [28]. X-ray photoelectron spectroscopy (XPS) was performed with a Physical Electronics PHI 1600 ESCA spectrometer equipped with a multi-channel detector and a spherical capacitor analyzer [25]. A magnesium anode at 15 kV and 400 W generated the X-ray source and the pressure in the analysis chamber was controlled to be below 2  106 Pa. Thermogravimetric analysis (TGA) was conducted with a TGA 2050 analyzer (TA Instruments) from 25 to 800 °C at 10 °C min1 on five samples [28]. The uniaxial tensile property of the NFM was determined by a materials testing machine (Tinius Olsen H1KT) using a 10 N load cell at room temperature [27]. The test specimen (1 cm  5 cm  200 lm) was mounted vertically between two mechanical gripping units, leaving a 3 cm gauge length for mechanical loading. Load–deformation data were recorded at a deforming rate of 5 mm min1. The ultimate tensile strength, elongation-at-break (ultimate tensile strain) and Young’s modulus were obtained from the stress–strain curves. The values were averaged over five tests performed on each specimen. To determine the equilibrium water content of PCL and PCL-gCS NFMs, a pre-weighed sample (200 lm thickness and 14 mm diameter) was immersed in 20 ml of DDI water at 25 °C. The weight of the wet NFM was recorded periodically after taking the sample from the water and removing its surface water with a filter paper. The water sorption (%) was calculated as:

Water sorption ð%Þ ¼



 wt  w0  100 w0

ð1Þ

where wt is the weight of the wet membrane measured at time t (h) and w0 is the weight of the dry membrane measured at time 0. The average values of six samples are reported. The wettability of the surface was determined using a sessile drop contact angle system with a camera (First Ten Ångstroms). The contact angles were measured after 10 s at 25 °C and calculated using an automated fitting program (FTA-125). All reported contact angles were averaged over three measurements for three replicate NFM samples using DDI water. 2.5. Permeability of serum albumin in electrospun NFM The permeability coefficients of bovine serum albumin (molecular weight = 68,000 Da) in the NFM were measured in sideby-side permeation cells at 37 °C [29]. The NFM was mounted between two half-cells acting as the donor cell and the receptor cell. A solution of the permeating solute (bovine serum albumin) prepared in phosphate-buffered saline (PBS) was added to both

the donor cell and the receptor cell. The entire content of the receptor cell was removed at intervals and replaced with fresh PBS. The solute concentration in the receptor cell was determined at different times. Protein concentrations were determined by a colorimetric method at 595 nm with a protein assay kit from Bio-Rad. Permeability coefficients P (cm s1) were calculated by

  Ct A ¼ 2 Pt ln 1  2 V C0

ð2Þ

where Ct is the solute concentration in the receptor cell at time t, C0 is the initial solute concentration in the donor cell, V is the volume of each half-cell and A is the effective area of the membrane available for solute permeation. The average values of six measurements are reported. 2.6. In vitro cell culture and analysis A disk-shaped NFM (1.4 cm in diameter) was sterilized with 75% ethanol overnight, rinsed three times with PBS and placed in a 24-well culture plate (Nunc). Human foreskin fibroblast (Hs68) cells (ATCC CRL-11372) were obtained from the American Type Culture Collection (Arlington, VA). Cells at passages 4–6 were used. An aliquot of 0.1 ml cells (1  105 cells ml1) was seeded onto the surface of the pre-wetted membrane in each well. Each cell-seeded membrane was incubated at 37 °C for 4 h to allow for cell adhesion. The membrane was transferred to a new well, and 1 ml culture medium (DMEM supplemented with 10 vol.% FBS and 1 vol.% antibiotic–antimycotic) was added to each well. Cell culture was carried out at 37 °C in a humidified 5% CO2 incubator. The viable cell number after cultured for 24 h was determined by MTS assays using the CellTiter 96Ò AQueous one solution kit. The kit contains a novel tetrazolium salt, which interacts with metabolically active cells to produce a soluble formazan dye. Colorimetric measurements of the formazan product were performed at 492 nm using an ELISA plate reader (BioTek Synergy HT). Cells cultured on tissue-culture polystyrene (TCPS) surfaces were used for comparison. The average values of five measurements are reported. To investigate the underlying mechanism of anti-adhesion, Hs68 cells were used to study the effects of NFM on cell viability and migration. The cells were cultured in DMEM containing 2 or 10% FBS in a double chamber dish separated by a porous membrane (Transwell cell culture inserts, Corning). The cells were inoculated at 2.5  105 per well in the upper chamber (containing 2% FBS), with an NFM (200 lm thickness) placed in the cell insert. Runs without an NFM in the cell insert were used as controls. After 24 h of culture, cell migration was determined by MTS assays and direct observations of cells moving to the lower chamber (containing 10% FBS). This movement is caused by the difference in serum concentration [30]. The average values of six measurements are reported.

Please cite this article in press as: Chen S-H et al. Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.08.030

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The cytoskeletal organization was visualized using an actin cytoskeleton and focal adhesion staining kit following the manufacturer’s protocol. The cells were washed in PBS, fixed in a 4% paraformaldehyde solution for 20 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Cells were incubated with TRITC-conjugated phalloidin, mouse anti-vinculin primary antibody and FITC-AffiniPure goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) for 1 h, then with 40 , 6-diamidino-2-phenylindole (DAPI) for 5 min, to stain actin filaments, focal adhesion complexes and nuclei, respectively. The fluorescence-stained cells were visualized using a confocal laser scanning microscope (Zeiss LSM 510 Meta). The actin cytoskeleton and vinculin fluoresced red and green, respectively, while the nuclei were stained blue. 2.7. Animal study Ninety-six 3-month-old New Zealand white rabbits (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, ROC) were used in this study. All procedures of the animal study were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chang Gung University. A rabbit flexor digitorum profundus (FDP) tendon model was used since its flexor mechanism is analogous to a human digit. The skin of hindpaw was shaved and sterilized after general anesthesia with xylazine and ketamine. The zone-II flexor tendons of the second and third digits from both hindpaws of rabbits were released from the tendon sheath. The flexor digitorum superficialis tendons were first removed. The FDP tendons were then divided and cut with a scalpel, just distal to the chiasm and proximal to the vincula, and then repaired with modified Kessler core sutures using 5-0 braided polyester. The animals were then randomly divided into four groups, with 24 animals per group. For each of the experimental group, a 6  10 mm piece of PCL NFM, PCL-g-CS NFM or Seprafilm™ was wrapped around the tendon repair site, while PBS solution was concurrently applied to the tendon repair site in the control group. Four different treatments (control, Seprafilm™, PCL NFM and PCL-g-CS) were performed randomly on each of the four flexor tendons of each animal. The operated leg was immobilized in a cast after skin closure to limit the interphalangeal joints movements. At 2, 4 and 8 weeks, eight animals from each group were euthanized with lethal doses of pentobarbital (0.5 g kg1 body weight). The toes were transected at the metatarsophalangeal joints. The skin incisions were opened for gross evaluation after removing the skin sutures along the original incisions [31]. The digits were assigned randomly for evaluation of peritendinous adhesions by measuring the range of motions (flexion angle) of the distal interphalangeal (DIP) and proximal interphalangeal (PIP) joints, the tendon gliding excursions and the maximum forces needed to pull the tendons out of the tendon sheath, and by histological analysis. To assess the state of tendon healing, the breaking strength of the healed tendon was measured 2 weeks post-operation.

to a load transducer in a home-made range-of-motion apparatus. The metacarpophalangeal joint was fixed by inserting a wire longitudinally through the metacarpal and the proximal phalanx. The proximal, middle and distal phalanges were fixed to T-shaped pins containing two reflective markers. The prepared digit was then mounted on the range-of-motion apparatus by fixing the proximal wire to a non-slip clamp. A 50 g weight was attached to the extensor tendon to apply an initial tension and to ensure full extension of the digit. The actuator pulled the tendon slowly at a rate of 3 mm s1 and caused digital flexion (angular range of motion). The angle measured between the distal phalangeal and the middle phalangeal determined the DIP joint flexion, and the angle between the middle phalangeal and the proximal phalangeal determined the PIP joint flexion [32]. For a functional evaluation based on tendon gliding displacement, we used two metal pins through the proximal phalanx to fix digits to a table and applied traction to the tendon to perform the flexion of the joints. The FDP tendon was exposed after removing skin, subcutaneous tissue and the other flexor tendons. The tendon sheath and FDP tendon was marked at the exit from the sheath while a counterweight from the distal phalanx was applied to fully extend the interphalangeal joints. A constant force of 1 N pulled the FDP tendon out of the sheath tunnel and the distance after pulling was measured with a micrometer caliper. This length of tendon gliding was recorded as the gliding excursion of the FDP tendon [33]. 2.10. Biomechanical tests To evaluate peritendinous adhesions, the stiffness and breaking strength were measured using a materials testing machine (Tinius Olsen H1KT) with a 50 N load cell. The FDP tendon was pulled at 5 mm min1 and the displacement (mm) and load (N) were recorded. The maximum force necessary to pull the tendon out of the tendon sheath was defined as the pull-out force (N). The stiffness (N mm1) was defined as the slope of the load– displacement curve over the linear range. The materials testing machine with 50 N load cell was also used to evaluate tendon healing after 2 weeks. Nonslip clamps (HT-51) fixed the distal and proximal ends of a repaired rabbit FDP tendon and pulled the tendon uniaxially at 5 mm min1 to rupture. The maximum tension force was recorded as the breaking force of the repaired tendon. 2.11. Statistics All data were expressed as the means ± standard deviations. The one-way analysis of variance least significant difference test was used for statistical analysis, with a p value of less than 0.05 being considered statistically significant. 3. Results and discussion

2.8. Histological analysis The second and third digits of the rabbits were fixed in 10% formaldehyde in PBS and sectioned into 4 lm slices. Hematoxylin & eosin and Masson trichrome stainings were carried out following standard protocols. The peritendinous adhesion formation, inflammatory reaction and remainder of the PCL and PCL-g-CS NFMs were evaluated at 2 weeks. 2.9. Range of motion and gliding excursion For the range of motion analysis, the FDP tendon was transected at the proximal metacarpal level and sutured to a cable connected

3.1. Preparation and characterization of electrospun PCL and PCL-g-CS NFMs As shown from SEM micrographs, bead-free and continuous PCL nanofibers could be prepared by ES (Fig. 1a). Subsequent chemical grafting of CS to PCL nanofibers retained the morphology of PCL nanofibers (Fig. 1b). From the histograms of fiber diameter distribution (Fig. 1c and d), CS grafting slightly increased the average fiber diameter of PCL NFM from 432 to 481 nm; however, these values were not statistically significant from each other (Table 1). The membrane pore size distribution shifted from 0.4–1.9 lm to 0.2–1.9 lm with the mode pore diameter changing from

Please cite this article in press as: Chen S-H et al. Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.08.030

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Fig. 1. SEM micrographs (magnification = 5000, bar = 2 lm) showing the (a, b), fiber diameter distribution (c, d) and pore size distribution (e, f) of electrospun PCL (a, c, e) and PCL-g-CS (b, d, f) nanofibrous membranes. The insets in (a, b) show the water contact angle measurements of the membranes.

0.85–0.90 to 0.60–0.65 lm after CS grafting (Fig. 1e and f). The average pore size also significantly reduced from 0.87 lm (PCL NFM) to 0.62 lm (PCL-g-CS NFM) (Table 1). An increase in fiber diameter and a decrease in pore size may arise from the space occupied by the surface-grafted CS molecules. The presence of a CS layer on the fiber surface is expected to influence the surface properties of the NFM, which could be studied by measuring the water contact angles of the membranes (Fig. 1a and b insets). As expected, the PCL NFM is highly hydrophobic, as evidenced by the 118.0° contact angle. After CS grafting, the contact angle of

the PCL-g-CS NFM drastically reduced to 86.8° due to the presence of hydrophilic CS molecules on the PCL nanofiber surface (Table 1). Additional chemical analysis of surface-bound CS indicates that the CS content in the PCL-g-CS NFM is at 706.5 ± 73.9 lg per cm2 of membrane, which is 8.5-fold higher than the value reported for grafting CS to a non-woven fabric composed of microfibers due to the increased surface area provided by the nanosized fibers [34]. One of the benefits of using NFM for anti-adhesion is its microporous structure, which enables nutrient permeation during tendon healing. Thus, permeation studies were carried out to

Table 1 The fiber diameter, pore size, contact angle and bovine serum albumin (BSA) permeability coefficient of nanofibrous membranes.

*

Membrane

Fiber diameter (nm)

Pore size (lm)

Contact angle (degrees)

Permeability coefficient of BSA (x 105cm s1)

PCL PCL-g-CS

432 ± 123 481 ± 157

0.87 ± 0.04 0.62 ± 0.01*

118.0 ± 4.1 86.8 ± 3.7*

4.3 ± 0.9 5.1 ± 1.0

p < 0.05 compared with PCL.

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simulate the diffusion of nutrients (bovine serum albumin) through the NFM. Indeed, increased fiber diameter and reduced pore size do not hinder the diffusion of the protein through the NFM because the permeability coefficient shows no significant difference between the two NFMs (Table 1). The FTIR spectra of different NFMs extracted within a range of 4000 to 600 cm1 are shown in Fig. S1. The ester stretching of PCL at 1728 cm1 was observed as a major peak in the spectra of the PCL and PCL-g-CS NFMs [35]. The appearance of characteristic peaks corresponding to the stretching of protonated amino groups (ANH+3) at approximately 1658 and 1547 cm1 suggests the presence of CS on the surface of CS-PCL nanofibers [36]. The thermal properties of NFMs and CS were analyzed by TGA and the derivative thermogravimetry (DTG) from the first derivative of TGA curves are shown in Fig. S1. A large weight loss between 200 and 500 °C was observed for all samples from the TGA curves due to thermal decomposition behavior of CS and PCL. CS showed an initial weight loss in the temperature range 60–80 °C from water evaporation, followed by a weight loss starting at approximately 200 °C and a maximum weight loss at 290 °C to give a residual weight of 37.2%. The PCL NFM showed an onset of weight loss at approximately 350 °C and the peak temperature was at 407 °C [28]. The curve for PCL-g-CS NFM showed that weight loss started at approximately 200 °C, with a broad peak at 263 °C (corresponding to CS) and a sharp peak at 411 °C (corresponding to PCL) from the DTG curves. The shifting of the peak decomposition temperature to a higher temperature may indicate increased thermal stability for PCL NFM after surface grafting of CS. The residual mass of PCL-g-CS NFM (4.3%) is also greater than that of PCL NFM (1.4%) due to the additional residual weight of surface-grafted CS. XPS was used to characterize the surfaces. The survey scan spectra and the high-resolution C1s peaks are shown in Fig. S2 for PCL NFM, plasma-treated PCL NFM and PCL-g-CS NFM. All XPS spectra showed two separated peaks corresponding to C1s (286 eV) and O1s (534 eV). The oxygen surface concentration substantially increased from 12.2 to 24.2% after plasma modification as a result of introducing oxygen-containing radicals (Table 2). A distinct N1s peak at 401 eV was observed only in the PCL-g-CS NFM spectrum; the nitrogen surface concentration is calculated to be 5.5% (Table 2). The fractions of different carbon functional groups could be calculated from high-resolution XPS C1s spectra (Table 3). The three components of the PCL NFM spectrum at 285.0, 286.5 and 289.0 eV could be assigned to CAC, CAO and O@CAO bonds in the PCL polymer chain, respectively. The fraction of the O@CAO carboxyl group increased from 7.0 to 15.4%, with a concomitant decrease in the fraction of the CAC saturated hydrocarbon group from 73.5 to 65.1% after plasma treatment. This increase in the carboxyl groups is due to the introduction of oxygen to the NFM [25]. The C1s spectrum revealed a new peak at 288.1 eV after CS grafting, which corresponds to the amide carbon in NHAC@O. This peak was not observed for PCL NFM and plasmatreated PCL NFM. Also, the fraction of O@CAO peak decreased from 15.4 to 4.5%. Taken together, the XPS results confirm that CS was successfully introduced onto the PCL nanofiber surface by forming amide bonds between the amino groups of the CS and the carboxyl groups on the plasma-treated PCL NFM surface [25].

Table 2 XPS analysis of surface compositions of nanofibrous membranes. Membrane

PCL Plasma-treated PCL PCL-g-CS

Atomic percent C

N

O

87.8 75.8 71.0

0.0 0.0 5.5

12.2 24.2 23.5

Table 3 Fraction of carbon functional groups from high-resolution C1s XPS peaks. Membrane

CAC 285.0 eV (%)

CAO 286.5 eV (%)

O@CAO 289.0 eV (%)

NHAC@O 288.1 eV (%)

PCL Plasma-treated PCL PCL-g-CS

73.5 65.1 48.9

19.5 19.5 33.6

7.0 15.4 4.5

0.0 0.0 13.0

The water uptake ability of the PCL NFM is compared with that of the PCL-g-CS NFM (Fig. 2a). Due to its hydrophobic property, the PCL NFM exhibited a relatively low water sorption. Grafting hydrophilic CS molecules onto the surface PCL NFM increased the water sorption after 24 h from 62.8 to 169.5%. Water sorption would cause the swelling of CS-PCL NFM and provides a hydrogel-like nature to the membrane surfaces, promoting interfacial tendon gliding at the membrane–tendon interface to exert anti-adhesion effects [19]. To clarify the effects of CS grafting on the mechanical properties of NFM, the stress–strain behaviors of the NFMs were measured and typical stress–strain curves are reported (Fig. 2b). Grafting CS to PCL NFM decreased the elongation-at-break value by 17%; however, there is no significant difference between the groups (Table 4). In contrast, the ultimate tensile stress and Young’s modulus for PCL-g-CS significantly increased to 1.65 and 2.85 times the values of PCL NFM, respectively (Table 4). There was no statistically significant increase in the fiber diameter after CS grafting, which could enhance the ultimate strength and Young’s modulus. Therefore, it could be postulated that the increase in hardness and strength offered by the PCL-g-CS NFM originates from the covalent binding of CS molecules to the PCL fiber surface. Undoubtedly, the better mechanical performance of the PCL-g-CS NFM, in terms of improved mechanical strength while retaining the ultimate tensile strain offered by the PCL NFM, will facilitate the use of the PCL-g-CS NFM by surgeons as an anti-adhesion barrier at the tendon surgery site. 3.2. In vitro cell culture An in vitro cell culture study was used to explore the underlying mechanism of the anti-adhesion effect of the PCL NFM and the improvement offered by the PCL-g-CS NFM. The cell migration test was carried out by determining the extent of cell movement from the upper compartment of the cell culture insert to the lower well of a cell culture plate due to the difference in serum concentrations (Fig. 3). The inhibition of cell migration by the PCL and PCL-g-CS NFMs was evident from the significant reduction in the number of penetrated viable cells according to MTS assays or from direct microscopic observation of the lower well surface. There is no significant difference between the PCL and PCL-g-CS groups from the MTS assays; however, the absorbance values in both groups are less than 20% of that in the control group without an NFM at the bottom of the cell culture insert. Therefore, the physical blockage of cell passage from the microstructure of the NFM hindered cell migration, which was not caused by the cytotoxicity of CS [30]. To exert an important underlying mechanism for an anti-adhesion effect, the micrometer-scaled pores (0.62–0.87 lm in diameter) of NFMs are expected to block the passage of extrinsic fibroblastic cells (>8–10 lm in diameter) that form peritendinous adhesions while allowing the diffusion of growth factors and cytokines for important tendon healing functions. In addition to the penetration of fibroblasts, the adhesion of fibroblasts on the surface of PCL, plasma-treated PCL and PCL-g-CS NFMs was compared. Many adhesive proteins present in extracellular matrices contain the tripeptide arginine–glycine–aspartic acid (RGD) as their cell recognition site. The RGD sequences of each

Please cite this article in press as: Chen S-H et al. Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.08.030

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Fig. 2. (a) Water sorption curves and (b) representative stress–strain curves of PCL and PCL-g-CS nanofibrous membranes.

Table 4 Mechanical properties of nanofibrous membranes.

*

Membrane

Ultimate tensile strength (MPa)

Elongation-atbreak (%)

Young’s modulus (MPa)

PCL PCL-g-CS

1.4 ± 0.1 2.2 ± 0.5*

71.8 ± 13.1 59.7 ± 1.1

7.1 ± 0.2 20.2 ± 3.0*

p < 0.05 compared with PCL.

of the adhesive proteins are recognized by at least one member of a family of structurally related receptors, integrins [37]. Some of these receptors bind to the RGD sequence of a single adhesion protein only, whereas others recognize groups of them. The conformation of the RGD sequence in the individual proteins may be critical to this recognition specificity. On the cytoplasmic side of the plasma membrane, the receptors connect the extracellular matrix to the cytoskeleton. Together, the adhesion proteins and their

receptors constitute a versatile recognition system for cell adhesion [38]. From MTS assays, the attached cell numbers on PCL, plasma-treated PCL and PCL-g-CS NFMs were significantly less than on TCPS. The value for PCL-g-CS NFM is also statistically less than those for PCL and plasma-treated PCL NFMs (Fig. 4a), which is consistent with the trend previously observed for CS film [39]. To further investigate the effect of CS on cell attachment, the morphology, cytoskeletal actin distribution and focal adhesion protein (vinculin) expression of fibroblasts were monitored on PCL and PCL-g-CS NFMs. As shown in Fig. 4b–d, when fibroblasts were cultured on PCL-g-CS NFM they maintained a rounded morphology, and showed minimal vinculin expression and diffused F-actin cytoskeletal distribution. In contrast, the cells cultured on PCL NFM demonstrated increased cellular spreading, enhanced vinculin expression and a well-distributed fibrous F-actin cytoskeleton. It has been reported that the isoelectric point of CS membrane occurs under physiological conditions, which

Fig. 3. Cell migration through PCL and PCL-g-CS nanofibrous membranes in 24 h by MTS assay and optical microscope observation. The run without the nanofibrous membrane was used as the control. Bar = 100 lm. ⁄p < 0.05.

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excludes the mechanism of cell adhesion to CS through the positive surface charge arising from protonated amino groups [40]. Although CS is a structural analogue of glycoaminoglycans, the negative charge of which is associated with its bioactivity via interactions with the positively charged amino groups of proteins, the lack of these groups in CS could be the reason for the poor cell adhesion on CS membranes [40]. On the other hand, CS has a poorer ability to absorb fibronectin from the serum-containing medium, which has been demonstrated to mediate fibroblast adhesion and proliferation. This resulted in decreased cell adhesion and a rounded morphology of human anterior cruciate ligament cells on CS film [41]. In addition, the fluorescence intensity of the b1 integrin subunit, which is involved in the recognition between integrin receptors and the cell-binding domain of fibronectin, was also lower on CS than PCL film [41]. Therefore, the CS layer on the PCL-g-CS NFM can prevent more non-specific cell adhesion than PCL NFM. The PCL-g-CS NFM was thus demonstrated to be inferior to PCL NFM for supporting cell attachment or spreading. The synergistic effect of preventing cell penetration and inhibiting fibroblasts attachment is expected to augment the anti-adhesion effect of the PCL-g-CS NFM in vivo. 3.3. In vivo animal study The peritendinous adhesions were evaluated by direct observation 2, 4 and 8 weeks after surgical exploration of the

repair sites, and Fig. 5 shows macroscopic views of representative tendons receiving different treatments at different times. Dense adhesion formations were noted around the tendons in the untreated control group at each time point, which required sharp dissection to separate the large fibrous tissue bundles bridging the tendon and the surrounding tissue [23,31]. For the tendons wrapped with Seprafilm™ and the PCL NFM, despite the adhesion area being separated by blunt dissection, small bundles of fibrous tissues still existed to loosely bridge the tendon and the surrounding tissue. In the tendons treated with the PCL-g-CS NFM, no adhesion was observed between the repaired tendon and the peritendinous tissue throughout the experiment. Representative histological sections of the tendons receiving each of the treatments were compared with the control group in Fig. 6. Severe adhesions were observed between the tendon and the surrounding vascular granulation tissue for the untreated control tendons [42]. Invasion of the epitenon and superficial layers of the repaired tendons by the fibrous adhesion tissue could be observed, with poor collagen maturation. In tendons wrapped with Seprafilm™, loose bundles of fibrous tissue bridging the repaired tendon and the surrounding tissue were observed. The surfaces of the repaired tendons were also very rough, which indicates partial healing of the injured tendon. In the PCL NFM treatment group, no adhesion formation was observed between the repaired tendons and the surrounding tissues to give a noticeable interface at the repaired sites only at 2 weeks post-operation. Furthermore,

Fig. 4. (a) Attachment of fibroblasts to TCPS, PCL, plasma-treated PCL (PT PCL) and PCL-g-CS nanofibrous membranes after 24 h, as shown by MTS assay. The cytoskeletal arrangement and focal adhesion protein expression of fibroblasts on (b) TCPS, (c) PCL and (d) PCL-g-CS nanofibrous membranes was determined by actin cytoskeleton and vinculin staining, and observed by confocal microscopy. Bar = 50 lm. ⁄p < 0.05. Vinculin focal adhesion, actin cytoskeleton and cell nucleus are shown in green, red and blue, respectively. Arrows indicate the expression of vinculin focal adhesion.

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Fig. 5. Gross evaluation of adhesion occurring in a rabbit model of flexor digitorum profundus tendon repair in untreated control, Seprafilm™, PCL NFM and PCL-g-CS NFM groups 2, 4 and 8 weeks post-operation. Tendon (T) and adhesion tissue (black arrows) are indicated in the figures.

Fig. 6. Hematoxylin & eosin (HE) and Masson trichrome (MT) staining of tissue sections of untreated control repair site, repair site wrapped with Seprafilm™, repair site wrapped with PCL NFM and repair site wrapped with PCL-g-CS NFM at 2 and 4 weeks post-operation. Arrows indicate the adhesion tissue surrounding the tendon. Subcutaneous tissue (SC), tendon (T) and residual membranes (M) could be detected. Bar = 400 lm.

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undegraded NFM residual fragments were observed between the repaired tendon and the surrounding tissue. In comparison, no adhesions between the repaired tendons and surrounding tissues were observed in the PCL-g-CS NFM treatment group at 2 and 4 weeks post-operation. At the repaired sites, the surfaces on the tendons were smooth and the organization of collagen in these tendons was good, indicating better tendon healing. The efficiency of the anti-adhesion effect of the PCL-g-CS NFM, which was prepared to recreate the anti-adhesive role of the tendon sheath to prevent tendon adhesion to the surrounding tissues, was supported by both the gross view (Fig. 5) and the histological assessments (Fig. 6). Previously, a perichondrium graft was shown to reduce peritendinous adhesion both macroscopically and histopathologically when wrapped around the tendon repair site. Although the base material in the NFM is synthetic, in contrast to a perichondrial autograft, a similar anti-adhesion effect could be

confirmed without eliciting an inflammatory response around the edge of the NFM [43]. To quantitatively evaluate the anti-adhesion effects of different treatments in vivo, we conducted a comprehensive range of motion, gliding excursion and biomechanical evaluations of rabbit FDP tendon when the surgery site was wrapped with Seprafilm™, PCL NFM or PCL-g-CS NFM and compared them with those of an untreated control group. The PIP and DIP joint range-of-motion assays were used because they are more physiologically and clinically relevant [44,45]. There was a statistical improvement (i.e. an increase in value) in the DIP joint flexion angle (Fig. 7a), PIP joint flexion angle (Fig. 7b) and sliding excursion (Fig. 7c) of the experimental groups compared with the control group. However, only the groups treated with the PCL-g-CS NFM could restore the values to those of the normal unoperated FDP tendons (dotted lines in Fig. 7). Tendon adhesion is thus prevented after each treatment,

Fig. 7. Evaluation of peritendinous adhesions at different time points post-operation from (a) DIP joint flexion angle, (b) PIP joint flexion angle, (c) tendon gliding excursion, (d) pull-out force and (e) pull-out stiffness. The dotted line represents the average value of normal unoperated flexor digitorum profundus tendon. (f) Comparison of the breaking strength of healed tendons 2 weeks post-operation. ⁄p < 0.05 compared with untreated control; #p < 0.05 compared with the group treated with Seprafilm™; d p < 0.05 compared with the group treated with PCL NFM. The data are expressed as mean ± standard deviation (each group, n = 8).

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as shown from the increase in the range-of-motion at joints and the gliding excursion of tendons from the surrounding tissue, though to different degrees with the different treatments. In general, the quantitative anti-adhesion assessments shows the order of treatment in preventing peritendinous adhesion to be PCLg-CS > PCL > Seprafilm™. Comparing all treatments, the DIP and PIP flexion and gliding excursion of the tendons treated with NFMs were better than those treated with Seprafilm™ at each time point. The DIP flexion of the PCL-g-CS NFM group was significantly different from other groups at week 2. The PIP flexion of the PCL-g-CS NFM group was significantly different from the other groups at weeks 2 and 8. The gliding excursion of PCL-g-CS NFM group was significantly different from the other groups at each time point. The pull-out force (i.e. the force required to completely remove the tendon from the tendon sheath; Fig. 7d) and stiffness from the pull-out test (i.e. the resistance to tendon gliding; Fig. 7e) correlate with the severity of peritendinous adhesion. As expected, the untreated control tendon required the highest force to remove the tendon from the tendon sheath. Among the three treatments, tendons treated with PCL-g-CS NFM required the least force and showed the lowest degree of stiffness; the PCL NFM treatment required an intermediate force and had an intermediate stiffness; and the Seprafilm™ treatment required the largest force and had the greatest stiffness. Overall, the Seprafilm™ and PCL NFM groups showed similar improvement over the control. However, the PCLg-CS group provides significantly lower values when compared with the other groups at weeks 4 and 8. PCL-g-CS was also the only treatment that restored the pull-out force and stiffness to the values of normal unoperated FDP tendons. After testing the mechanical strength of tendons healed for 2 weeks, we noted an increase in the breaking strength of the healed tendon in the group treated with the PCL-g-CS NFM compared with the other groups (Fig. 7f). However, this breaking force was not significantly different among the four groups. As an anti-adhesion barrier, Seprafilm™ could be difficult to handle during surgery, especially when wrapped around an injured tendon, because of its weak mechanical properties and the relative dimensions of the tendon compared with the abdominal cavity. Furthermore, because the healing time after tendon surgery is normally longer than 6 weeks, the fast degradation rate of Seprafilm™ in vivo cannot offer a residence time long enough to allow adhesion-free healing to occur during the critical first 3 week period. In contrast, the PCL and PCL-g-CS NFMs are robust, elastic and easy to handle, and could maintain their integrity during positioning. For the prevention of postoperative peritoneal adhesion, a soft anti-adhesion barrier membrane like Seprafilm™ may be beneficial for the abdominal environment: during peritoneal application, Seprafilm™ may adhere well to the irregular tissue surface with less motion. However, it may not work well for peritendinous application, since the tissue surface will experience frequent gliding motion. The PCL-g-CS NFM is expected to have greater biomechanical strength and an improved ability to isolate the moving tendon from the surrounding tissue during healing, thereby preventing peritendinous adhesion.

4. Conclusion Electrospun PCL and PCL-g-CS NFMs were successfully prepared to act as biomimetic tendon sheaths to prevent peritendinous adhesion. With its microporous structure and good mechanical properties, the NFM could allow nutrients and waste transport through the membrane to allow for normal tendon healing while providing a robust barrier preventing the penetration of fibrotic cells responsible for post-surgical adhesion. Surface grafting CS to the plasma-treated PCL NFM provides a facile method to introduce

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CS to the NFM without affecting the fiber diameter, microstructure and permeability of the NFM, while enhancing its mechanical strength. The grafted CS layer on the PCL nanofiber provides a molecular mechanism to reduce fibroblast attachment to the PCL-g-CS NFM in vitro. Using the rabbit FDP tendon in vivo model, we demonstrated that the PCL-g-CS NFM is the best peritendinous anti-adhesion barrier film compared with both the PCL NFM and a commonly used commercial anti-adhesion product (Seprafilm™) by macroscopic observation, histological analysis, and functional (joints flexion and tendon gliding) and biomechanical (pull-out force and stiffness) evaluation. Acknowledgements We would like to express our appreciation of the financial assistance provided by grants from the National Science Council, ROC (NSC102-2320-B-182-004-MY3), the Department of Health, ROC (DOH102-TD-PB-111-NSC004) and Chang Gung Memorial Hospital (CMRPG3A1431-2). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 2, 4–7 are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2014.08.030. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.08. 030. References [1] Hellebrekers BW, Trimbos-Kemper TC, Trimbos JB, Emeis JJ, Kooistra T. Use of fibrinolytic agents in the prevention of postoperative adhesion formation. Fertil Steril 2000;74:203–12. [2] diZerega GS, Campeau JD. Peritoneal repair and post-surgical adhesion formation. Hum Reprod Update 2001;7:547–55. [3] Taras JS, Lamb MJ. Treatment of flexor tendon injuries: surgeons’ perspective. J Hand Ther 1999;12:141–8. [4] Lilly SI, Messer TM. Complications after treatment of flexor tendon injuries. J Am Acad Orthop Surg 2006;14:387–96. [5] Boyer MI. Flexor tendon biology. Hand Clin 2005;21:159–66. [6] Yeo Y, Kohane DS. Polymers in the prevention of peritoneal adhesions. Eur J Pharm Biopharm 2008;68:57–66. [7] Choung HK, Hwang JM. The use of SurgiWrap™ in delayed adjustable strabismus surgery. Am J Ophthalmol 2005;140:433–6. [8] Hagberg L, Heinegard D, Ohlsson K. The contents of macromolecule solutes in flexor tendon sheath fluid and their relation to synovial fluid; a quantitative analysis. J Hand Surg Br 1992;17:167–71. [9] Peterson WW, Manske PR, Dunlap J, Horwitz DS, Kahn B. Effect of various methods of restoring flexor sheath integrity on the formation of adhesions after tendon injury. J Hand Surg Am 1990;15:48–56. [10] Bolgen N, Vargel I, Korkusuz P, Menceloglu YZ, Piskin E. In vivo performance of antibiotic embedded electrospun PCL membranes for prevention of abdominal adhesions. J Biomed Mater Res Part B 2007;81:530–43. [11] Lo HY, Kuo HT, Huang YY. Application of polycaprolactone as an anti-adhesion biomaterial film. Artif Organs 2010;34:648–53. [12] Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosan – a versatile semisynthetic polymer in biomedical applications. Prog Polym Sci 2011;36: 981–1014. [13] Silva SS, Luna SM, Gomes ME, Benesch J, Pashkuleva I, Mano JF, et al. Plasma surface modification of chitosan membranes: characterization and preliminary cell response studies. Macromol Biosci 2008;8:568–76. [14] Li J, Yun H, Gong Y, Zhao N, Zhang X. Investigation of MC3T3-E1 cell behavior on the surface of GRGDS-coupled chitosan. Biomacromolecules 2006;7: 1112–23. [15] Diamond MP, Luciano A, Johns DA, Dunn R, Young P, Bieber E. Reduction of postoperative adhesions by N,O-carboxymethylchitosan: a pilot study. Fertil Steril 2003;80:631–6. [16] Lauder CIW, Garcea G, Strickland A, Maddern GJ. Use of a modified chitosan– dextran gel to prevent peritoneal adhesions in a rat model. J Surg Res 2011;171:877–82.

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Please cite this article in press as: Chen S-H et al. Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.08.030