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Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis
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Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis Tae Hoon Leea,b,1, Sung Eun Kimc,1, Jae Yong Leed , Jae Gyoon Kime, Kyeongsoon Parkf,* , Hak-Jun Kimc,* a
Department of Orthopedic Surgery, College of Medicine, Korea University, Anam-dong, Seongbuk-gu, 02841, Korea 9988Hospital, 269 Wangsimin-ro, Seongdong-gu, Seoul, 04745, Korea c Department of Orthopedic Surgery and Rare Diseases Institute, Korea University Medical College, Korea University Guro Hospital 148, Gurodong-ro, Guro-gu, Seoul, 08308, Korea d Department of Biomedical Science, College of Medicine, Korea University, Anam-dong, Seongbuk-gu, Seoul, 02841, Korea e Department of Orthopedic Surgery, Korea University, College of Medicine, Korea University Ansan Hospital 123, Jeokgeum-ro, Danwon-gu, Ansan-si, Gyeonggi-do, 15355, Korea f Department of Systems Biotechnology, Chung-Ang University, Anseong-si, Gyeonggi-do, 17546, Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 December 2018 Received in revised form 7 January 2019 Accepted 17 January 2019 Available online xxx
We fabricated diclofenac-immobilized polycaprolactone (DFN/PCL) fibrous sheets to improve tendon healing of rabbits with collagenase-induced Achilles tendinitis. DFN/PCL have anti-inflammatory effects related to suppression of mRNA levels of pro-inflammatory factors including COX-2, IL-6, TNF-α, and MMP-3 in inflamed tenocytes and tendon tissues of rabbits with Achilles tendinitis as well as those of anti-inflammatory cytokines including IL-4, IL-10, and IL-13. In vivo studies, wrapping of tendon tissues with DFN/PCL fibrous sheets may afford superior tendon healing, with decreased inflammatory cells, increased collagen content, restored collagen fiber organization, and improved mechanical strength. This approach will be a promising treatment from Achilles tendinitis. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Achilles tendinitis Fibrous sheet Diclofenac Polycaprolactone Tendon healing
Introduction Tendons are unique forms of connective tissue that transmit muscular forces to bone, permitting joint motion and subsequent body movement [1]. Achilles tendons are ruptured more often than any other because they are constantly subjected to large mechanical loads [2,3]. Too much mechanical stress can cause the Achilles tendon to become inflamed, and potentially to tear or rupture. Local inflammation responses in Achilles tendinitis are characterized by the presence of multiple proinflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2), as well as matrix metalloproteinases (MMPs; MMP-2, -3, -9, and -13). Eventually, various pathological processes lead to cell damage
* Corresponding authors. E-mail addresses: [email protected] (K. Park), [email protected] (H.-J. Kim). 1 These authors contributed equally to this paper.
and loss of tissue integrity with full or partial rupture of the tendon, ultimately resulting in reduced biomechanical properties (i.e., tensile strength) [4–7]. Thus, the restoration of normal structure and function of injured tendons by suppressing inflammation responses is considered a potential therapeutic approach in orthopedic medicine [8]. Given the inflammatory nature of injured tendons, nonsteroidal inflammatory drugs (NSAIDs) are often used to treat Achilles tendinitis. Indeed, NSAIDs relieve pain in the acute phase, reduce the possibility of leg stiffness, and reduce modest symptoms of Achilles tendinitis due to their anti-inflammatory properties [9]. However, systemic treatment with NSAIDs has not been successful due to several limitations: (1) insufficient bioavailability of drugs to alter the cellular and molecular milieu in which the tendon heals, (2) the multiple administrations required to achieve prolonged therapeutic effects, and (3) the side effects (i.e., gastrointestinal bleeding, ulceration, and perforation) induced by their long-term use [10]. Furthermore, several studies have indicated that systemically administered NSAIDs may inhibit tendon cell migration and proliferation and impair tendon healing
https://doi.org/10.1016/j.jiec.2019.01.018 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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[11], and they showed little or no effect on the clinical outcome [12]. As an alternative, local injections of anti-inflammatory drugs can reduce pro-inflammatory factors and improve tendon healing in vivo [13,14]. However, the therapeutic effects of locally injected drugs with much higher dosages were not as effective as those of long-term drug delivery systems [13,14]. Therefore, drug delivery systems with controlled drug release ability have been used to overcome these limitations. We recently developed injectable drug delivery systems such as porous microspheres for the local treatment of Achilles tendinitis [13,14]. Porous microspheres have attracted considerable attention for controlled, long-term drug delivery systems [15]. Recently, using a simple fluidic device, we fabricated porous microspheres with drugs and used them to treat Achilles tendinitis [13,14]. Drug-loaded porous microspheres have more beneficial effects on suppression of inflammation and enhancement of tendon restoration in vivo compared to small-molecule drugs. Interestingly, we found that injectable porous microspheres were not fully effective in collagenase-treated tendon tissues. Herein, in order to enhance restorative effects on the entire area of tendon tissues, we fabricated polycaprolactone (PCL) fibrous sheets using electrospinning, which is a simple technique used to produce ultrafine and continuous submicron fibers and nanofibers [16,17]. The PCL fibrous sheet can be wrapped around the entire area of rabbit tendon tissue. Diclofenac (DFN), which is an NSAID that has been used widely to relieve pain and suppress inflammation [18], was immobilized on the surface of PCL fibrous sheets following dopamine (DOPA) coating to improve antiinflammatory and tendon restorative effects. In this study, we evaluated the in vitro anti-inflammatory effects on inflamed tenocytes by determining pro-inflammatory factors. For in vivo experiments, after the collagenase-treated tendon tissues in the rabbits were wrapped with DFN-immobilized PCL (DFN/PCL) fibrous sheet via surgical operation, the in vivo anti-inflammatory factors and tendon healing effects were quantified by measuring pro- and anti-inflammatory factors, histology, collagen content, and performing biomechanical tests. Materials and methods Materials PCL (Mw = 70,000–90,000), dichloromethane (DCM), tetrahydrofuran (THF), dopamine hydrochloride (DOPA), and lipopolysaccharide (LPS, from Escherichia coli) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphatebuffered saline (PBS), and penicillin-streptomycin were purchased from Gibco-BRL (Rockville, MD, USA). DFN sodium was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Cell counting kit (CCK)-8 was purchased from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). All other chemicals used were of analytical grade and were used as received without any further purification. Fabrication of DFN-immobilized PCL (DFN/PCL) fibrous sheet PCL fibrous sheets were fabricated using the electrospinning method [19]. Briefly, 2 g of PCL was clearly dissolved in 10 mL of THF, and the PCL solution was loaded into a 10 mL glass syringe with an 18-gauge needle. Then, the syringe containing PCL solution was placed in a syringe pump (KD-200, KD Scientific, Inc., Holliston, MA, USA) with 10 cm between the syringe needle tip and the collector. The syringe tip was connected to the positive output of a high-voltage power supply (12 kV), and the flow rate was set to 1 mL/h.
Prior to immobilization of DFN on a PCL fibrous sheet with width length thickness = 3 cm 5 cm 0.3 cm, the surface of the PCL fibrous sheet was modified with DOPA, which has a positively charged amine group. The PCL fibrous sheet [3 cm (width) 5 cm (length)] was placed in 15 mL of 10 mM TriHCl buffer (pH 8.0) solution containing DOPA (2 mg/mL) and gently shaken overnight under dark conditions. The DOPA-immobilized PCL (termed ‘aminated PCL’) sheet was washed with deionized water (DW) and dried under N2 gas. Next, the aminated PCL fibrous sheet (3 cm 5 cm) was immersed in 15 mL of 0.1 M MES buffer (pH 5.6) containing 1 or 5 mg of DFN. The DFN-immobilized PCL fibrous sheet was washed again with DW and dried under N2 gas. The 1 and 5 mg/ mL DFN-immobilized PCL fibrous sheets are hereafter referred to as DFN (1 mg)/PCL and DFN (5 mg)/PCL, respectively. Characterization of PCL and DFN/PCL fibrous sheets The surface morphologies of PCL, aminated-PCL, and DFN (1 mg or 5 mg)/PCL with were investigated via scanning electron microscope (SEM, S2300, Hitachi, Japan) at 3 kV. All samples were coated with gold using a sputter-coater (Eiko IB, Japan). To determine the amount of immobilized DFN on the surface of the fibrous sheets, each fibrous sheet (width length = 3 cm 5 cm) such as DFN (1 mg)/PCL and DFN (5 mg)/PCL were each dissolved in 2 mL of DCM. Then, PBS (15 mL) was added to each DCM solution and gently vortexed. After 3 h, the mixtures were centrifuged and aqueous PBS solutions were collected. The amount of DFN was determined at 285 nm using the UV/Vis spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). In vitro drug release study To evaluate in vitro DFN release from DFN (1 mg or 5 mg)/PCL, each sample (width length = 3 cm n5 cm) was placed in a 15-mL conical tube containing PBS buffer (pH 7.4, 15 mL), followed by gentle shaking in a water bath (37 C) oscillating at 100 times/min. At scheduled time intervals, the PBS was collected and replaced with an equal volume of fresh PBS. The collected PBS solutions were stored at 20 C before analysis. The released amount of DFN from the PCL fibrous sheets was determined by measuring the absorbance at 285 nm using the UV/Vis spectrophotometer. In vitro cytotoxicity test The cytotoxicity of PCL, aminated PCL, and DFN (1 mg or 5 mg)/ PCL with 1 cm (width) 1 cm (length) on tenocytes was estimated by the indirect method based on the ISO/EN 10993 Part 5 guidelines. To do this, tenocytes were isolated from the Achilles tendon tissues of rats as previously described [13]. To obtain the extraction medium, each fibrous sample was immersed in a 15 mL tube containing DMEM medium (1 mL), followed by gentle shaking in a water bath (37 C) oscillating 100 times/min. Tenocytes (5 104 cells/well) were incubated with DMEM containing 10% FBS and 1% antibiotics. After 24 h, the cells were washed with PBS and the extraction medium or diclofenac (500 mg/mL) was used to treat the cells. After Days 1 and 3 of incubation, DMEM medium was removed and CCK-8 reagent was added to each well, followed by further incubation for 1 h. The optical density of the live cells was determined at 450 nm using a Flash Multimode Reader (Thermo Scientific, CA, USA). In vitro anti-inflammatory effects on inflamed tenocytes The in vitro anti-inflammatory effects of DFN/PCL fibrous sheets on tenocytes were investigated by measuring the mRNA expression levels of the pro-inflammatory cytokines COX-2, IL-6, MMP-3, and TNF-α using real-time polymerase chain reaction (real-time
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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PCR). Tenocytes (1 105 cells/mL) were carefully seeded on PCL, diclofenac (500 mg/mL), DFN (1 mg)/PCL (real dose = 63.48 1.01 mg of DFN), and DFN (5 mg)/PCL (real dose = 313.19 5.82 mg of DFN) fibrous sheets in 24-well tissue culture plates. The dimension of PCL, DFN (1 mg)/PCL, and DFN (5 mg)/PCL fibrous sheets was 1 cm width 1 cm length. After 24 h of incubation, all groups were treated with LPS (1 mg/mL). After Days 1 and 3, the cells were harvested and the total RNA was isolated from the harvested cells in each group using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). The total RNA concentration was measured using an ND1000 spectrometer (NanoDrop Technologies Inc., Montchanin, DE, USA). Then, the total RNA (1 mg) was reverse-transcribed into cDNA using AccuPower RT PreMix (Bioneer, Daejeon, Korea) according to the manufacturer’s protocols. The primers used for amplification of COX-2, IL-6, MMP-3, and TNF-α are as follows: COX-2, (F) 50 -CAG CCA TAC TAT GCC TCG GA-30 , (R) 50 -GGA TGT CTT GCT CGT CGT TC-30 ; IL-6, (F) 50 - CCG TTT CTA CCT GGA GTT TG -30 , (R) 50 - GTT TGC CGA GTA GAC CTC AT -30 ; MMP-3, (F) 50 -ACC TGT CCC TCC AGA ACC TG-30 , (R) 50 -AAC TTC ATA TGC GGC ATC CA-30 ; and TNF-α, (F) 50 - CTC CCA GAA AAG CAA GCA AC -30 , (R) 50 - CGA GCA GGA ATG AGA AGA GG-30 . PCR amplification and detection were carried out using an ABI7300 Real-Time Thermal Cycler (Applied Biosystems, Foster, CA, USA). The mRNA levels of these pro-inflammatory cytokines were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and are expressed as relative values.
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Induction of Achilles tendinitis in a rabbit model and in vivo treatment with fibrous sheets In vivo animal experiments were performed and approved by the Institutional Animal Care and use Committee of the Korea University Medical Center (KOREA-2018-0052). New Zealand White rabbits (DooYeol Biotech, Seoul, Korea) were anesthetized with isoflurane (1% w/v in 2 L oxygen). As seen in Fig. 1B, after shaving the rabbit’s right hind limb, Achilles tendon tissue was carefully exposed, and 50 mL of collagenase type I [50 mg/ mL in PBS (pH 7.4)] was injected into the exposed Achilles tendon tissues. Then, collagenase-treated tendon tissue was carefully wrapped with PCL, DFN (1 mg)/PCL, or DFN (5 mg)/PCL fibrous sheets (width length = 3 cm 5 cm, Fig. 1A). The wrapped fibrous sheet was fixed using an absorbable 4-0 Vicryl (Ethicon, Somerville, NJ, USA), and the remnant sheet was trimmed with scissors. After trimming the remnant fibrous sheet with scissors, the dimension of wrapped fibrous sheet on tendon tissues was approximately 3 cm (width) 2 cm (length). The real drug contents of DFN (1 mg)/PCL [3 cm (width) 2 cm (length)] and DFN (5 mg)/PCL (width length = 3 cm 2 cm) were approximately 0.380 0.006 mg and 1.880 0.524 mg, respectively. Finally, the subcutaneous tissue and skin were sutured using an absorbable 4-0 Vicryl. In the positive drug control group, diclofenac (5 mg) solution was injected into the collagenase-injected Achilles tendon. After four weeks of
Fig. 1. (a) Photo of DFN/PCL scaffolds (width length = 3 cm 5 cm). (b) Serial photographs show the wrapping of DFN/PCL fibrous scaffold to treat Achilles tendinitis. After shaving the area, Achilles tendon tissue was carefully exposed, and 50 mL of collagenase type I (50 mg/mL) was injected into tendon tissue. Collagenase-treated tendon tissue was carefully wrapped with PCL or DFN/PCL fibrous sheets. The wrapped fibrous sheets were fixed by suturing and the remnant sheet was trimmed. Then, the subcutaneous tissue and skin were sutured.
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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treatment, rabbits were euthanized and tendon specimens were harvested for the evaluation of histopathology, in vivo antiinflammatory effects, and biomechanics. Experimental subjects were randomly divided into six groups: (I) control (no treatment), (II) collagenase (collagenase injection), (III) Col (I) and PCL, (IV) Col (I) and diclofenac (5 mg) solution, (V) Col (I) and DFN (1 mg)/PCL (real dose = 0.380 0.006 mg) and (VI) Col (I) and DFN (5 mg)/PCL (real dose = 1.880 0.524 mg). In vivo anti-inflammatory effects on Achilles tendinitis rabbit models The in vivo anti-inflammatory effects of DFN/PCL fibrous sheets on Achilles tendinitis rabbit models were evaluated by determining the gene expression of pro-inflammatory cytokines (COX-2, IL-6, MMP-3, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) using real-time PCR. The total RNA from the isolated tendon tissues from rabbits in each group was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The total RNA concentration was measured using an ND-1000 spectrometer (NanoDrop Technologies Inc.). The total RNA (1 mg) was reverse-transcribed into cDNA using AccuPower RT PreMix (Bioneer). The same primer sequences for the proinflammatory cytokines were used as described above in the in vitro cell study. The primer sequence of the anti-inflammatory cytokines IL-4, IL-10, and IL-13 were as follows: IL-4, (F) 50 -ACA GGA GAA GGG ACG CCA T-30 , (R) 50 -GAA GCC CTA CAG ACG AGC TCA-30 ; IL10, (F) 50 -GGT TGC CAA GCC TTA TCG GA-30 , (R) 50 -ACC TGC TCC ACT GCC TTG CT-30 ; and IL-13, (F) 50 -AGA CCA GAC TCC CCT GTG CA-30 , (R) 50 -TGG GTC CTG TAG ATG GCA TTG-30 . PCR amplification and detection were performed on an ABI7300 Real-Time Thermal Cycler (Applied Biosystems) using a DyNAmoTM SYBR1 Green qPCR Kit (Finnzymes, Espoo, Finland). The relative mRNA levels of pro- and anti-inflammatory cytokines were normalized to those of GAPDH.
2.10. Hydroxyproline assay To evaluate the Achilles tendon healing effect, the collagen contents of Achilles tendon tissues in each group were measured by hydroxyproline assay. In short, Achilles tendon tissues (5 mg) were hydrolyzed with 12 N HCl on a hot plate at 120 C for 3 h, and DW (20 mL) was added to the hydrolyzed tissues. Then, NaOH solution was slowly added to the hydrolyzed tissue solution until the solution was neutralized. To initiate hydroxyproline oxidation, 50 mL of 60 mM chloramine T solution was mixed with 100 mL standard of hydroxyproline or samples in a 96-well plate. After 20 min of incubation at room temperature, the chloramine T was destroyed by adding 50 mL of perchloric acid to each well. Finally, the absorbance was recorded at 550 nm using a Flash Multimode Reader. Biomechanical test The tendon specimens (width length = 2 2 cm) were securely fixed to a specially designed device, which allowed the specimens to be oriented such that a tensile load could be applied along the axis of the tendon. The fixed tendon specimen from each group was tested on an Instron Mechanical Tester (AG-10KNX, Shimadzu, Kyoto, Japan), which was operated at 5 mm/min velocity traction speed. The ultimate tensile strength (defined as maximum stress or force per unit area) and stiffness (the force per unit displacement) were measured. Statistical analysis Data are represented as mean standard deviation (n = 5). Statistical comparisons were carried out via one-way analysis of variance (ANOVA) using Systat software (Chicago, IL, USA). Differences were considered statistically significant at *P < 0.05 and **P < 0.01.
Histopathological study
Results
The harvested calcaneus-Achilles tendon tissues were fixed in 3.7% paraformaldehyde and embedded in paraffin. The tissues were crosssectioned to a thickness of 5 mm in the longitudinal parallel direction using a rotary microtome (HM 355S automatic microtomes, Thermo Scientific). Hematoxylin and eosin (H&E) and Masson’s Trichrome staining were done to confirm the in vivo anti-inflammatory and tendon restoration effects of DFN/PCL fibrous sheets.
Characterization of PCL and DFN/PCL fibrous sheets The surface morphologies of the fabricated fibrous sheets were examined by SEM. All fibrous sheets (PCL, aminated PCL, and DFN (1 mg or 5 mg)/PCL) had irregular structures and smooth surfaces with diameters of approximately 200 nm to 3 mm (Fig. 2). There were no substantial differences in diameter among the fibrous
Fig. 2. SEM images of PCL, aminated PCL, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Scale bar: (a)–(d) = 100 mm, and (e)–(h) = 10 mm.
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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Fig. 3. (a) In vitro release profiles of diclofenac from DFN (1 mg)/PCL and DFN (5 mg)/PCL (n = 5). (b) Cytotoxicity of PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL against tenocytes at Days 1 and 3 (n = 5).
sheets. The immobilization efficiency of DFN on the surface of DFN (1 mg)/PCL and DFN (5 mg)/PCL was 95.23 1.51% and 93.95 1.74%, respectively. Also, the immobilized DFN contents of DFN (1 mg)/PCL and DFN (5 mg)/PCL were 952.26 15.20 mg and 4697.79 87.30 mg, respectively. In vitro drug release and cytotoxicity studies The in vitro DFN release profiles from the DNF/PCL fibrous sheets are seen in Fig. 3(a). On Day 1, the released amounts of DFN were 248.25 0.07 mg (24.83 0.01%) for DFN (1 mg)/PCL and 812.59 0.07 mg (16.25 0.01%) for DFN (5 mg)/PCL. At Day 28, 749.12 0.22 mg (74.91 0.02%) and 2187.50 0.82 mg (43.75 0.02%) of DFN were released from DFN (1 mg)/PCL and DFN (5 mg)/PCL, respectively. The prepared DFN/PCL fibrous sheets have no high initial burst release, and they also showed sustained drug release up to 28 days.
The cytotoxicity of PCL, diclofenac, DFN (1 mg or 5 mg)/PCL, and DFN (5 mg)/PCL against tenocytes was evaluated at Days 1 and 3. As seen in Fig. 3(b), no significant cytotoxic effects are observed and the cell viabilities in all groups were maintained over 95% relative to that of the control group. These results indicate that PCL, diclofenac and DFN/PCL fibrous sheets were non-toxic and safe on tenocytes. In vitro anti-inflammatory effects of DFN/PCL on inflamed tenocytes To determine whether DFN/PCL inhibits inflammation responses in inflamed tenocytes, we incubated tenocytes with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Then, we stimulated the cells in all groups with LPS and assessed the mRNA expression levels of COX-2, IL-6, MMP-3, and TNF-α using real-time PCR (Fig. 4). PCL alone had no effect in decreasing the mRNA levels of these cytokines in inflamed tenocytes. However, diclofenac and
Fig. 4. Relative mRNA expression levels of pro-inflammatory cytokines in inflamed tenocytes. (a) COX-2, (b) IL-6, (c) MMP-3, and (d) TNF-α in LPS-stimulated tenocytes cultured on PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL at Days 1 and 3. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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Fig. 5. Relative mRNA expression levels of pro-inflammatory cytokines in collagenase-treated tendon tissues. (a) COX-2, (b) IL-6, (c) MMP-3, and (d) TNF-α in collagenaseinjected tendon tissues treated with PCL, diclofenac (5 mg), DFN (1 mg)/PCL, and DFN (5 mg)/PCL at four weeks. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
DFN/PCL fibrous sheets were comparably and dose-dependently effective in attenuating the mRNA levels of inflammatory cytokines on Days 1 and 3 compared to LPS or PCL alone (*P < 0.05 or ** P < 0.01). In cell culture condition, DFN/PCL fibrous sheets showed less anti-inflammatory effect compared to diclofenac due to the sustained release of DFN from the fibrous sheet. In vivo anti-inflammatory effects in Achilles tendinitis rabbit model To further investigate the in vivo anti-inflammatory effects of DFN/PCL fibrous sheets in Achilles tendinitis rabbit models, we determined the mRNA levels of pro-inflammatory cytokines (COX2, IL-6, MMP-3, and TNF-α) as well as anti-inflammatory cytokines (IL-4, IL-10, and IL-13) in tendon tissues isolated from the rabbits in each group. Fig. 5 shows that treatment of tendon tissues with collagenase markedly increased the mRNA expression levels of all of pro-inflammatory cytokines up to over 7-fold compared to the normal group. No significant differences of these inflammatory
cytokines in tendon tissue treated with PCL were observed compared to those in the collagenase-treated group. Two DFN/ PCL groups were much more effective in decreasing the mRNA levels of these cytokines than diclofenac in a dose-dependent manner (**P < 0.01). To further confirm whether DFN/PCL influences the expression of the anti-inflammatory cytokines, the mRNA expression levels of IL-4, IL-10, and IL-13 were analyzed using real-time PCR (Fig. 6). Compared to the normal group, collagenase treatment markedly decreased the mRNA levels of IL-4, IL-10, and IL-13. The PCL group did not increase the mRNA levels of these anti-inflammatory cytokines. Diclofenac and two DFN/PCL groups increased their mRNA levels. However, two DFN/PCL groups displayed much higher mRNA levels of anti-inflammatory cytokines in a dosedependent manner than did the diclofenac-treated group (**P < 0.01), suggesting that the DFN/PCL system was more effective in suppressing inflammation responses and enhancing healing and restoration of tendon tissues.
Fig. 6. The relative mRNA expression levels of anti-inflammatory cytokines in collagenase-treated tendon tissues. (a) IL-4, (b) IL-10, and (c) IL-13 in collagenase-injected tendon tissues treated with PCL, diclofenac (5 mg), DFN (1 mg)/PCL, and DFN (5 mg)/PCL at four weeks. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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Histological validation of tendon tissues To assess the anti-inflammatory and tendon healing effects of DFN/PCL in vivo, we performed histopathological examinations with H&E (Fig. 7A) and Masson’s trichrome staining (Fig. 7B). In the
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control group, normal tendon tissues showed typical spindleshaped tenocytes arranged parallel to the fiber pattern as well as uniform, compact, and longitudinally well-aligned collagen fibers (Fig. 7A(a) and B(a)). In contrast, collagenase-treated tendon tissues exhibited a large number of inflammatory cells and clear
Fig. 7. Histological evaluations with (A) H&E and (B) Masson’s trichrome staining at four weeks after collagenase (Col (I)) injection into tendon tissues and treatments with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Groups are categorized as follows: (a) control (no treatment), (b) Col (I), (c) Col (I) + PCL, (d) Col (I) + diclofenac, (e) Col (I) + DFN (1 mg)/PCL, and (f) Col (I) + DFN (5 mg)/PCL. Scale bar: 50 mm.
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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Fig. 8. Hydroxyproline contents in collagenase-injected tendon tissues at four weeks after treatment with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
increased the hydroxyproline levels compared to the collagenasetreated group (**P < 0.01). Significant increases of the hydroxyproline content in Achilles tendon in two DFN/PCL groups were observed in a dose-dependent manner compared to those in diclofenac (*P < 0.05 or **P < 0.01). In particular, DFN (5 mg)/PCL group had the highest hydroxyproline content among the treatment groups (**P < 0.01). To further demonstrate the tendon restorative effects of DFN/ PCL, biomechanical studies were performed to determine stiffness and tensile strength of tendon tissues. As shown in Fig. 9(a) and (b), the stiffness and tensile strengths of the tendon tissues treated with collagenase or PCL were much lower than those of normal tendon tissue. Diclofenac and both DFN/PCL groups increased the stiffness and tensile strength of the tendon tissues. DFN (1 mg)/PCL showed similar or moderate effects in increasing the stiffness and tensile strength compared to diclofenac. However, DFN (5 mg)/PCL had much higher stiffness and tensile strength than diclofenac and DFN (1 mg)/PCL (*P < 0.05 or **P < 0.01). Discussion
degenerative changes including the entire fiber disruption and fiber mal arrangement (Fig. 7A(b) and B(b)). PCL treatment alone still showed many inflammatory cells and overall loss of collagen fibers (Fig. 7A(c) and B(c)). In the diclofenac (5 mg) treated group, the inflammatory cells, the collagen fiber disruption, and fiber mal arrangement were still observed (Fig. 7A(d) and B(d)) although the number of inflammatory cells was decreased and the collagen fibers were sporadically restored (Fig. 7B(d)). Two DFN/PCL groups decreased the number of inflammatory cells and restored the collagen fiber rearrangement (Fig. 7A(e and f) and B(e and f)) compared to the collagenase- and diclofenac-treated groups. Although the DFN (1 mg)/PCL group seemed to be have better attenuation of inflammatory cells and repair of collagen fiber organization, its efficacy was moderate compared to that of the diclofenac-treated group. However, DFN (5 mg)/PCL showed a significant decrease of inflammatory cells and exhibited overall restoration of collagen fibrillary organization (Fig. 7A(f) and B(f)), indicating that DFN (5 mg)/PCL has the best anti-inflammatory effects and tendon restoration effects. Determination of hydroxyproline content and biomechanical tests To quantify changes in collagen content of Achilles tendinitis models, the hydroxyproline content was determined after the treatments (Fig. 8). Compared to the control (normal) group, the hydroxyproline content was significantly decreased in collagenase- and PCL-treated groups, with similar hydroxyl hydroxyproline content. Diclofenac- and two DFN/PCL-treated groups greatly
Achilles tendinitis commonly occurs during athletic activity or in the course of daily life and causes pain, inflammation, and impaired tendon function in its early stage [20], eventually leading to partial or total tendon ruptures. Recently, emerging evidence has indicated that a strong inflammatory component is involved in the pathogenesis of tendon disease with inflammatory cells and cytokines as important regulators of the tendon extracellular matrix (ECM) [21]. High levels of cytokines may result in collateral tissue damage and impaired tendon healing [22]. Indeed, tendon disruption and injuries provoke local inflammation responses by inducing pro-inflammatory cytokines (i.e., IL-1β, TNF-α, and COX2), ECM degrading enzymes (i.e., MMPs), and ECM disintegration [5]. Therefore, the suppression of inflammation in Achilles tendinitis is considered as a therapeutic approach for Achilles tendinitis. DFN is a widely used non-steroidal anti-inflammatory drug (NSAID) [23]. The mechanism of DFN action is intervened in habiting COX, an enzyme converting arachidonic acid into thromboxane and prostacyclin, and thereby the suppression of prostaglandin synthesis [18,24–27]. However, when orally administered, DFN causes adverse effects (i.e., gastrointestinal damage and disorders and stomach ulceration) when taken at high dosages for a long period and undergoes first pass metabolism (approximately 50%). Due to these drawbacks, DFN has been used in several topical formulations such as gel, spray, lotion, and patch [18]. Several groups have suggested that topical delivery of DFN is a safer, more effective method for reducing pain and improving
Fig. 9. (a) Stiffness and (b) tensile strength of tendon tissues isolated from rabbits in each group at four weeks after collagenase (Col (I)) injection into tendon tissues and treatment with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
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physical function for the treatment of several painful diseases including rheumatoid arthritis, osteoarthritis, and Achilles tendinitis [18,23,28]. Recently, the Paula group reported that topical delivery of DFN using gold nanoparticles and phonophoresis was more effective in controlling the inflammatory cytokines such as IL-1β and TNF-α in a tendinopathy model [29]. In previous studies, our group developed porous microsphere systems for long-term drug delivery to treat Achilles tendinitis [13,14]. The anti-inflammatory drug-delivering porous microsphere systems effectively suppressed inflammation and restored tendon tissues. However, histological studies showed that the tendon restorative effect of these microsphere systems was only partial. Considering these outcomes, we attributed the partially restorative effect on the tendon to the local injection of porous microsphere systems by syringe due to their residence of limited area at around injected areas [13,14]. In order to improve tendon restorative effects in overall tendon tissues for the treatment of Achilles tendinitis, we fabricated DFN-immobilized PCL fibrous sheets (width length = 3 5 cm) capable of wrapping the overall area of collagenase-treated tendon tissues. DFN/PCL fibrous sheets were prepared by easy surface modification of DOPA molecules with catechol functional group on PCL fibrous sheet to introduce the positively charged amine group [30], followed by the immobilization of DFN with negatively charged carboxyl group on the surface of the aminated-PCL via electrostatic interactions. In vitro release and cytotoxicity studies demonstrate that DFN/PCL systems are suitable for long-term delivery due to its sustained drug release profiles for four weeks without high initial burst release and they are safe materials without toxic effects. Cytokines are well known as major mediators of inflammatory responses. To demonstrate the anti-inflammatory effects of DFN/ PCL fibrous sheet systems, we determined the mRNA levels of proinflammatory and anti-inflammatory factors in inflamed tenocytes or collagenase-treated tendon tissues. The in vitro anti-inflammatory effects of the DFN/PCL system were assessed against tenocytes, which are fibroblast-like differentiated cells that form the mature tendon. Previous studies reported that the tenocytes after LPS treatment increased the expression of several cytokines such as TNF-α, IL-6, and IL-1β. Also, these cytokines further stimulated tenocytes to produce pro-inflammatory cytokines (i.e., TNF-α, IL-6, IL-1β, and COX-2) and proteases (i.e., MMP-1, -3, and -13), which are thought to drive disease progression [31,32]. This study showed that DFN/PCL systems remarkably and dosedependently suppressed the mRNA levels of pro-inflammatory cytokines in inflamed tenocytes. The in vivo studies consistently supported that DFN/PCL systems markedly decreased the mRNA levels of pro-inflammatory cytokines (i.e., COX-2, IL-6, and TNF-α) and protease (i.e., MMP-3), which were expressed in collagenasetreated tendon tissues, whereas they could enhance the mRNA expressions of anti-inflammatory cytokines (i.e., IL-4, IL-10, and IL13). Mature tendons normally have low cellular density. Approximately 90-95% of the cellular content of tendons comprises tendon-specific cell types including tenoblasts and tenocytes [33]. Tenocytes play an important role in producing tendon ECM [34]. Tendon ECM components are composed predominantly of collagen fibrils (>95% of collagen type I, and other collagen types (type III and type V)) and a small fraction of elastin embedded in hydrated proteoglycans [35]. The parallel organization of collagen fibrils in tendons results in optimal stiffness and tendon tensile strength and biomechanical stability [35]. Histological examinations with H&E and Masson’s trichrome staining showed that collagenase injection into rabbit tendon tissues resulted in the recruitment of a large number of inflammatory cells and severe disruption of the well-aligned collagen fiber organization. These phenomena might be attributed to an inflammatory response by expressing
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inflammatory cytokines as well as mechanical degradation by producing protease after collagenase injection into tendon tissues. Although diclofenac suppressed the number of inflammatory cells and collagen matrix degeneration, DFN/PCL systems, in particular DFN (5 mg)/PCL, exerted more beneficial effects in preventing inflammation responses and collagen disruption as well as in repairing the parallel collagen fiber organizations. Also, the greatest tendon restorative effects of DFN (5 mg)/PCL group can be explained by the increased hydroxyproline content in tendon tissues because the hydroxyproline content is directly correlated to early maturation of fibroblasts, early parallel arrangement of collagen fibers, and bundle formation [36]. Moreover, we suggest that more significant enhancement of mechanical properties such as stiffness and tensile strength by DFN (5 mg)/PCL is closely related with the increase of both collagen fiber organization and the absolute amount of collagen. In the present study, we demonstrated that our long-term drug delivery system of DFN/PCL fibrous sheets suppresses inflammation reactions in both inflamed cells and collagenase-treated tendon tissues, as well as enhancing tendon healing, as demonstrated by restoration of collagen fibril structures and improved mechanical properties of tendon tissues. Treatments of small molecular drugs also have promising therapeutic effects. However, due to the relatively short residence time at injured sites, multiple injections are required to achieve the greater therapeutic effects. In contrast, the use of a long-term drug delivery system such as a DFN/PCL fibrous sheet could achieve more beneficial therapeutic outcomes due to the sustainable delivery of drug molecules for a long time to injured sites without multiple treatments. Furthermore, compared to porous microsphere systems for long-term drug delivery, PCL fibrous sheet systems may have an advantage because they can cover the overall area of tendon tissues via wrapping, leading to homogeneous distribution of diclofenac molecules into collagenase-treated tendon tissues. Therefore, DFN/ PCL fibrous sheet systems have great potential for Achilles tendinitis treatment. Conclusion Herein, we demonstrated robust anti-inflammatory and tendon restoration effects of a long-term diclofenac delivery system using PCL fibrous sheets on rabbit models with collagenase-induced Achilles tendinitis. DFN/PCL systems significantly suppressed inflammation responses that occurred in inflamed tenocytes and/or tendon tissues of Achilles tendinitis models by reducing the expression of pro-inflammatory cytokine genes (i.e., COX-2, IL6, and TNF-α) and protease such as MMP-3 as well as by increasing the mRNA levels of anti-inflammatory cytokines (i.e., IL-4, IL-10, and IL-13). Also, we confirmed that the inflammation inhibition of DFN/PCL systems resulted in increased collagen content and restoration of collagen fiber organization in tendon tissues. This study shows that wrapping collagenase-treated tendon tissues with DFN/PCL fibrous sheets improves tendon healing of Achilles tendinitis. Acknowledgement This study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI13C1501). References [1] H. Aslan, N. Kimelman-Bleich, G. Pelled, D. Gazit, J. Clin. Invest. 118 (2) (2008) 439. [2] K.M. Khan, N. Maffulli, Clin. J. Sport Med. 8 (3) (1998) 151.
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Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis Tae Hoon Leea,b,1, Sung Eun Kimc,1, Jae Yong Leed , Jae Gyoon Kime, Kyeongsoon Parkf,* , Hak-Jun Kimc,* a
Department of Orthopedic Surgery, College of Medicine, Korea University, Anam-dong, Seongbuk-gu, 02841, Korea 9988Hospital, 269 Wangsimin-ro, Seongdong-gu, Seoul, 04745, Korea c Department of Orthopedic Surgery and Rare Diseases Institute, Korea University Medical College, Korea University Guro Hospital 148, Gurodong-ro, Guro-gu, Seoul, 08308, Korea d Department of Biomedical Science, College of Medicine, Korea University, Anam-dong, Seongbuk-gu, Seoul, 02841, Korea e Department of Orthopedic Surgery, Korea University, College of Medicine, Korea University Ansan Hospital 123, Jeokgeum-ro, Danwon-gu, Ansan-si, Gyeonggi-do, 15355, Korea f Department of Systems Biotechnology, Chung-Ang University, Anseong-si, Gyeonggi-do, 17546, Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 December 2018 Received in revised form 7 January 2019 Accepted 17 January 2019 Available online xxx
We fabricated diclofenac-immobilized polycaprolactone (DFN/PCL) fibrous sheets to improve tendon healing of rabbits with collagenase-induced Achilles tendinitis. DFN/PCL have anti-inflammatory effects related to suppression of mRNA levels of pro-inflammatory factors including COX-2, IL-6, TNF-α, and MMP-3 in inflamed tenocytes and tendon tissues of rabbits with Achilles tendinitis as well as those of anti-inflammatory cytokines including IL-4, IL-10, and IL-13. In vivo studies, wrapping of tendon tissues with DFN/PCL fibrous sheets may afford superior tendon healing, with decreased inflammatory cells, increased collagen content, restored collagen fiber organization, and improved mechanical strength. This approach will be a promising treatment from Achilles tendinitis. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Achilles tendinitis Fibrous sheet Diclofenac Polycaprolactone Tendon healing
Introduction Tendons are unique forms of connective tissue that transmit muscular forces to bone, permitting joint motion and subsequent body movement [1]. Achilles tendons are ruptured more often than any other because they are constantly subjected to large mechanical loads [2,3]. Too much mechanical stress can cause the Achilles tendon to become inflamed, and potentially to tear or rupture. Local inflammation responses in Achilles tendinitis are characterized by the presence of multiple proinflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2), as well as matrix metalloproteinases (MMPs; MMP-2, -3, -9, and -13). Eventually, various pathological processes lead to cell damage
* Corresponding authors. E-mail addresses: [email protected] (K. Park), [email protected] (H.-J. Kim). 1 These authors contributed equally to this paper.
and loss of tissue integrity with full or partial rupture of the tendon, ultimately resulting in reduced biomechanical properties (i.e., tensile strength) [4–7]. Thus, the restoration of normal structure and function of injured tendons by suppressing inflammation responses is considered a potential therapeutic approach in orthopedic medicine [8]. Given the inflammatory nature of injured tendons, nonsteroidal inflammatory drugs (NSAIDs) are often used to treat Achilles tendinitis. Indeed, NSAIDs relieve pain in the acute phase, reduce the possibility of leg stiffness, and reduce modest symptoms of Achilles tendinitis due to their anti-inflammatory properties [9]. However, systemic treatment with NSAIDs has not been successful due to several limitations: (1) insufficient bioavailability of drugs to alter the cellular and molecular milieu in which the tendon heals, (2) the multiple administrations required to achieve prolonged therapeutic effects, and (3) the side effects (i.e., gastrointestinal bleeding, ulceration, and perforation) induced by their long-term use [10]. Furthermore, several studies have indicated that systemically administered NSAIDs may inhibit tendon cell migration and proliferation and impair tendon healing
https://doi.org/10.1016/j.jiec.2019.01.018 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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[11], and they showed little or no effect on the clinical outcome [12]. As an alternative, local injections of anti-inflammatory drugs can reduce pro-inflammatory factors and improve tendon healing in vivo [13,14]. However, the therapeutic effects of locally injected drugs with much higher dosages were not as effective as those of long-term drug delivery systems [13,14]. Therefore, drug delivery systems with controlled drug release ability have been used to overcome these limitations. We recently developed injectable drug delivery systems such as porous microspheres for the local treatment of Achilles tendinitis [13,14]. Porous microspheres have attracted considerable attention for controlled, long-term drug delivery systems [15]. Recently, using a simple fluidic device, we fabricated porous microspheres with drugs and used them to treat Achilles tendinitis [13,14]. Drug-loaded porous microspheres have more beneficial effects on suppression of inflammation and enhancement of tendon restoration in vivo compared to small-molecule drugs. Interestingly, we found that injectable porous microspheres were not fully effective in collagenase-treated tendon tissues. Herein, in order to enhance restorative effects on the entire area of tendon tissues, we fabricated polycaprolactone (PCL) fibrous sheets using electrospinning, which is a simple technique used to produce ultrafine and continuous submicron fibers and nanofibers [16,17]. The PCL fibrous sheet can be wrapped around the entire area of rabbit tendon tissue. Diclofenac (DFN), which is an NSAID that has been used widely to relieve pain and suppress inflammation [18], was immobilized on the surface of PCL fibrous sheets following dopamine (DOPA) coating to improve antiinflammatory and tendon restorative effects. In this study, we evaluated the in vitro anti-inflammatory effects on inflamed tenocytes by determining pro-inflammatory factors. For in vivo experiments, after the collagenase-treated tendon tissues in the rabbits were wrapped with DFN-immobilized PCL (DFN/PCL) fibrous sheet via surgical operation, the in vivo anti-inflammatory factors and tendon healing effects were quantified by measuring pro- and anti-inflammatory factors, histology, collagen content, and performing biomechanical tests. Materials and methods Materials PCL (Mw = 70,000–90,000), dichloromethane (DCM), tetrahydrofuran (THF), dopamine hydrochloride (DOPA), and lipopolysaccharide (LPS, from Escherichia coli) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphatebuffered saline (PBS), and penicillin-streptomycin were purchased from Gibco-BRL (Rockville, MD, USA). DFN sodium was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Cell counting kit (CCK)-8 was purchased from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). All other chemicals used were of analytical grade and were used as received without any further purification. Fabrication of DFN-immobilized PCL (DFN/PCL) fibrous sheet PCL fibrous sheets were fabricated using the electrospinning method [19]. Briefly, 2 g of PCL was clearly dissolved in 10 mL of THF, and the PCL solution was loaded into a 10 mL glass syringe with an 18-gauge needle. Then, the syringe containing PCL solution was placed in a syringe pump (KD-200, KD Scientific, Inc., Holliston, MA, USA) with 10 cm between the syringe needle tip and the collector. The syringe tip was connected to the positive output of a high-voltage power supply (12 kV), and the flow rate was set to 1 mL/h.
Prior to immobilization of DFN on a PCL fibrous sheet with width length thickness = 3 cm 5 cm 0.3 cm, the surface of the PCL fibrous sheet was modified with DOPA, which has a positively charged amine group. The PCL fibrous sheet [3 cm (width) 5 cm (length)] was placed in 15 mL of 10 mM TriHCl buffer (pH 8.0) solution containing DOPA (2 mg/mL) and gently shaken overnight under dark conditions. The DOPA-immobilized PCL (termed ‘aminated PCL’) sheet was washed with deionized water (DW) and dried under N2 gas. Next, the aminated PCL fibrous sheet (3 cm 5 cm) was immersed in 15 mL of 0.1 M MES buffer (pH 5.6) containing 1 or 5 mg of DFN. The DFN-immobilized PCL fibrous sheet was washed again with DW and dried under N2 gas. The 1 and 5 mg/ mL DFN-immobilized PCL fibrous sheets are hereafter referred to as DFN (1 mg)/PCL and DFN (5 mg)/PCL, respectively. Characterization of PCL and DFN/PCL fibrous sheets The surface morphologies of PCL, aminated-PCL, and DFN (1 mg or 5 mg)/PCL with were investigated via scanning electron microscope (SEM, S2300, Hitachi, Japan) at 3 kV. All samples were coated with gold using a sputter-coater (Eiko IB, Japan). To determine the amount of immobilized DFN on the surface of the fibrous sheets, each fibrous sheet (width length = 3 cm 5 cm) such as DFN (1 mg)/PCL and DFN (5 mg)/PCL were each dissolved in 2 mL of DCM. Then, PBS (15 mL) was added to each DCM solution and gently vortexed. After 3 h, the mixtures were centrifuged and aqueous PBS solutions were collected. The amount of DFN was determined at 285 nm using the UV/Vis spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). In vitro drug release study To evaluate in vitro DFN release from DFN (1 mg or 5 mg)/PCL, each sample (width length = 3 cm n5 cm) was placed in a 15-mL conical tube containing PBS buffer (pH 7.4, 15 mL), followed by gentle shaking in a water bath (37 C) oscillating at 100 times/min. At scheduled time intervals, the PBS was collected and replaced with an equal volume of fresh PBS. The collected PBS solutions were stored at 20 C before analysis. The released amount of DFN from the PCL fibrous sheets was determined by measuring the absorbance at 285 nm using the UV/Vis spectrophotometer. In vitro cytotoxicity test The cytotoxicity of PCL, aminated PCL, and DFN (1 mg or 5 mg)/ PCL with 1 cm (width) 1 cm (length) on tenocytes was estimated by the indirect method based on the ISO/EN 10993 Part 5 guidelines. To do this, tenocytes were isolated from the Achilles tendon tissues of rats as previously described [13]. To obtain the extraction medium, each fibrous sample was immersed in a 15 mL tube containing DMEM medium (1 mL), followed by gentle shaking in a water bath (37 C) oscillating 100 times/min. Tenocytes (5 104 cells/well) were incubated with DMEM containing 10% FBS and 1% antibiotics. After 24 h, the cells were washed with PBS and the extraction medium or diclofenac (500 mg/mL) was used to treat the cells. After Days 1 and 3 of incubation, DMEM medium was removed and CCK-8 reagent was added to each well, followed by further incubation for 1 h. The optical density of the live cells was determined at 450 nm using a Flash Multimode Reader (Thermo Scientific, CA, USA). In vitro anti-inflammatory effects on inflamed tenocytes The in vitro anti-inflammatory effects of DFN/PCL fibrous sheets on tenocytes were investigated by measuring the mRNA expression levels of the pro-inflammatory cytokines COX-2, IL-6, MMP-3, and TNF-α using real-time polymerase chain reaction (real-time
Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018
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PCR). Tenocytes (1 105 cells/mL) were carefully seeded on PCL, diclofenac (500 mg/mL), DFN (1 mg)/PCL (real dose = 63.48 1.01 mg of DFN), and DFN (5 mg)/PCL (real dose = 313.19 5.82 mg of DFN) fibrous sheets in 24-well tissue culture plates. The dimension of PCL, DFN (1 mg)/PCL, and DFN (5 mg)/PCL fibrous sheets was 1 cm width 1 cm length. After 24 h of incubation, all groups were treated with LPS (1 mg/mL). After Days 1 and 3, the cells were harvested and the total RNA was isolated from the harvested cells in each group using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). The total RNA concentration was measured using an ND1000 spectrometer (NanoDrop Technologies Inc., Montchanin, DE, USA). Then, the total RNA (1 mg) was reverse-transcribed into cDNA using AccuPower RT PreMix (Bioneer, Daejeon, Korea) according to the manufacturer’s protocols. The primers used for amplification of COX-2, IL-6, MMP-3, and TNF-α are as follows: COX-2, (F) 50 -CAG CCA TAC TAT GCC TCG GA-30 , (R) 50 -GGA TGT CTT GCT CGT CGT TC-30 ; IL-6, (F) 50 - CCG TTT CTA CCT GGA GTT TG -30 , (R) 50 - GTT TGC CGA GTA GAC CTC AT -30 ; MMP-3, (F) 50 -ACC TGT CCC TCC AGA ACC TG-30 , (R) 50 -AAC TTC ATA TGC GGC ATC CA-30 ; and TNF-α, (F) 50 - CTC CCA GAA AAG CAA GCA AC -30 , (R) 50 - CGA GCA GGA ATG AGA AGA GG-30 . PCR amplification and detection were carried out using an ABI7300 Real-Time Thermal Cycler (Applied Biosystems, Foster, CA, USA). The mRNA levels of these pro-inflammatory cytokines were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and are expressed as relative values.
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Induction of Achilles tendinitis in a rabbit model and in vivo treatment with fibrous sheets In vivo animal experiments were performed and approved by the Institutional Animal Care and use Committee of the Korea University Medical Center (KOREA-2018-0052). New Zealand White rabbits (DooYeol Biotech, Seoul, Korea) were anesthetized with isoflurane (1% w/v in 2 L oxygen). As seen in Fig. 1B, after shaving the rabbit’s right hind limb, Achilles tendon tissue was carefully exposed, and 50 mL of collagenase type I [50 mg/ mL in PBS (pH 7.4)] was injected into the exposed Achilles tendon tissues. Then, collagenase-treated tendon tissue was carefully wrapped with PCL, DFN (1 mg)/PCL, or DFN (5 mg)/PCL fibrous sheets (width length = 3 cm 5 cm, Fig. 1A). The wrapped fibrous sheet was fixed using an absorbable 4-0 Vicryl (Ethicon, Somerville, NJ, USA), and the remnant sheet was trimmed with scissors. After trimming the remnant fibrous sheet with scissors, the dimension of wrapped fibrous sheet on tendon tissues was approximately 3 cm (width) 2 cm (length). The real drug contents of DFN (1 mg)/PCL [3 cm (width) 2 cm (length)] and DFN (5 mg)/PCL (width length = 3 cm 2 cm) were approximately 0.380 0.006 mg and 1.880 0.524 mg, respectively. Finally, the subcutaneous tissue and skin were sutured using an absorbable 4-0 Vicryl. In the positive drug control group, diclofenac (5 mg) solution was injected into the collagenase-injected Achilles tendon. After four weeks of
Fig. 1. (a) Photo of DFN/PCL scaffolds (width length = 3 cm 5 cm). (b) Serial photographs show the wrapping of DFN/PCL fibrous scaffold to treat Achilles tendinitis. After shaving the area, Achilles tendon tissue was carefully exposed, and 50 mL of collagenase type I (50 mg/mL) was injected into tendon tissue. Collagenase-treated tendon tissue was carefully wrapped with PCL or DFN/PCL fibrous sheets. The wrapped fibrous sheets were fixed by suturing and the remnant sheet was trimmed. Then, the subcutaneous tissue and skin were sutured.
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treatment, rabbits were euthanized and tendon specimens were harvested for the evaluation of histopathology, in vivo antiinflammatory effects, and biomechanics. Experimental subjects were randomly divided into six groups: (I) control (no treatment), (II) collagenase (collagenase injection), (III) Col (I) and PCL, (IV) Col (I) and diclofenac (5 mg) solution, (V) Col (I) and DFN (1 mg)/PCL (real dose = 0.380 0.006 mg) and (VI) Col (I) and DFN (5 mg)/PCL (real dose = 1.880 0.524 mg). In vivo anti-inflammatory effects on Achilles tendinitis rabbit models The in vivo anti-inflammatory effects of DFN/PCL fibrous sheets on Achilles tendinitis rabbit models were evaluated by determining the gene expression of pro-inflammatory cytokines (COX-2, IL-6, MMP-3, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) using real-time PCR. The total RNA from the isolated tendon tissues from rabbits in each group was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The total RNA concentration was measured using an ND-1000 spectrometer (NanoDrop Technologies Inc.). The total RNA (1 mg) was reverse-transcribed into cDNA using AccuPower RT PreMix (Bioneer). The same primer sequences for the proinflammatory cytokines were used as described above in the in vitro cell study. The primer sequence of the anti-inflammatory cytokines IL-4, IL-10, and IL-13 were as follows: IL-4, (F) 50 -ACA GGA GAA GGG ACG CCA T-30 , (R) 50 -GAA GCC CTA CAG ACG AGC TCA-30 ; IL10, (F) 50 -GGT TGC CAA GCC TTA TCG GA-30 , (R) 50 -ACC TGC TCC ACT GCC TTG CT-30 ; and IL-13, (F) 50 -AGA CCA GAC TCC CCT GTG CA-30 , (R) 50 -TGG GTC CTG TAG ATG GCA TTG-30 . PCR amplification and detection were performed on an ABI7300 Real-Time Thermal Cycler (Applied Biosystems) using a DyNAmoTM SYBR1 Green qPCR Kit (Finnzymes, Espoo, Finland). The relative mRNA levels of pro- and anti-inflammatory cytokines were normalized to those of GAPDH.
2.10. Hydroxyproline assay To evaluate the Achilles tendon healing effect, the collagen contents of Achilles tendon tissues in each group were measured by hydroxyproline assay. In short, Achilles tendon tissues (5 mg) were hydrolyzed with 12 N HCl on a hot plate at 120 C for 3 h, and DW (20 mL) was added to the hydrolyzed tissues. Then, NaOH solution was slowly added to the hydrolyzed tissue solution until the solution was neutralized. To initiate hydroxyproline oxidation, 50 mL of 60 mM chloramine T solution was mixed with 100 mL standard of hydroxyproline or samples in a 96-well plate. After 20 min of incubation at room temperature, the chloramine T was destroyed by adding 50 mL of perchloric acid to each well. Finally, the absorbance was recorded at 550 nm using a Flash Multimode Reader. Biomechanical test The tendon specimens (width length = 2 2 cm) were securely fixed to a specially designed device, which allowed the specimens to be oriented such that a tensile load could be applied along the axis of the tendon. The fixed tendon specimen from each group was tested on an Instron Mechanical Tester (AG-10KNX, Shimadzu, Kyoto, Japan), which was operated at 5 mm/min velocity traction speed. The ultimate tensile strength (defined as maximum stress or force per unit area) and stiffness (the force per unit displacement) were measured. Statistical analysis Data are represented as mean standard deviation (n = 5). Statistical comparisons were carried out via one-way analysis of variance (ANOVA) using Systat software (Chicago, IL, USA). Differences were considered statistically significant at *P < 0.05 and **P < 0.01.
Histopathological study
Results
The harvested calcaneus-Achilles tendon tissues were fixed in 3.7% paraformaldehyde and embedded in paraffin. The tissues were crosssectioned to a thickness of 5 mm in the longitudinal parallel direction using a rotary microtome (HM 355S automatic microtomes, Thermo Scientific). Hematoxylin and eosin (H&E) and Masson’s Trichrome staining were done to confirm the in vivo anti-inflammatory and tendon restoration effects of DFN/PCL fibrous sheets.
Characterization of PCL and DFN/PCL fibrous sheets The surface morphologies of the fabricated fibrous sheets were examined by SEM. All fibrous sheets (PCL, aminated PCL, and DFN (1 mg or 5 mg)/PCL) had irregular structures and smooth surfaces with diameters of approximately 200 nm to 3 mm (Fig. 2). There were no substantial differences in diameter among the fibrous
Fig. 2. SEM images of PCL, aminated PCL, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Scale bar: (a)–(d) = 100 mm, and (e)–(h) = 10 mm.
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Fig. 3. (a) In vitro release profiles of diclofenac from DFN (1 mg)/PCL and DFN (5 mg)/PCL (n = 5). (b) Cytotoxicity of PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL against tenocytes at Days 1 and 3 (n = 5).
sheets. The immobilization efficiency of DFN on the surface of DFN (1 mg)/PCL and DFN (5 mg)/PCL was 95.23 1.51% and 93.95 1.74%, respectively. Also, the immobilized DFN contents of DFN (1 mg)/PCL and DFN (5 mg)/PCL were 952.26 15.20 mg and 4697.79 87.30 mg, respectively. In vitro drug release and cytotoxicity studies The in vitro DFN release profiles from the DNF/PCL fibrous sheets are seen in Fig. 3(a). On Day 1, the released amounts of DFN were 248.25 0.07 mg (24.83 0.01%) for DFN (1 mg)/PCL and 812.59 0.07 mg (16.25 0.01%) for DFN (5 mg)/PCL. At Day 28, 749.12 0.22 mg (74.91 0.02%) and 2187.50 0.82 mg (43.75 0.02%) of DFN were released from DFN (1 mg)/PCL and DFN (5 mg)/PCL, respectively. The prepared DFN/PCL fibrous sheets have no high initial burst release, and they also showed sustained drug release up to 28 days.
The cytotoxicity of PCL, diclofenac, DFN (1 mg or 5 mg)/PCL, and DFN (5 mg)/PCL against tenocytes was evaluated at Days 1 and 3. As seen in Fig. 3(b), no significant cytotoxic effects are observed and the cell viabilities in all groups were maintained over 95% relative to that of the control group. These results indicate that PCL, diclofenac and DFN/PCL fibrous sheets were non-toxic and safe on tenocytes. In vitro anti-inflammatory effects of DFN/PCL on inflamed tenocytes To determine whether DFN/PCL inhibits inflammation responses in inflamed tenocytes, we incubated tenocytes with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Then, we stimulated the cells in all groups with LPS and assessed the mRNA expression levels of COX-2, IL-6, MMP-3, and TNF-α using real-time PCR (Fig. 4). PCL alone had no effect in decreasing the mRNA levels of these cytokines in inflamed tenocytes. However, diclofenac and
Fig. 4. Relative mRNA expression levels of pro-inflammatory cytokines in inflamed tenocytes. (a) COX-2, (b) IL-6, (c) MMP-3, and (d) TNF-α in LPS-stimulated tenocytes cultured on PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL at Days 1 and 3. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
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Fig. 5. Relative mRNA expression levels of pro-inflammatory cytokines in collagenase-treated tendon tissues. (a) COX-2, (b) IL-6, (c) MMP-3, and (d) TNF-α in collagenaseinjected tendon tissues treated with PCL, diclofenac (5 mg), DFN (1 mg)/PCL, and DFN (5 mg)/PCL at four weeks. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
DFN/PCL fibrous sheets were comparably and dose-dependently effective in attenuating the mRNA levels of inflammatory cytokines on Days 1 and 3 compared to LPS or PCL alone (*P < 0.05 or ** P < 0.01). In cell culture condition, DFN/PCL fibrous sheets showed less anti-inflammatory effect compared to diclofenac due to the sustained release of DFN from the fibrous sheet. In vivo anti-inflammatory effects in Achilles tendinitis rabbit model To further investigate the in vivo anti-inflammatory effects of DFN/PCL fibrous sheets in Achilles tendinitis rabbit models, we determined the mRNA levels of pro-inflammatory cytokines (COX2, IL-6, MMP-3, and TNF-α) as well as anti-inflammatory cytokines (IL-4, IL-10, and IL-13) in tendon tissues isolated from the rabbits in each group. Fig. 5 shows that treatment of tendon tissues with collagenase markedly increased the mRNA expression levels of all of pro-inflammatory cytokines up to over 7-fold compared to the normal group. No significant differences of these inflammatory
cytokines in tendon tissue treated with PCL were observed compared to those in the collagenase-treated group. Two DFN/ PCL groups were much more effective in decreasing the mRNA levels of these cytokines than diclofenac in a dose-dependent manner (**P < 0.01). To further confirm whether DFN/PCL influences the expression of the anti-inflammatory cytokines, the mRNA expression levels of IL-4, IL-10, and IL-13 were analyzed using real-time PCR (Fig. 6). Compared to the normal group, collagenase treatment markedly decreased the mRNA levels of IL-4, IL-10, and IL-13. The PCL group did not increase the mRNA levels of these anti-inflammatory cytokines. Diclofenac and two DFN/PCL groups increased their mRNA levels. However, two DFN/PCL groups displayed much higher mRNA levels of anti-inflammatory cytokines in a dosedependent manner than did the diclofenac-treated group (**P < 0.01), suggesting that the DFN/PCL system was more effective in suppressing inflammation responses and enhancing healing and restoration of tendon tissues.
Fig. 6. The relative mRNA expression levels of anti-inflammatory cytokines in collagenase-treated tendon tissues. (a) IL-4, (b) IL-10, and (c) IL-13 in collagenase-injected tendon tissues treated with PCL, diclofenac (5 mg), DFN (1 mg)/PCL, and DFN (5 mg)/PCL at four weeks. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
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Histological validation of tendon tissues To assess the anti-inflammatory and tendon healing effects of DFN/PCL in vivo, we performed histopathological examinations with H&E (Fig. 7A) and Masson’s trichrome staining (Fig. 7B). In the
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control group, normal tendon tissues showed typical spindleshaped tenocytes arranged parallel to the fiber pattern as well as uniform, compact, and longitudinally well-aligned collagen fibers (Fig. 7A(a) and B(a)). In contrast, collagenase-treated tendon tissues exhibited a large number of inflammatory cells and clear
Fig. 7. Histological evaluations with (A) H&E and (B) Masson’s trichrome staining at four weeks after collagenase (Col (I)) injection into tendon tissues and treatments with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Groups are categorized as follows: (a) control (no treatment), (b) Col (I), (c) Col (I) + PCL, (d) Col (I) + diclofenac, (e) Col (I) + DFN (1 mg)/PCL, and (f) Col (I) + DFN (5 mg)/PCL. Scale bar: 50 mm.
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Fig. 8. Hydroxyproline contents in collagenase-injected tendon tissues at four weeks after treatment with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
increased the hydroxyproline levels compared to the collagenasetreated group (**P < 0.01). Significant increases of the hydroxyproline content in Achilles tendon in two DFN/PCL groups were observed in a dose-dependent manner compared to those in diclofenac (*P < 0.05 or **P < 0.01). In particular, DFN (5 mg)/PCL group had the highest hydroxyproline content among the treatment groups (**P < 0.01). To further demonstrate the tendon restorative effects of DFN/ PCL, biomechanical studies were performed to determine stiffness and tensile strength of tendon tissues. As shown in Fig. 9(a) and (b), the stiffness and tensile strengths of the tendon tissues treated with collagenase or PCL were much lower than those of normal tendon tissue. Diclofenac and both DFN/PCL groups increased the stiffness and tensile strength of the tendon tissues. DFN (1 mg)/PCL showed similar or moderate effects in increasing the stiffness and tensile strength compared to diclofenac. However, DFN (5 mg)/PCL had much higher stiffness and tensile strength than diclofenac and DFN (1 mg)/PCL (*P < 0.05 or **P < 0.01). Discussion
degenerative changes including the entire fiber disruption and fiber mal arrangement (Fig. 7A(b) and B(b)). PCL treatment alone still showed many inflammatory cells and overall loss of collagen fibers (Fig. 7A(c) and B(c)). In the diclofenac (5 mg) treated group, the inflammatory cells, the collagen fiber disruption, and fiber mal arrangement were still observed (Fig. 7A(d) and B(d)) although the number of inflammatory cells was decreased and the collagen fibers were sporadically restored (Fig. 7B(d)). Two DFN/PCL groups decreased the number of inflammatory cells and restored the collagen fiber rearrangement (Fig. 7A(e and f) and B(e and f)) compared to the collagenase- and diclofenac-treated groups. Although the DFN (1 mg)/PCL group seemed to be have better attenuation of inflammatory cells and repair of collagen fiber organization, its efficacy was moderate compared to that of the diclofenac-treated group. However, DFN (5 mg)/PCL showed a significant decrease of inflammatory cells and exhibited overall restoration of collagen fibrillary organization (Fig. 7A(f) and B(f)), indicating that DFN (5 mg)/PCL has the best anti-inflammatory effects and tendon restoration effects. Determination of hydroxyproline content and biomechanical tests To quantify changes in collagen content of Achilles tendinitis models, the hydroxyproline content was determined after the treatments (Fig. 8). Compared to the control (normal) group, the hydroxyproline content was significantly decreased in collagenase- and PCL-treated groups, with similar hydroxyl hydroxyproline content. Diclofenac- and two DFN/PCL-treated groups greatly
Achilles tendinitis commonly occurs during athletic activity or in the course of daily life and causes pain, inflammation, and impaired tendon function in its early stage [20], eventually leading to partial or total tendon ruptures. Recently, emerging evidence has indicated that a strong inflammatory component is involved in the pathogenesis of tendon disease with inflammatory cells and cytokines as important regulators of the tendon extracellular matrix (ECM) [21]. High levels of cytokines may result in collateral tissue damage and impaired tendon healing [22]. Indeed, tendon disruption and injuries provoke local inflammation responses by inducing pro-inflammatory cytokines (i.e., IL-1β, TNF-α, and COX2), ECM degrading enzymes (i.e., MMPs), and ECM disintegration [5]. Therefore, the suppression of inflammation in Achilles tendinitis is considered as a therapeutic approach for Achilles tendinitis. DFN is a widely used non-steroidal anti-inflammatory drug (NSAID) [23]. The mechanism of DFN action is intervened in habiting COX, an enzyme converting arachidonic acid into thromboxane and prostacyclin, and thereby the suppression of prostaglandin synthesis [18,24–27]. However, when orally administered, DFN causes adverse effects (i.e., gastrointestinal damage and disorders and stomach ulceration) when taken at high dosages for a long period and undergoes first pass metabolism (approximately 50%). Due to these drawbacks, DFN has been used in several topical formulations such as gel, spray, lotion, and patch [18]. Several groups have suggested that topical delivery of DFN is a safer, more effective method for reducing pain and improving
Fig. 9. (a) Stiffness and (b) tensile strength of tendon tissues isolated from rabbits in each group at four weeks after collagenase (Col (I)) injection into tendon tissues and treatment with PCL, diclofenac, DFN (1 mg)/PCL, and DFN (5 mg)/PCL. Data represent mean SD (n = 5), (*P < 0.05 and **P < 0.01).
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physical function for the treatment of several painful diseases including rheumatoid arthritis, osteoarthritis, and Achilles tendinitis [18,23,28]. Recently, the Paula group reported that topical delivery of DFN using gold nanoparticles and phonophoresis was more effective in controlling the inflammatory cytokines such as IL-1β and TNF-α in a tendinopathy model [29]. In previous studies, our group developed porous microsphere systems for long-term drug delivery to treat Achilles tendinitis [13,14]. The anti-inflammatory drug-delivering porous microsphere systems effectively suppressed inflammation and restored tendon tissues. However, histological studies showed that the tendon restorative effect of these microsphere systems was only partial. Considering these outcomes, we attributed the partially restorative effect on the tendon to the local injection of porous microsphere systems by syringe due to their residence of limited area at around injected areas [13,14]. In order to improve tendon restorative effects in overall tendon tissues for the treatment of Achilles tendinitis, we fabricated DFN-immobilized PCL fibrous sheets (width length = 3 5 cm) capable of wrapping the overall area of collagenase-treated tendon tissues. DFN/PCL fibrous sheets were prepared by easy surface modification of DOPA molecules with catechol functional group on PCL fibrous sheet to introduce the positively charged amine group [30], followed by the immobilization of DFN with negatively charged carboxyl group on the surface of the aminated-PCL via electrostatic interactions. In vitro release and cytotoxicity studies demonstrate that DFN/PCL systems are suitable for long-term delivery due to its sustained drug release profiles for four weeks without high initial burst release and they are safe materials without toxic effects. Cytokines are well known as major mediators of inflammatory responses. To demonstrate the anti-inflammatory effects of DFN/ PCL fibrous sheet systems, we determined the mRNA levels of proinflammatory and anti-inflammatory factors in inflamed tenocytes or collagenase-treated tendon tissues. The in vitro anti-inflammatory effects of the DFN/PCL system were assessed against tenocytes, which are fibroblast-like differentiated cells that form the mature tendon. Previous studies reported that the tenocytes after LPS treatment increased the expression of several cytokines such as TNF-α, IL-6, and IL-1β. Also, these cytokines further stimulated tenocytes to produce pro-inflammatory cytokines (i.e., TNF-α, IL-6, IL-1β, and COX-2) and proteases (i.e., MMP-1, -3, and -13), which are thought to drive disease progression [31,32]. This study showed that DFN/PCL systems remarkably and dosedependently suppressed the mRNA levels of pro-inflammatory cytokines in inflamed tenocytes. The in vivo studies consistently supported that DFN/PCL systems markedly decreased the mRNA levels of pro-inflammatory cytokines (i.e., COX-2, IL-6, and TNF-α) and protease (i.e., MMP-3), which were expressed in collagenasetreated tendon tissues, whereas they could enhance the mRNA expressions of anti-inflammatory cytokines (i.e., IL-4, IL-10, and IL13). Mature tendons normally have low cellular density. Approximately 90-95% of the cellular content of tendons comprises tendon-specific cell types including tenoblasts and tenocytes [33]. Tenocytes play an important role in producing tendon ECM [34]. Tendon ECM components are composed predominantly of collagen fibrils (>95% of collagen type I, and other collagen types (type III and type V)) and a small fraction of elastin embedded in hydrated proteoglycans [35]. The parallel organization of collagen fibrils in tendons results in optimal stiffness and tendon tensile strength and biomechanical stability [35]. Histological examinations with H&E and Masson’s trichrome staining showed that collagenase injection into rabbit tendon tissues resulted in the recruitment of a large number of inflammatory cells and severe disruption of the well-aligned collagen fiber organization. These phenomena might be attributed to an inflammatory response by expressing
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inflammatory cytokines as well as mechanical degradation by producing protease after collagenase injection into tendon tissues. Although diclofenac suppressed the number of inflammatory cells and collagen matrix degeneration, DFN/PCL systems, in particular DFN (5 mg)/PCL, exerted more beneficial effects in preventing inflammation responses and collagen disruption as well as in repairing the parallel collagen fiber organizations. Also, the greatest tendon restorative effects of DFN (5 mg)/PCL group can be explained by the increased hydroxyproline content in tendon tissues because the hydroxyproline content is directly correlated to early maturation of fibroblasts, early parallel arrangement of collagen fibers, and bundle formation [36]. Moreover, we suggest that more significant enhancement of mechanical properties such as stiffness and tensile strength by DFN (5 mg)/PCL is closely related with the increase of both collagen fiber organization and the absolute amount of collagen. In the present study, we demonstrated that our long-term drug delivery system of DFN/PCL fibrous sheets suppresses inflammation reactions in both inflamed cells and collagenase-treated tendon tissues, as well as enhancing tendon healing, as demonstrated by restoration of collagen fibril structures and improved mechanical properties of tendon tissues. Treatments of small molecular drugs also have promising therapeutic effects. However, due to the relatively short residence time at injured sites, multiple injections are required to achieve the greater therapeutic effects. In contrast, the use of a long-term drug delivery system such as a DFN/PCL fibrous sheet could achieve more beneficial therapeutic outcomes due to the sustainable delivery of drug molecules for a long time to injured sites without multiple treatments. Furthermore, compared to porous microsphere systems for long-term drug delivery, PCL fibrous sheet systems may have an advantage because they can cover the overall area of tendon tissues via wrapping, leading to homogeneous distribution of diclofenac molecules into collagenase-treated tendon tissues. Therefore, DFN/ PCL fibrous sheet systems have great potential for Achilles tendinitis treatment. Conclusion Herein, we demonstrated robust anti-inflammatory and tendon restoration effects of a long-term diclofenac delivery system using PCL fibrous sheets on rabbit models with collagenase-induced Achilles tendinitis. DFN/PCL systems significantly suppressed inflammation responses that occurred in inflamed tenocytes and/or tendon tissues of Achilles tendinitis models by reducing the expression of pro-inflammatory cytokine genes (i.e., COX-2, IL6, and TNF-α) and protease such as MMP-3 as well as by increasing the mRNA levels of anti-inflammatory cytokines (i.e., IL-4, IL-10, and IL-13). Also, we confirmed that the inflammation inhibition of DFN/PCL systems resulted in increased collagen content and restoration of collagen fiber organization in tendon tissues. This study shows that wrapping collagenase-treated tendon tissues with DFN/PCL fibrous sheets improves tendon healing of Achilles tendinitis. Acknowledgement This study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI13C1501). References [1] H. Aslan, N. Kimelman-Bleich, G. Pelled, D. Gazit, J. Clin. Invest. 118 (2) (2008) 439. [2] K.M. Khan, N. Maffulli, Clin. J. Sport Med. 8 (3) (1998) 151.
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Please cite this article in press as: T.H. Lee, et al., Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j. jiec.2019.01.018