Effect of scopoletin on fascia-wrapped diced cartilage grafts

Purpose: To evaluate the effect of scopoletin (SL) on fascia-wrapped diced cartilage grafts in rhinoplasty surgery. Methods: Cartilage grafts (2 × 2 cm) from the ears of New Zealand rabbits were diced into sections (1 mm) and then wrapped in muscle fascia taken from the right rear leg. Each graft was placed on the back of the animal after measuring its weight, and then the rabbits were separated into two groups, viz, control and the SL-treated groups {10 mg/kg, per os (p.o.)}. The treatments were administered for 3 months, the rabbits were sacrificed, and the histopathological features and weight of the grafts were examined. Results: The weight of the grafts in the two groups did not significantly (p < 0.05) differ but the histopathological results suggested that there was a pronounced increase in the viability of the graft tissues in the SL-treated group compared to the control group. Treatment with SL decreased the resorption rate and enhanced basophilia relative to the control group. However, fibrosis, inflammation, and bone metaplasiaand calcification-like factors did not significantly differ (p < 0.05). Conclusion: Treatment with SL significantly enhances the viability of the grafts, and thus may have a beneficial effect on fascia-wrapped diced cartilage grafts.


INTRODUCTION
The term 'cartilage graft' was first coined at the end of the 19 th century in association with nasal defect reconstruction surgeries [1].Subsequently, various types of cartilage graft have been used by surgeons to maintain the structural integrity of a patient's body [2].The graft resorption rate is affected by a variety of parameters, including surgical trauma, graft preparation, and shape; anaesthetic deformities and defective contours can result from excessive resorption rates [3,4].
Long-term permanent results and easily shaped grafts can be obtained by the application of several surgical techniques.For example, block form and fascia-wrapped grafts are easy to implement and have produced successful outcomes for nasal dorsal augmentation and surgically wrapped diced cartilage grafts [5].Although several current techniques can be used to reduce the resorption rate and form a graft that can be easily shaped, not all surgeons accept a particular model as being ideal for increasing the viability of diced cartilage grafts despite the use of novel surgical techniques.Thus, the present study aimed to determine if specific drugs could enhance the viability of cartilage grafts without requiring modification of current surgical techniques.
Scopoletin (SL) is isolated from a Chinese medicinal plant known as Aster tataricus [6], which contains a coumarin compound that is reported to have antitumor, antioxidant, and anti-inflammatory effects [7][8][9].These effects are likely due to the inhibitory effect of this compound on tube formation, endothelial cell migration, cytokinin production, COX-2 expression, and nitric oxide synthesis [10][11][12].Intraperitoneal administration of SL significantly reduces the thickness of synovium and attenuates inflammation in the joints [6].Moreover, SL activates caspase-3 and subsequently induces apoptosis and cell arrest in tumour cells [13,14].Thus, the present study evaluated the effects of SL on fascia-wrapped diced cartilage grafts.

EXPERIMENTAL Animals
The present study used New Zealand rabbits (age, 2 months; weight, 800-1,200 g) that were housed under standard laboratory conditions in a room maintained under a temperature of 20-25°C and a 12-h light-dark cycle.The rabbits were housed in the lab, under environmental conditions according to the relevant guidelines, for 10 days prior to the experiment to acclimatise them to the environment.All protocols used in the present study were approved by the animal ethical committee of Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, China (ref.no.TJM/ 2015/ 19) and the given study followed the guidelines of Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) for experimentation and animal use [15].

Animal studies
All animals were anaesthetised with an intramuscular injection of xylazine (5 mg/kg) and ketamine (35 mg/kg) and treated with cefuroxime, which was injected at a prophylactic dose of 30 mg/kg (intramuscular) prior to surgery.The shaving was done at the donor site of each animal and an iodine solution was used to clean the exposed skin.Subsequently, the skin was pulled away from the auricular tissue and the cartilage sections were diced into squares (2 × 2 cm) with the perichondrium protected in the cartilage tissue.Next, a scalpel was used to cut the sections into 2-3 mm pieces; then, the cartilage graft was wrapped in a 2 × 2 cm section of fascia obtained from the right hind leg and the incision was sutured with 5/0 vicryl (Figure 1).
A sensitive scale was used to measure the weights of the fascia-wrapped cartilage grafts, which were implanted by preparing a subcutaneous cavity 5 cm lateral to the midline on the back of each animal.Next, the incision was sutured with 4/0 silk and the animals were divided into two groups based on treatment: the control group received a saline solution and the SL-treated group received daily administrations of SL {10 mg/kg, per os (p.o.)} for 3 months.At the end of the protocol, all animals were sacrificed with thiopental sodium (150 mg/kg) and the grafts were removed.Next, the cleaned grafts were weighed and placed into a 10 % formalin solution for further histopathological study (see Figure 2).

Histopathology examination
The tissue samples were dehydrated with ethyl alcohol and then cleaned with a solution of xylol.Next, the tissues were embedded in molten paraffin to obtain paraffin blocks and a microtome was used to cut the tissue into sections (5-mm thick); then they were stained with Masson's trichrome, toluidine blue, and hematoxylin and eosin (H&E).Histopathology of the tissue sections was performed under a light microscope; the chondroid tissues and viability of the chondrocytes were assessed with H&E staining, the connective tissues were examined with Masson's trichrome staining, and the viabilities of the chondrocytes and the chondroid tissue matrix metachromasia were estimated using toluidine blue staining.The grafts were evaluated based on the loss of chondrocyte nuclei in the lacuna, chondroid matrix status, and the life of the cartilage graft.Non-viable chondroid tissues were also evaluated for chondrocyte nuclei and loss of the matrix metachromasia.
The pathologist who evaluated the grafts was not aware of the group assignment when examining various factors such as fibrosis, basophilia, inflammation, metaplasia of bone, calcification, resorption, and graft life.Viable chondrocytes were assessed for viability in each specimen and considered as a percentage of the whole specimen.All parameters were scored on the following scale: 0 = negative, 1 = weak; 2 = moderate, and 3 = excessive.

Statistical analysis
All data are presented as means ± standard deviation (SD; n = 10) and were statistically analysed with Student's t-tests (GraphPad Prism 6.1.,CA, USA).P < 0.05 was considered to indicate statistical significance.

RESULTS
The effects of SL on the weights of the grafts are shown in Table 1.Compared to the application of saline, SL treatment increased the weights of the grafts following implantation.However, the preand post-implantation weights of the fasciawrapped cartilage grafts did not significantly differ between the groups.The effects of SL treatment on fibrosis, basophilia, inflammation, metaplasia of bone, calcification, resorption, and viability were also determined (Table 2).The SL group did not significantly differ from the control group in terms of fibrosis, inflammation, bone metaplasia, or calcification, but basophilia were up to 2.25-fold higher in the SL-treated group compared to the control group (0.85).Additionally, the resorption value significantly decreased in the SL-treated group (0.45) compared to the control group (2.1) and the SL group had a viability percentage of 82.9 %, while that of the control group was 42.1 %.The viability of the tissue sections was also significantly higher in the SL-treated group compared to the control group (Figure 3).

DISCUSSION
A variety of factors, including a patient's current systemic status, oxygenation in the local tissue, surgical technique, graft size, and vascularised host bed, contribute to the survival of a cartilage graft.However, the resorption rate may be unpredictable even if the cartilage graft is placed under optimal conditions [16].There are several techniques that can increase the survival of diced cartilage grafts, including the Turkish delight method described by Erol, which has proven effective and popular around the world [3].Surgically wrapped diced cartilages exhibit various degrees of resorption; thus, it has been recommended that fascia be used to wrap diced cartilages.However, few surgeons suggest the use of AlloDerm for wrapping because the harvesting of fascia may cause hematoma and alopecia [17].Additionally, block grafts have a higher viability than diced cartilage grafts and several medical agents, such as steroids and dimethyl sulfoxide, can be used to increase the resorption rate and viability of cartilage grafts [18].
It has been reported that SL exerts antitumor, antioxidant, and anti-inflammatory activities, inhibits the migration of endothelial cells, alters the production of cytokines, and changes the expression levels of COX-2.The intraperitoneal administration of SL significantly reduces the thickness of synovium and attenuates inflammation in joints.Additionally, SL activates caspase-3, which in turn induces apoptosis and cell arrest in tumour cells.
The viability of chondrocytes is higher following application of diced cartilage wrapped in fascia [19].The present findings showed that SL significantly increased the viability of chondrocytes and decreased the resorption rate compared to a control group receiving saline.The present study also demonstrated that there was a post-implantation increase in the weights of the grafts in the SL-treated group compared to the control group.Thus, the present findings suggest that fascia-wrapped diced cartilage grafts may benefit from the application of SL.

CONCLUSION
The findings of the study demonstrate that SL has beneficial and positive effects on fasciawrapped diced cartilage grafts and may be of potential prophylactic application in cases of foreign matter reaction where there is an increase in resorption rate.

Figure 1 :
Figure 1: Cartilage implants.(A) Diced cartilage for wrapping with the fascia.(B) Fascia graft folded around the diced cartilage

Table 1 :
Effects of SL on the weights of fasciawrapped diced cartilage grafts before and after implantation