The Effect of Distraction Osteogenesis on Peripheral Nerve Regeneration in Rats: A Preliminary Study In Vivo

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Introduction
Distraction osteogenesis (DO) has been widely used in the treatment of limb discrepancies and deformity correction [1,2]. Intramembranous bone formation is induced by gradual mechanical distraction via an external fxator during DO, and this osteogenic process is usually regulated by the diferentiation of bone mesenchymal stem cells, hypoxiainducible factors, and dynamic mechanical microenvironment [3,4]. Previous studies have reported that a moderate mechanical microenvironment has benefts in promoting bone formation by activating the HIF pathway and subsequently augmenting the osteogenic-angiogenic coupling [5]. However, few studies have focused on the phenomenon of peripheral nerve apraxia and self-repair during DO, which may involve the process of bone regeneration and remodelling.
Previous studies have reported the subclinical damage of peripheral nerves around the distraction area caused by DO [6][7][8]. Teoretically, nerves and soft tissues around the distracted area are inevitably afected during the distraction phase. However, there is a layer of undulating bundles of collagen fbres and elastic fbres in the interfascicular epineurium which makes the peripheral nerve naturally elastic and allows the fascicles to glide against one another [9,10]. Although the collagen fbres of peripheral nerves can exhibit self-repair over a low-deformation range, they will be irreversibly damaged if the velocity exceeds the elastic limitation (>1 mm/day) [11,12]. For instance, studies on mandibular DO have reported that a higher stress load may severely afect the synthesis and proliferation of neural cells, which may lead to neuroma and neurodegeneration [6,13].
Mitogen-activated protein kinase (MAPK) cascade is composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K/MEK/MKK), and MAPK in response to various extracellular stimuli. P38 MAPK is one of the major members of the MAPK family and is involved in the regulation of cellular proliferation, diferentiation, and death of all eukaryotic cells from yeast to humans. Te MAP3K-MAP2K (MKK)-MAPK pathway is the most common pathway that can be activated, representing a tertiary enzyme-linked reaction. Studies have confrmed that p38 MAPK is involved in the regulation of cellular diferentiation at the MAPK cascade, which is closely related to the development of neural and skeletal systems in humans [14,15]. Although p38 MAPK is often silent, ischemic, hypoxic, and infammatory factors can activate it and lead to its phosphorylation (p-p38) and subsequent entry into the cellular membrane or nucleus. Beyond this, some studies have found that p38 MAPK can be further involved in the process of cellular growth in the human body and is involved in the response to infammation and cellular stress. Moreover, it can be involved in the repair and regeneration of cells and the related processes of its regulation [16,17], such as the MAPK/NF-κB [18] and MAPK/ERK signalling pathways [19]. P38 MAPK not only transmits and transforms a variety of bio signals in the process of cellular regulation, but also interacts with diferent signalling pathways in the cell, ultimately converging various signal transmission information and becoming the fnal intersection point [20], making it an important target in the feld of neural and bone tissue regeneration [5,21].
Terefore, the purpose of this study was to observe the histomorphological and ultrastructural changes of peripheral nerves around the distraction area during DO and to investigate the self-repair mechanism of peripheral nerves in a rat DO model.

Materials and Methods
2.1. Animals. Sixty 12-week-old specifc-pathogen-free (SPF) male Sprague-Dawley (SD) rats weighing 300 to 400 grams were utilized in this study, which was provided by the Laboratory Animal Center of Xinjiang Medical University (certifcate number: SYXK (Xin) 2018-0003). All experimental rats were fed for two weeks in an appropriate environment (22 ± 2°C temperature; 40-60% relative humidity) before undergoing DO surgery, with access to healthy food and water. Te ethical standards of animal medicine in China and the ethical standards of the Laboratory Animal Center of Xinjiang Medical University (approval number: IACUC-20200318-82) were strictly followed.

Surgical Technique.
After successful general anesthesia with 2% pentobarbital sodium (3 mg/100 g, Sigma, US), the animals were placed on the experimental operation table in the left lateral decubitus position. Skin preparation was performed in the operation area. According to a previous study [3], a longitudinal incision was made on the lateral middle segment of the right thigh. Te skin and subcutaneous tissue were carefully incised layer by layer and the space between the vastus lateralis and rectus femoris muscles was opened. Te middle line of the right femur was then exposed, which was chosen as the osteotomy level. A preoperatively designed microexternal fxator was placed on the lateral side of the femur and two Kirschner wires (1.2 × 250 mm) were inserted into the proximal and distal parts of the femur, respectively. Osteotomy was then performed using a micro-oscillating saw, and the microexternal fxator was assembled and adjusted for distraction and compression. Finally, the surgical area was fushed with 0.9% saline and the incision was sutured.

Postoperative
Care. After surgery, each rat was fed in a single clean cage. Intramuscular injection of benzylpenicillin (200,000 IU/kg) was performed daily for three days to prevent infection. Te skin incision was disinfected with 75% alcohol once a day for fve days postoperatively. Te latency phase lasted for fve days, and the distraction phase was started manually with a rate of 0.5 mm/day for ten days. Te consolidation phase started upon the completion of the distraction phase. Control (n � 10) was defned as no distraction after DO surgery. Group 0-week (n � 10), Group 2week (n � 10), Group 4-week (n � 10), Group 6-week (n � 10), and Group 8-week (n � 10) were defned as different time points during the consolidation phase. After a short period of isofurane anesthesia, all experimental rats underwent weekly anterior-posterior X-ray imaging of the distraction area until they were sacrifced (n � 10 per group).

Electrophysiology.
Electrophysiology analysis (Alpine/ Biomed-Keypoint-4, Dantec Dynamics A/S, Denmark) of the sciatic nerve was, respectively, performed for all groups under general anesthesia (hypnorm and diazepam, n � 10 per group). Te stimulating electrode was placed at the proximal side of the femur (approximately 2 cm from the distraction area), and an inductive electrode was placed on the innervated muscle. Te electromyography rectangular pulse was designed with the following pulse parameters: 0.1 ms, 15.8 mA, 1.5 Hz, and 6 consecutive pulses. Te time taken for the conduction of the stimulus was measured and the motor nerve conduction velocity (MNCV) was calculated as the distance between the two stimulating points divided by the diference in the latency of the two points' stimulus.

Sciatic Function Index.
A self-made rat walking box (50 × 15 cm) was used to record the four to fve footprints of rats during crawling with ink dipped in dye on both feet, and clear footprints on the experimental (E) and normal (N) sides were selected to measure three variables: footprint length (PL), defned as the longest distance of the footprint; toe width (TW), the distance from the frst toe to the ffth toe; and intermediate toe distance (IT), the distance from the second toe to the fourth toe. Te sciatic function index (SFI, n � 10 per group) was calculated by taking the above three variables into account with Bain's formula, SFI � −38.3 (EPL − NPL)/NPL + 109. 5 (ETW − NTW)/NTW + 13.3 (EIT − NIT)/NIT − 8.8. A normal functioning limb was considered to have values of SFI closer to 0.

Histomorphometric Analyses of Nervous Tissue.
Static histomorphological observation of the sciatic nerve was conducted using Hematoxylin and Eosin staining (H&E, n � 3 per group). Specifcally, specimens were randomly selected from each group and then fxed in 10% neutral bufered formalin for twenty-four hours. After parafn embedding and molding, all selected specimens were sliced into ultrathin sections with an ultramicrotome (Leica, Germany) with a thickness of 5 μm, which were stained with H&E stain. Te slides were then washed three times with distilled water and dehydrated using a series of increasing concentrations of ethanol. Finally, the slides were air-dried at room temperature and mounted with neutral gum for later observation under an optical inverted microscope (DP26, OLYMPUS Corporation, Japan).
Ultrastructural changes of the sciatic nerve around the distraction area (n � 2 per group) were assessed using transmission electron microscopy (Hitachi, Japan). Tree samples from each group were fxed in a 2.5% glutaraldehyde solution (Millipore Sigma, US) for transmission electron microscopy analysis. All selected nerve specimens were fxed in 1% osmium tetroxide for one hour and then diluted to 2% for refxation. Te selected specimens were rinsed thrice with PBS and dehydrated in a series of absolute ethanol, which were then placed in pure acetone and Epon 812 epoxy solution for thirty minutes and embedded in Epon 812 epoxy at 60°C for forty-eight hours. Ultrathin (70 nm) sections were cut and stained with lead and uranium.
Te remaining specimens (n � 5 per group) were fxed in 10% bufered formalin. After deparafnization in xylene and rehydration in a graded series of alcohol, immunohistochemistry staining was performed using a primary antibody against p38MAPK (1 : 100, ab216842, Abcam, UK) at 4°C overnight. Te sections were then incubated with secondary rabbit anti-rat IgG (DS-0003, 1 : 100, Zhongshan Golden Bridge Biotechnology, China) for 30 minutes at 37°C. Diaminobenzidine and hematoxylin were used to stain these sections, which were then sealed with neutral resins. Images were captured under an optical inverted microscope (magnifcation, ×200; DP26, OLYMPUS Corporation, Japan), and the integrated optical density of p38MAPK expression in three randomly selected felds of distraction area was measured using Image Pro-Plus 6.0 software (Media Cybernetics Inc., US). Te results were presented as the area percentage of positive staining.

Statistical Analysis.
Te data were expressed as mean-± standard deviation (SD). Te Kolmogorov-Smirnov test was employed to assess the normality of the data. Student's ttest was used to compare diferences between the two groups, while one-way ANOVA was used for multiple comparisons. In cases where the test of homogeneity of variances yielded a P value greater than 0.05, Tukey's multiple comparison post hoc test was applied. Otherwise, Dunnett's T3 post hoc test was used. Statistical signifcance was considered when the P value was less than 0.05 between groups. All analyses were performed by the SPSS 20.0 software package (IBM, US). Graphs were plotted by GraphPad Prism 8.0 software (GraphPad, US).

Histomorphometric Analysis.
In the HE staining view of the Control (Figure 3(a)), the sciatic nerve fbres showed a wavy course in the nerve fascicle, and the fascicle was smooth and closely arranged. Te axons were also arranged in parallel with good continuity, and nodes of Ranvier were observed prominently. A few darkly stained blue and fattened nuclei of Schwann cells were seen scattered. However, the sciatic nerve fbres of the distraction area were disorganized and nodes of Ranvier were widened when the distraction phase fnished (Figure 3(b)). A few lipophilic structures produced by Schwann cell disintegration were observed between the nerve fbres, and the myelin sheath Journal of Tissue Engineering and Regenerative Medicine was swollen. Even the nerve fbre expansive demyelination was observed and myeloid-like changes formed by demyelination were noticed as well. Over the consolidation phase, mild reparative changes of sciatic nerve fbres in the distraction area were observed (Figures 3(c)-3(e)). After 8 weeks of consolidation, the arrangement of injured sciatic nerve fbres recovered to a natural and neat wavy shape without disturbance (Figure 3(f )). Te structure of nerve axons and nodes of Ranvier was clear and the myelin swelling disappeared, which was approximately similar to the Control.
In the results of transmission electron microscopy, the myelin of the sciatic nerve in the Control was a lamellar structure arranged closely in several layers (Figure 4). Te Schwann cells could be seen on one side of the myelin sheath, and their nuclei were large with relatively little cytoplasm. Moreover, an intact axonal membrane was observed, containing a large number of microtubules and microflaments within the axoplasm. Both myelinated and unmyelinated nerve fbres showed signs of neurodegeneration in the Group 0-week, which was similar to the HE staining. Specifcally, there was loose separation of the lamellar structure at the sciatic nerve in the distraction area, with separation of the axonal membrane from the innermost layer of the myelin. Te lamellar structure was patchy and loose, exhibiting scattered vacuolated myelin degeneration, compressing and swelling of axons, and even disintegrated myelin fragments. Ten, new nerve myelination, active Schwann cell proliferation, and mitochondrial proliferation in the axoplasm were observed in Group 2-week, Group 4week, and Group 6-week, suggesting that the regenerative phase had started. Finally, in Group 8-week, the injured nerve almost completely recovered to its normal state, and the new myelin was still visible.

Discussion
Peripheral nerves are very sensitive to tension stress. Previous studies have reported that the peripheral nerve around the distraction area is usually damaged by the mechanical traction force during the distraction phase, thus hindering the further application of DO [6,7,22]. Te DO model of the mandible has been well-established and extensively utilized to investigate the mechanism of bone regeneration and remodelling. Studies on mandibular DO models in rabbits have found that denervation is not conducive to bone regeneration in the distraction area. For instance, Zhao et al. [8] successfully established a rabbit mandibular DO model and found that distraction rates of 1.0 and 1.5 mm/d had regenerative efects on the inferior alveolar nerve. Cao et al. [6] observed that loss of the sensory nerves could result in decreased new bone quality during mandibular DO, utilizing their rabbit mandibular DO model. Although these studies have reported changes in peripheral nerves due to diferent distraction rates during mandibular DO, more attention has focused on the efects of peripheral nerve dysfunction on bone regeneration, rather than the gliding mechanism and repair process of peripheral nerves during DO. Hence, the mechanism of injury and self-repair of peripheral nerves during DO is still unclear, which impedes exploring the development of neural self-repair and regenerative medicine. Tis study aims to investigate the changes in peripheral nerve self-repair via a rat DO model. DO technique has been validated for more than three decades, and a DO rate of 1 mm/day is generally accepted in clinical practice for humans, while 0.5 mm/day is widely used for rodent experimental animals, such as SD rats weighing 300 to 400 grams. Ilizarov [23] showed various diameters of nerve axons and irregular cytoplasm in dog tibial peripheral nerve with a lengthening rate of 1 mm/day. However, fewer injurious changes of the nerve were observed when using a DO rate of 0.25 mm/6 h, and the nerve  structure was even maintained normal with a total DO rate of 1 mm/day for 60 small turns of the fxator [24,25]. Namgung et al. [26] reported that nerve rupture occurred when the rabbit sciatic nerve was lengthened by an amplitude of more than 30%-55% of its original length. In this study, a DO rate of 0.5 mm/day was applied in all models, and it was noticed that signifcant injurious changes in the sciatic nerve were observed near the distraction area via ultrastructure observation. Te nerve fbres were disorganized and the nodes of Ranvier were widened after the distraction phase. Lipodrop-like structures produced by the disintegration of Schwann cells were found between nerve fbres, and the myelin sheaths were swollen, which could lead to demyelination of the myelin fbres. Te nerve fbres returned to their normal state and the myelin oedema disappeared over time. Terefore, the injurious changes occur during the process of distraction, when the glide of the sciatic nerve is close to the limit of its existing elasticity mechanism. However, the continuity of axons and lamellar structure of the myelin is repaired after 8 weeks of consolidation. We believe that the paraneurium around the sciatic nerve allows it to glide during the distraction phase to reduce the development of injurious changes, which is also the reason why the sciatic nerve can self-repair after the distraction phase, instead of sustaining irreversible damage. Schwann cells are glial cells of peripheral nerves, which wrap around axons to form myelin in the peripheral nervous system and participate in nerve regeneration [27]. Schwann cells respond rapidly and exhibit plasticity when exposed to peripheral nerve injury, guiding the growth of regenerated axons and promoting peripheral nerve repair and regeneration. It has been reported that the ERK signalling pathway plays an important role in Schwann cell proliferation and migration in the repair process of peripheral nerve, activating the p38 MAPK to regulate Schwann cell elongation and alignment around axons for myelination [28,29]. It can also upregulate the expression of the transcription factor c-Jun to promote Schwann cells' plasticity and demyelination, transforming them into a repairpromoting phenotype [21,30]. In this study, the expression of p38MAPK was higher in Group 2-week, Group 4week, and Group 6-week, compared with the Control (P < 0.05), indicating that self-repair of the sciatic nerve had begun after the distraction phase.
Te self-repair process of the peripheral nerve gradually progresses during the consolidation phase. However, there is controversy regarding the repair duration of the peripheral nerve after an injury caused by DO. Hirofuji et al. [31] reported that sciatic injurious changes caused by femoral lengthening could be repaired by themselves within 8-12 weeks. Furthermore, the rabbit tibial DO model established by Simpson et al. [7] found that peripheral nerve dysfunction caused by the distraction phase could be recovered approximately 12 weeks after the distraction phase. In this study, the injured nerve recovered after 8 weeks of consolidation. Tere was no statistically signifcant diference in MNCV and SFI of the sciatic nerve between the Group 8-week and the Control (P > 0.05). We believe that the self-repair duration of the sciatic nerve is related to the distraction rate. Nerves cannot achieve self-repair when the rate and magnitude of distraction exceed the limits of nerve sliding (DO rate > 1.5 mm/day), which can impede bone regeneration and remodelling. Although this study did not compare the repair process after nerve injury caused by diferent DO rates, based on the observations of this study, we conclude that a distraction rate of 0.5 mm/day is a safe DO rate that contributes to bone regeneration for the rat femoral DO model.   Figure 5: Immunohistochemistry images of p38MAPK in diferent groups (n � 5 per group). (a, b) Te semiquantitative analysis indicated that p38MAPK was signifcantly expressed in Group 2-week, Group 4-week, and Group 6-week compared to the Control (black arrows, P < 0.05). However, the expression of p38MAPK was downregulated in the Group 8-week, and there was no statistically signifcant diference compared to the control group.
Limitations exist in this study. Te physical mechanism of the mechanical force stimulating Schwann cell proliferation and diferentiation is still unclear. A vitro experiment is not performed to verify the efects of mechanical forces on Schwann cells. Randomized controlled trials are further needed to elucidate the molecular mechanism between the distraction rate with diferent amplitudes and nerve elongation in rat peripheral nerves during DO.

Conclusion
Femoral DO in rats with a rate of 0.5 mm/day may cause sciatic neurapraxia, which can be self-repaired after 8 weeks of consolidation. Te paraneurium around the sciatic nerve enables it to glide during the distraction phase to reduce the occurrence of injurious changes. In addition, the plasticity of Schwann cells has been enhanced, leading to a repairpromoting phenotype and forming a microenvironment that is benefcial for sciatic nerve repair and regeneration.

DO:
Distraction osteogenesis E: Experimental HE: Hematoxylin-eosin staining IT: Intermediate toe distance MNCV: Motor nerve conduction velocity N: Normal PL: Footprint length SD: Sprague -Dawley SFI: Sciatic function index SPF: Specifc-pathogen-free TW: Toe width UK: United Kingdom US: United States.

Data Availability
Te original data presented in the study were included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

Disclosure
Kai Liu and Yuanxin Chen are co-frst authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest.