Effect of methoxatin loaded chitosan conduit on deep digital flexor tendon healing in rabbits: An animal model study

Chitosan is of great interest in regenerative medicine because of its plentiful properties, like biocompatibility, biodegradability and non-toxicity. The objective of the present study was histopathological and biomechanical survey on effect of methoxatin loaded chitosan conduit on Deep Digital Flexor Tendon (DDFT) healing in rabbit models. Eighteen healthy male white New Zealand rabbits were randomized into three groups of six animals each. In CONTROL group the DDF tenotomy was performed and the sumps were sutured. In CTN (chitosan) group the DDF tenotomy was performed and the sumps were sutured and CTN conduit was wrapped around the damaged area. In CTN/METHO (chitosan/methoxatin) group the procedure was the same as CTN group as well as local administration of 100 μL methoxatin (100 μg/Rabbit) into the CTN conduit. The histopathological assessments including infl ammation, angiogenesis and collagen fi bers arrangement, and biomechanical assessments were performed after 8 weeks. Histopathological observations showed that the conduit was absorbed and adhesion around the tendon was deceased in CTN and CTN/ METHO groups. There were no noticeable signs of infection and tissue reaction in the granulation tissue in CTN/METHO group compared to other groups (P<0.05). Local administration of methoxatin in combination with chitosan conduit could accelerate deep digital fl exor tendon healing via decrease in adhesion around the tendon with no signs of excessive tissue reaction or infection in rabbits. Research Article Effect of methoxatin loaded chitosan conduit on deep digital fl exor tendon healing in rabbits: An animal model study Negin Mozafari1*, Mohammad Velayati2, Azad Bahrampour3, Erfan Yarahmadi2, Roghaiyeh Neisari4 and Rahim Mohammadi2 1Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran 2Department of Surgery and Diagnostic Imaging, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran 3Young Researchers and Elite Club, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran 4Department of Anesthesiology, Emam Khomeini Hospital, Urmia University of Medical Sciences, Urmia, Iran Received: 10 December, 2020 Accepted: 26 December, 2020 Published: 28 December, 2020 *Corresponding author: Negin Mozafari, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran, Tel: +98 41 33340081; Fax: +98 44 33344013; E-mail:


Introduction
Functional association among the dynamic and the static parts of the musculoskeletal system transferring muscle contraction to the skeletal system are tendons, hence, ending up motion. Accordingly, function and motion are compromised in tendon damage ranging from acute traumatic ruptures to chronic overuse and degenerative tendinopathy. However, still with amended therapeutic approaches including nonsurgical, surgical, and rehabilitation techniques, results are not satisfactory because repaired tendon tissue infrequently attains functionality the same as pre-dameged condition [1,2].
Tendon damages result in extensive morbidity, and ensued debility can remain for several months in spite of what is called proper treatment [3].
Injuries to tendons may be acute or chronic, and are produced by alone or in combination of intrinsic or extrinsic causes. In case of acute trauma, extrinsic factors prevail and in chronic cases intrinsic reasons also play a crucial role. In chronic tendon injuries there are associations between intrinsic and extrinsic factors. It has been demonstrated that in two-thirds of athletes with Achilles tendon injuries, intrinsic factors like alignment and biomechanical shortcomings play a crucial role [3].
Chitosan is a linear polysaccharide and is linked with scarless repair of soft tissues and has been demonstrated to prevent adhesion formation within tendon repair following surgery [4,5]. Chitosan inclines to precipitate in physiologic pH that explains its effectiveness. A chitosan solution that does not precipitate in physiologic settings was recently produced [6].
Therefore, no precipitation allows it to adhere to the healing site for enough time to take effect. These properties pave the way for intimate contact between chitosan and tendon, hence, enabling guided-tissue regeneration and avoiding adhesion formation. Other biological mediators like platelet-rich plasma are administered as fl uid rather than gel and are therefore more prone to diffuse from the repair site, modifying their effects. Therefore, chitosan seems to be unique among other mediators [7].
The restricted capacity of tendon for self regeneration and the overall inadequacies of existing treatment schedules have augmented the enthusiasm to develop tissue engineering approaches for tendon healing. Recently, one of the specifi c attitudes has been adoption of variuos types of scaffold to renew functionality of tendons and ligaments. It is important to scheme and formulate a appropriate scaffold for application in specifi c tissue repair, because it contacts with tissue cells, and delivers structural upkeep and regulation for successive tissue development. Towards this, more attention has been paid to the design of scaffolds for guiding cell behaviors and tissue regeneration, and the design of scaffolds should be based on knowledge learned from native tissues, such as their anatomic structures, compositions and functions [8].
The development of infl ammation normally ends up the discharge of biologically active mediators to draw neutrophils, leucocytes and monocytes to the area of wound in order to invade foreign debris and microorganisms via phagocytosis.
This progression results in the generation of oxygen-free radicals like hydrogen peroxide, superoxide anion, and hydroxyl anion, that their excess, leads to tissue injury where they devastate the natural antioxidant enzymes of the host like catalase, superoxide dismutase, and glutathione peroxidase.
Hence, antioxidants avoid the free radicals activity and avoid cells and tissues injury, and also augment healing of wounds with or without infection [9,10].
In tissue damage, free oxygen radicals react with DNA and generate 8-hydroxyguanine (8-OHGua) that is DNA damaged product. Production of free oxygen radicals takes place uninterruptedly in cells and existence of defense systems for endogenous antioxidant protects tissues from detrimental impacts of free oxygen radicals [11]. It has been demonstrated that there are different anti-infl ammatory and antioxidant free radical scavengers that bear constructive effects to avoid ischemic/reperfusion damages in tissues [12][13][14]. It has been indicated that methoxatin performs like an antioxidant, and it is able to prevent lipid peroxidation damage, enhance thymidine incorporation into fi broblasts and augment growth factors generation [15].
The aim of theour research was to histopathologically and biomechanically explore infl uence of methoxatin loaded chitosan conduit on deep digital fl exor tendon healing in rabbit models. The assessments were based on macroscopic, histopathological and biomechanical criteria.

Materials and methods
Preparation and fabrication of chitosan conduit, animal grouping and procedures CTN (85% deacetylated medium molecular weight) was supplied from Fluka, Sigma-Aldrich (USA). Acetic acid and Glycerol were purchased from Merck (Germany) and Sigma Chemical Co. (USA). The aqueous solution (1% V/V) of glacial acetic acid was prepared at fi rst, then CTN solution (2% W/V) was prepared by adding 2 g CTN to 100 ml acetic acid (1% V/V) while stirring on a magnetic stirrer-hot plate, The solution was stirred with low heat (at 50°C) for 1 hour. The resultant CTN solution was fi ltered through a Whatman No. 3 fi lter paper (UK) to remove any un-dissolved particles and to prevent the fragility of CTN, glycerol was added in amount of 30% of the total solid weight in solution [16,17].
The conduit was fabricated according to works of other researchrs [18]. The mold with CTN solution was put in a -80 ˚C freezer for 12 h. The frozen molds were placed at room temperature and after 5 min; the outer layers of frozen molds were removed. The frozen solutions were dried in a freezedryer (model Alpha 1-4 LDplus; Martin Christ, Osterode, Germany). The main drying temperature was -40 ˚C and the main drying pressure was 12 Pa for 15 hr. Then, the scaffolds were immersed into 2.00% (w/v) sodium hydroxide solution (Merck) and equilibrated for 20 min to exclude the residual acetic acid. Scaffolds were cleaned using deionized water until the rinsing solution was neutral, and then equilibrated in 0.20 mol L-1 phosphate buffered saline (pH: 7.40) for half an hour and fi nally scaffolds were dried at room temperature for 6 hr. 19,20 The fabricated conduit was 0.20 mm thick and 3.50 ± 0.50 mm in inner diameter. All of the conduits were sterilized with formaldehyde tablets in airtight containers for 24 h.
Forty eight male New Zealand white rabbits, weighing 2.5-3.0 kg, were included into the present study. The rabbits were placed in standard cages and fed with commercial rabbit pellet and water freely. All processes were perfomed based on the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The research project received the confi rmation of the Institution Ethics Committee.
The rabbits were divided into three groups of 6 animals each, randomly. They were anesthetized intramuscularly using xylazine hydrochloride 10 mg/kg (Alfasan, The Netherlands) and ketamine hydrochloride 40 mg/kg (Ketaset, Germany). The right hind limb of each rabbit was prepped and plantar skin incision was made longitudinally and DDF tenotomy was pattern using 3-0 monofi lament nylon (Ethilon, Ethicon, Inc., Somerville, NJ, USA). In CONTROL group, the DDF tenotomy was performed and the sumps were sutured. In CTN group the DDF tenotomy was performed and the sumps were sutured and CTN conduit was wrapped around the damaged area. In CTN/METHO group the procedure was the same as CTN group as well as local administration of 100 μL Methoxatin (100 μg/ Rabbit) into the CTN conduit.  (Table 1) [19]. from each group) and fi xed in 10% formalin solution. They were then dehydrated and embedded in paraffi n wax, sectioned at 5 μm and stained with and stained with Hematoxylin and Eosin (H&E) and Masson's trichrome stains. Images were taken using light microscope to assess infl ammation, angiogenesis and collagen fi brils arrangement.

Statistical analysis
Kruskal-Wallis variance analysis were used to evaluate differences among groups. Multiple comparison tests were adopted to know differences when the P-value from the Kruskal-Wallis test statistics was signifi cant statistically. SPSS 18 (SPSS Inc., Chicago, IL, USA) was adopted for statistical analysis. A p-value < 0.05 was set as statistical signifi cance.

Macroscopic fi ndings
There were no signs of local infection around tendons in all experimental groups. The conduit was absorbed in the CTN/ METHO and CTN groups. Remarkable peritendinous adhesions were found in the CONTROL group that needed sharp dissection for detachments. The adhesion scores in CTN/METHO and CTN groups were signifi cantly lower than that of the CONTROL group (P=0.001) ( Table 2).

Histological assessments
Following 8 week post operation and after macroscopic assessments the tendon samples were taken (three samples  (Figure 1).  2-4).

Discussion
Findings of tendon handling for repair are not satisfactory The objective in management of tendon pathologies, acute or chronic, should be as close as to a natural tendon injury with analogous characteristics and in this regard degenerated tissues are great challenges [18][19][20].
Infl ammation in tendon drops few days after injury, and syntheses of fi broblasts proliferation, extracellular matrix and mostly collagen type III after fi ve day. The newly synthetized collagen fi brils are arranged in the extracellular matrix in a random fashion and after 3-4 weeks are aggregated in organized bundles. Diminution in collagen type III contents and escalation in collagen type I synthesis are considered as a key properties of tendon healing remodeling phase starting within two month post injury. In spite of immature and weak nature of collagen type III fi bers and their random orientation, they are responsible for neotendon stability [20][21][22]. Furthermore, high expression of type I collagens and longitudinal orientation of these fi bers are thought to be indispensable to get to the    the time in which the tendon would be at the risk of re-injury [23][24]. Therefore, the neotendons were evaluated within two months. It has been approved that extreme infl ammatory response will interfere with the proliferative phase of healing and the tensile strength of the wound repair will decline as a result of scar formation [25].
Histopathological results of our study showed signifi cant reduction in infl ammatory cells was observed in CTN/METHO group, indicating benefi cial effect of methoxatin loaded chitosan conduit in tendon repair.
Chitosan that is a natural polymer from deacetylation of chitin (poly-N-acetylglucosamine), has been extensively used as local dressing in wound healing because it bears antimicrobial and nontoxic, biocompatible and biodegradable characteristics [26]. At the end of the study period, the conduits were totally absorbed in CTN/METHO and CTN groups indicating biocompatibility and biodegradability of the conduit.
Adhesion formation following trauma to tendon still is challenging in practice and no satisfactory preventive measure has been established. It could be possible to formulate improved approaches to prevent adhesion formation due to advances in the understanding of the mechanisms involved [27]. Trauma is considered as the most important factor involved in adhesion formation is [28]. Key cells in tendon healing are tenocytes and tenoblasts. The actin isoform has been identifi ed in tendons and ligaments [29]. Tenocytes that express -smooth muscle actin are known as myofi broblasts. Stress fi bers (actin microfi laments), well-developed cell-stroma attachment sites (fi bronexus) and intercellular gap junctions are three essential morphological elements that defi ne myofi broblasts [30].
Tensile forces are transferred to extracellular matrix network by fi bronexus [31]. Extracellular matrix network homeostasis in tendons and ligaments is achieved by myofi broblasts that are responsible for tendon adhesions formation [32]. Efforts have been made to diminish formation of adhesion by usage of materials acting as mechanical barriers like polyethylene or silicone or by usage of pharmacological agents like ibuprofen and indomethacin, however no simple method is widely adopted [33][34][35].
Other investigators showed that chitosan avoids proliferation in sheath cells of tendon and production of collagen [36][37]. Chitosan prevents survival of fi broblasts that may be one of the explanations for the augmentation of gliding of tendon. The adhesion formation inhibits the gliding function of tendon and therefore, limits the range of motion of affected limb [37]. In our fi ndings there was no peritendinous adhesions in CTN/METHO and CTN groups indicating that tendon gliding was achieved in the injured tendons.
The collagen fi bers are considered as the leading structural components of tendon in charge of its mechanical strength

Conclusion
Local administration of methoxatin loaded conduit improved tendon healing in rabbits. It could be concluded that use of the chitosan conduit could be of clinical benefi t due to reduced peritendinous adhesion formation around injured site of tendon during repairing period and also the conduit could be used as a carrier for drug delivery to improve and accelerate tendon healing.