Animals
Forty-eight skeletally-mature, male or female New Zealand white rabbits, weighing 2.5-3.0 kg (mean 2.7 ± 0.2), were included in the study. The animals were transferred to the experimental animal center of Zunyi medical University at least one week before the surgery and kept in separate cages to help them adapt to the new environment as well as to ensure their health. This study was performed by the regulations of the Animal Management Regulations and Administrative Measures on Experimental Animal and approved by the medical ethics committee of Zunyi Medical University (Approval No. 2018. 246).
Establishment of rabbit fracture model
Forty-eight healthy rabbits were used to establish fracture models by the following surgery under aseptic conditions. All rabbits were randomly selected and placed in the supine position, and their submandibular region was prepared individually. They were anesthetized with an ear vein injection of 3% pelltobarbitalum natricum (1 ml/kg body weight), and 2% lidocaine (2 ml) was then injected for intensive local anesthesia. After submandibular incision and dissection of the periosteum, both sides of the mandible anterior to the masseter muscle were exposed by blunt and sharp dissection, and the neurovascular bundle of the mandible of 36 rabbits was transected. Then, incomplete fractures (about 5 mm×2 mm) were made in front of the mental foramen of the mandible through the buccal and lingual using diamond burs, and the zone was fully rinsed and cooled by physiological saline at the same time. All fracture models need no reduction and fixation.
Experimental groups
Forty-eight New Zealand white rabbits were randomly assigned to the nerve growth factor group (NGF group), gelatin sponge group (GS group), blank group, and intact group with 12 rabbits in each group. In the NGF group, the mental neurovascular bundle was cut off and implanted l ml nerve growth factor (10 μg/ml) with gelatin sponge as the carrier. The mental neurovascular bundle was cut off and implanted with normal saline equivalent to NGF with gelatin sponge as the carrier in the GS group. The mental neurovascular bundle was cut off without implanting material serving as the blank group. The intact group retained the neurovascular bundle intact and no material was implanted.
Postoperative care
After the rabbits were fully awakened from the operation, they were returned to separate cages, and the lower lip response to acupuncture was performed on the same day. Penicillin (0.4 million units) was administered intramuscularly to each rabbit twice a day for three days. Before the stitches were removed on the 7th day, the wound was cleaned and the healing status was observed every day.
Observation of lower lip reaction to acupuncture
The mental nerve innervated the skin of the lower lip area in front of one side of the chin foramen. If the lower lip did not respond to acupuncture on the first day after the operation, the nerve fracture models were successfully established. Besides, the recovery of nerve reflex was assessed by observing the number of rabbits with lower lip responses to acupuncture at the 2nd, 4th, 6th, and 8th weeks after the operation.
Visual observation and CBCT examination of the fracture zone
At the 2nd, 4th, 6th, and 8th weeks after the operation, three animals in each group were sacrificed and fracture healing was determined by observing and comparing the size of the bone gaps and the clarity of the osteotomy line using cone-beam computed tomography (CBCT) systems. The operated mandible of each executed rabbit was dissected subperiosteally to visually observe the formation of healing tissue at the fracture site as well as the amount of healing tissue formation and degree of fusion. The healing tissues were immediately collected for subsequent hematoxylin and eosin (HE) staining observation and qRT-PCR analysis.
Histological observation
Callus tissues from the mandibular fracture area were collected using rongeur, rinsed in physiological saline, and then immediately put into 10% neutral buffered formalin fixative for 48 hours. Subsequently, the samples were rinsed in running tap water for ten minutes and incubated with 10% ethylenediaminetetraacetic acid (EDTA) (pH = 7.2). The decalcifying solution was changed every three days until the decalcification was completed. The decalcification process was finished when the specimen was easily penetrated by a needle without any force. Next, samples were washed in 0.01 M phosphate-buffered saline (PBS) for ten minutes and then followed by routine dehydration and paraffin embedding. The paraffin wrapped tissues were cut into 4 µm sections using a Leica microtome (Leica, Germany). The tissue sections were soaked by xylene solution two times with twenty minutes to dewax the sections, and then soaked in 100%, 95%, 80%, and 75% alcohol for 5 min, and finally rinsed in running tap water.
After deparaffinization and rehydration, the sections were stained with hematoxylin dye for five minutes and then rinsed in running tap water. Soak the sections with 100%, 95%, 80%, and 75% alcohol to dehydrate the sections. The sections were soaked in xylene until the sections were clear, and then the tablet was sealed. Finally, the pathological changes of callus tissue between all groups were observed and photographed using an Olympus BX53 fluorescence microscope (Tokyo, Japan) by the following criteria: (1) the distribution of bone matrix collagen; (2) the distribution and number of osteoblasts; (3) the distribution and number of vascular endothelial cells; (4) the arrangement of trabecular.
RNA extraction and quantitative real-time PCR
To determine whether exogenous NGF could improve the expression of BMP-9 and VEGF during mandibular fracture by detecting the expression levels of BMP-9 mRNA and VEGF mRNA in four groups of healing tissues at the 2nd, 4th, 6th, and 8th weeks after the operation. For RNA extraction, callus tissue in the mandibular fracture area was collected using rongeur and immediately frozen in liquid nitrogen. The frozen tissue was ground to a fine powder in liquid nitrogen using a freezer mill (Bone Mill; SPEX CertiPrep, Metuchen, NJ, USA). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. The quantity, degradation, and contamination of total RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis, respectively.
RNAs were reverse-transcribed by oligo (dT) primer using the ThermoScriptTM RT–PCR system (Invitrogen, Carlsbad, CA, USA). QRT-PCR analysis was carried out using an ABI PRISM7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The published sequences of BMP-9, VEGF, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from GeneBank and these oligonucleotide primers for the rabbit-specific genes were designed using the Primer Express Software (Applied Biosystems), as shown in Table 1. GAPDH, a constitutively-expressed housekeeping gene, was used as a control gene, and all gene expression data was calibrated to those for GAPDH. Gene expression quantitation was calculated with the comparative cross-threshold (Ct) method. The difference between the average Ct value of the gene of target and the GAPDH was expressed as (ΔCt), and ∆∆Ct equals the difference between the ΔCt and the Ct value of the calibrator sample. The 2-ΔΔCt gave the relative quantitation value of gene expression.
Statistical analysis
The descriptive values for BMP-9 and VEGF in different periods in the NGF group, GS group, blank group, and intact group were presented in mean and standard deviations. The statistical analysis was analyzed with SPSS 20.0, (IBM, Armonk, NY, USA). Statistical differences among groups were detected by one-way ANOVA followed by Bonferroni’s multiple comparison test. A p-value of <0.05 was considered statistically significant.