Histopathological assessment of healing process using an amniotic membrane in osteotomy defects caused by piezosurgery and conventional surgery in rats


 There is a paucity of studies aimed to compare the healing process of piezosurgery with conventional surgery. This study evaluated the histopathological assessment of healing process in osteotomy defects caused by these surgical methods in rats using an amniotic membrane (hAM). In this randomized controlled experimental study, there were 2 main groups: group 1 (Piezosurgery method-right tibia) and group 2 (Conventional surgery method-left tibia) and subgroups determined by the sacrification periods on the 7th (n = 20) and 21st days (n = 20). hAM was used in all groups. The primary outcome variable was new bone formation, while inflammation, necrosis, fibrotic tissue formation in the defective zone were secondary outcomes. 40 male Sprague Dawley rats were used. The 7th day fibrosis levels and the 7th new bone-building level of the group who underwent piezosurgery were found to be significantly higher than those of the group undergoing conventional surgery (p < 0.05) and the 21st new bone-building level was significantly lower than conventional surgery. In all rats, on the 21st day, there were statistically significant decreases in inflammation, fibrosis, and increases in the healing score (p < 0.05). Also, there was no statistically difference in new bone formation between 7st and 21st days (p > 0.05). The choice of conventional surgery when applying with hAM increases the new bone building in the late period.


Introduction
Traditionally, rotating instruments like conventional burs were the only option for hard tissue surgery until 1988. 1 Considering the tendency toward minimally invasive surgery, piezosurgery-a new osteotomy technique-has been introduced in oral and maxillofacial surgery. 2 Piezosurgery devices use microvibrations of scalpels at ultrasonic frequencies to perform osteotomies. 3 The use of it has become widespread in oral and maxillofacial surgery, with its selective cutting feature and not damaging tissues.
Also, studies are indicating that it has positive effects on soft tissue compared with conventional surgery.
In a meta-analysis, Liu et al., 4 described that there might be some advantages in terms of pain, mouth opening, swelling on third mandibular molar extraction with piezosurgery compared to conventional surgery. Similar results were reported in mandibular cyst enucleation. 3 Also, these two techniques were compared in crest preparation before implant application 5 , inferior alveolar nerve and transposition 6 , osteotomy and maxillary sinus augmentation procedure 7 , and alveolar distraction 8 in the literature. It is described that the use of ultrasonic devices as piezosurgery for osteotomies has several advantages, including less surgical injury, cutting selectively, postoperative pain, and neurological complications 9,10 in these studies. However, the effects of these two techniques on bone healing using an amniotic membrane (hAM) are histopathologically controversial.
hAM is an intrauterine tissue that is widely used as a cell culture substrate in various surgical therapies because it facilitates the proliferation, differentiation, and functional organization of various cell types. It is a source of various factors and enzymes such as epidermal growth factor (EGF), keratinocyte growth factor (CGF), hepatocyte growth factor (HGF), changing growth factor (TGF), steroid hormones (estrogen, progesterone), hydrolytic enzymes, and oxidation-reduction enzymes. 11,12 It is used as a physical barrier between the ap and the bone defect to promote the increase of regenerative cells in the area of the defect. 11 Today, the use of hAM has become common in repairing bone defects. 13,14 Although piezosurgery has been reported to have positive effects healing process histopathologically and clinically, its use with the hAM is uncertain. The purpose of the present study was to compare the healing process in osteotomy defects caused by piezosurgery and conventional surgery using hAM in rats. The investigators hypothesized that piezosurgery would increase bone healing compared with conventional surgery in defects used hAM.The speci c aims of the study were to measure 1) in ammatory levels, 2) brosis level, 3) healing scale, 4) new bone-building level, and 5) necrosis.

Results
All the rats survived during and after the surgery. No fractures or infections were observed in any of the rats post-operatively.
Light microscope images 7 th day: Left tibia: More brous and bone tissues were observed compared with the right tibia ( Figure 4 A-B). Also, more compact bone tissue was seen in the right tibia (Figure 4 B). 21 st day: Left tibia: More compact bone tissue was seen ( Figure 4C). Right: Thin, irregular cortical bone formation was present in the defect region ( Figure 4D).

Comparison of groups
There was statistically signi cant difference between the piezosurgery and conventional surgery techniques on the 7 th days in terms of brosis level (p=0.028) and new bone-building level (p=0.038), on 21 s t day in terms of new bone-building level (p=0.001) ( Table 1). While brosis and new bone formation were higher in the piezosurgery group on the 7th day, new bone formation on the 21st day was higher in the conventional surgery group ( Figure 5). Comparison of sacri cation periods in piezosurgery group (Right tibias) The in ammation and brosis levels on the 21 st day were signi cantly lower than those on the 7 th day. (p = 0.001, p = 0.001). Healing scores on the 21 st day were found to be signi cantly higher than those on the 7 th day in (p = 0.001). There was no statistically signi cant difference in the new bone building (p = 0.648) and necrosis (p = 0.474) ( Table 2) ( Figure 6).

Comparison of sacri cation periods in conventional burs group (Left tibias)
The in ammation and brosis levels on the 21 st day were signi cantly lower than on the 7 th (p = 0.001, p=0.008). The new bone building and healing score were signi cantly higher on the 21 st (p = 0.001).
Healing scores on the 21 st day were found to be signi cantly higher than on the 7 th day (p = 0.001). There was no signi cant difference in necrosis (p = 0.677) ( Table 2) ( Figure 6). The results of the present study revealed that new bone building was higher in the piezosurgery group on the 7 th day (p = 0.038) and on the 21 st day new bone formation was higher in the conventional surgery group (p = 0.001). However, Preti et al., 21 in the study where they investigated the biomolecular and histologic analysis of implants' osteointegration and they reported that on the 7 th and 14 th days, the new bone level and amount of osteoblast in the bone surgery site was higher than in the conventional drill surgery technique. Another study found that as a result of their study on dogs, bone level in osteotomy/osteoplasty with piezzo increased after 8 weeks compared to the conventional rotary instruments. 22 Bone healing probably related to various factors including searching methods, the size of the bone defect, and timing in histopathological examination. 23,24 Furthermore, Gomes et al., 13 in their study on animals found that healing was delayed as shown by histological follow-up, in the group that used only the hAM. The reason for higher bone building levels in the conventional surgery on 21 st day likely that the hAM maintains its tissue integrity by performing its barrier function in the present study.
It was stated that piezosurgery had better results in the early period of bone healing because as an ultrasound technique, it results in successful induction of osteoblast differentiation and proliferation. 25,26 This result may due to the increase in BMP4 and TGF-β2 in the rst period in piezosurgery. It was reported that BMPs and TGF are important factors for chondrocyte differentiation and osteogenic differentiation in the healing process. 21 Similarly, in the present study, there were statistically signi cant increases in the new bone-formation level on the 7 th day for the piezosurgery than the conventional technique. The early effect of hAM application in piezosurgery is not yet available in the literature, and studies on this subject are recommended.
Extreme tissue damage and necrotic cells at the operation area occurring due to traumatic surgeries result in an intense in ammatory response and provide a high level of edema due to the release of proin ammatory mediators such as bradykinin and prostaglandin E2. 22,27 However, in the present study, there was no signi cant difference between groups on in ammation, healing score, necrosis, and brosis. The reason for this may be the use of an amnion membrane.
It is stated that the hAM can be used as an alternative to the oral mucosa membrane because it causes rapid healing 28 , less patient morbidity 29 , and minimum complications 30 . Also, it can stimulate healing and improve the epithelialization of the oral mucosa because it contains growth factors such as TGF-α, TGF-β1, -2, -3, EGF, IGF, and VEGF. 11,30,31 Velez et al., 32 conducted the rst study in the eld of dental implants using hAM and reported that amnion group was better compared to the group where no material was applied after surgery in terms of epithelialization and pain scoring on the 6-day during the early period. The reason it was used in the present study was to speed up and increase the healing process.
To our knowledge, this is the rst study to address the relationship between piezosurgery and conventional rotary instruments histopathologically evaluating new bone formation, in ammation, necrosis, brotic tissue formation, recovery score, and foreign body reaction using an hAM. One limitation of this study was that the sample size was relatively small compared to human studies. Therefore, necrosis is not detected in some subgroups. Also, this is an animal study, and the results of animal studies cannot easily be extrapolated to a human situation.
This study showed that the selection of conventional surgical procedure in defects used hAM affects new bone formation positively in the late period. Ultrasonic or conventional devices should be chosen based on their safety and e ciency. We would recommend the use of conventional surgery for defects using hAM. Future studies with larger samples are required to identify additional histopathologic features of both surgery methods used hAM.

Animal model
Fourty (n = 40) male Sprague Dawley rats (13 weeks old, weighing 240 ± 20 g) were used in the present study. The rats were kept in separate metal cages in an automated environment at a temperature of 21ºC ± 1ºC, a relative humidity of 40%-60%, and a lighting period of 12 hours light and 12 hours dark for the duration of the experiment. All the rats were fed a standard diet and allowed free mobilization during the Animal groups 2 main groups were created (n = 40) according to the surgical methods (Piezosurgery method and conventional burs method). Osteotomy defect was applied to the right tibia of the rats by piezosurgery and the left tibia by conventional method. Randomization was achieved by assigning the rats according to a sequential order of enrolment. Subgroups (n = 20) were also created according to the time of sacri ce (7 th or 21 st day).

Surgical Procedure
The rats were anesthetized by an intraperitoneal injection mixture of 5 mg/kg Xylazine hydrochloride and 6 mg/kg Ketamine HCL. The medial surfaces of the right and left hind legs of the rats xed in the standard posture were shaved and prepped with betadine solution.
A 20-25 mm bilateral skin incision was made (the right and left tibia) extending to the periosteum and then, soft tissues around the tibial bones were dissected using a periosteal elevator. The periosteum surrounding the bone was also incised and exposed by gentle retraction and dissection. Also, 5-mm long and 3-mm wide defects containing cortical and cancerous bones were created (Figure 1) in all the groups using the SL4 tip piezzo instruments (23 W) in the right tibia ( Figure 2A) and conventional instruments (2 mm round burs) ( Figure 2B) in the left tibia, under irrigation with sterile physiological saline solution to minimize thermal damage. All the osteotomies were performed by the same surgeon, using all the aforementioned ultrasonic devices in the automatic scansion mode (from 24 kHz to 36 kHz). The Sterishield amnion patch (20mm x 20mm, Bonemank Allografts, Texas, USA) was used under the periosteum to cover the defect areas on the right and left iliac regions of all the rats ( Figure 3A). For all the groups, the aps were sutured with 3.0 silk (Permahand® Silk Suture, Ethicon, Johnson & Johnson, Livingston, Scotland) ( Figure 3B).

Light microscopy monitoring and evaluation criteria
The operated rats were sacri ced successively at intervals of 1 and 3 weeks. After the rats were sacri ced using high doses of diethyl ether, the right and left iliac bones were removed in all the groups. The tibias, already xed in formalin for one month, were immersed in formic acid-sodium nitrate solution for 15 days to attain complete decalci cation. Then, each decalci ed specimen was cut and shaped to t into the defect sites and embedded separately in para n blocks. During the embedding, the positioning of the piece of tibia was standardized in an attempt to ensure that the same region was evaluated in all specimens. Serial sections of 5-7 µm thickness were taken from the para n blocks, placed on the slide, and depara nized in an oven at 70ºC. It was then stained with hematoxylin-eosin (H&E) and examined under different magni cations using a light microscope.
Cross sections obtained from the 7 th and 21 st days in all the groups were evaluated according to the following criteria: In ammation, necrosis, brosis, and new bone formation described as covering in an area of 0%-5% (0), 5%-30% (1), 30%-60% (2), and 60% (3). Healing score was evaluated as Fibrous tissue (0), Incomplete cartilage structure (cartilage structure with brous tissue) (1), Completed cartilage structure (2), Incomplete bone structure in the ossi cation phase (3), Incomplete bone structure in the middle phase of ossi cation (5), Incomplete bone structure in the advanced phase of ossi cation or the completed bone structure (6). Foreign body reaction described as yes or no.
Statistical Analysis IBM SPSS Statistics 22 software (IBM SPSS, Türkiye) was used for statistical analysis. Results were expressed as mean ± standard deviation. Test for normality of the different variables was done using the Shapiro Wilks test. Mann Whitney U test was used to compare parameters that's were not normally distributed between the groups. Fisher's Exact Chi-Square test was used to compare qualitative data.
Differences were considered signi cant when p value was <0.05.

Declarations Data Availability Statement
All data generated or analysed during this study are included in this published article.