Distraction osteogenesis (DO) has been developed and performed successfully for many years, but a long consolidation period and potential damage to peripheral nerves function has hindered the spread of this technique in the clinic. In some particular studies, up to 30% of patients who underwent limb-lengthening procedures encountered neurosensory disturbances (Galardi et al., 1990; Aqerreta et al., 1994). Long-lasting neurosensory disturbance of the inferior alveolar nerve is also the most common complication after DO in the mandible with a reported incidence of ∼23% (Wijbenga et al., 2009). For this reason, it is important to further understand how to protect the distracted nerves and/or shorten nerve repair time during DO.
A remarkable part of the process of distraction is the ability of the soft tissue to adapt to the incremental mechanical stress by apparent regeneration. With tension, the peripheral nerve fibers and the perineurium, the protective sheath, are straightened with increasing stretching force. When these nerve fibers are stretched to the limit of their elasticity, neural degeneration is observed (Haftek, 1970). Tang et al. (2004) reported that DO and secondary Wallerian degeneration triggers the proliferation of Schwann cells, with subsequent synthesis of NGF and other neurotrophic factors which play roles in nerve repair.
Nerve growth factor (NGF) was originally identified as a member of the neurotrophic factor family and has diverse functions in the normal growth and development of the nervous system (Lee et al., 2001). NGF can be detected in mature secretory glands (Watson et al., 1985). NGF acts at two distinct receptors, TrkA (low-/high-affinity receptor) and p75NGFR (low-affinity receptor) (Shengli and Kerong, 2005). p75NGFR receptor is normally present at low concentrations in adult peripheral nerves. p75 receptor can also be detected in immune system cells, such as the spleen (Perez-Perez et al., 2003). But in newborn rats, levels of NGF and NGF receptor (NGFR) in peripheral nerves are from 10 and 120 times higher than in adult animals, respectively. NGFR levels decreased steadily from birth and approached adult levels by the third postnatal week, whereas NGF levels decreased only after the first postnatal week and reached adult levels by the third week (Heumann et al., 1987). When the peripheral nerves were damaged, NGF levels rapidly increase within 6 hr in the Schwann cells and persist for at least 2 weeks after injury, then return to baseline level (Levi-Montalcini, 1987). p75NGFR is undetectable in intact adult peripheral nerves, but its expression increases following nerve injury (You et al., 1997). p75NGFR was found to be increased and an induction of Schwann cells proliferation was observed at 7 days after the sciatic nerve was severed (Taniuchi et al., 1986). At the proximal and distal nerve segments, higher NGFR expression was not maintained until nerve fiber regeneration occurred (Heumann et al., 1987). Although a similar process should control neurotrophic factors expression between different types of nerve regeneration, it is important to determine whether nerve regeneration in DO is the same as that in other injuries.
Although many neurotrophic factors are known to play critical roles in nerve recovery, there have been few studies on the mechanisms of peripheral nerve recovery after DO. This is especially true during lower limb DO, where the changes in expression of neurotrophic factors have not been well described. The aim of the current study is to describe the time dependent changes in cellular factors that occur in distracted peripheral nerve injury/recovery after DO and provide the groundwork for further exploration of the cellular mechanism of peripheral nerves adaptation during DO.
Materials and Methods
Animal model and surgical protocol
Twenty mature male Japanese white rabbits were used in this study. All procedures were conducted in accordance to protocol approved by the animal ethics review board. Unilateral external fixators used in the animal bone lengthening technique were purchased from Tian Jin Xin Zhong Medical Devices (Tianjin, P.R. China). Animals were first anesthetized with intraperitoneal injection of the mixture of 0.1 mL/kg ketamine hydrochloride and 0.1 mL/kg Sumianxin. Once anesthetized, an anteromedial middle incision was made along the left hind limb. The fixator was installed on the medial side of tibia. A transverse osteotomy was performed at the midshaft of the tibia between the second and third screws (Fig. 1) and care was taken not to damage or distract the tibial nerve during the procedure. The rabbits were allowed to recover for 7 days before any additional surgical operations. To lengthen the tibia, the osteotomy site was distracted manually at a rate of 1.0 mm/day (two adjustments/day; 0.5 mm/adjustment) for 10 days.
X-Ray photos were captured of the distracted limb at different times throughout the surgical procedure (just after osteotomy, at 28 and 56 days after distraction) to show the recovery status of operation. At time 0 (immediately after distraction), 7, 14, 28, 56 days after completing distraction (hereafter referred to C0, C7, C14, C28, C56 groups), 4 animals were sacrificed by an overdose of anesthesia and the elongated nerve tissues surrounding the distracted area just beneath the soleus were carefully dissected and separated. Normal tibial nerves of the contralateral side were harvested as control group. Nerve specimens were fixed in 10% neutral buffered formalin and embedded in paraffin blocks. Thick sections (5 μm) were cut using standard techniques.
Sections were deparaffinized and rehydrated. Endogenous peroxidase was neutralized with 0.3% hydrogen peroxide in 50% methanol. Antigen retrieval was performed using a conventional heating method. The slides were placed in a preheated (95°C) 0.01 M citraconic anhydride buffer solution (pH 6.0) for 15 min and then cooled to RT. The sections were blocked with normal fetal bovine serum for 20 min. A 1:200 dilution of primary rabbit polyclonal anti-human nerve growth factor (NGF) (sc-548, Santa Cruz, CA) was used to visualize NGF expression and reacted overnight at 4°C. Goat anti-rabbit immunoglobulin solution (1:5,000 dilution) was incubated with the sections at 37°C for 40 min, followed by incubating with horseradish peroxidase-conjugated streptavidin (1:200 dilution) incubation at 37°C for 40 min. 3,3′-Diaminobenzidin was used as a substrate-chromogen system. Finally, nuclei were counterstained with hematoxylin. Negative control sections were treated by PBS instead of the primary antibody. Immunohistochemical staining of NGFR was conducted in a similar manner as explained above using a primary mouse monoclonal anti-human p75NGFR (sc-13577, Santa Cruz).
Histological evaluation and statistical analysis
NGF and p75NGFR expression results were analyzed by Image-Pro Plus 6.0 and integrated optical density (IOD) and area of positive area for each measured field of view was counted. A minimum of five sections per animal were evaluated at each time point for each immunohistochemical expression.
A one-sided t test was used to determine significant differences between the groups (SPSS 17.0).
Tibial DO proceeded without postoperative infection or failure, and the average tibial lengthening was 10 mm according to X-ray photographs of the distracted limb. New bone formation at the distracted tibia was observed by radiography (Fig. 2) at different times throughout the surgical procedure. The active movements of the distracted limb (dorsiflexion and lateral rotation of the foot) indicated the tibial nerve was not damaged during the operation. From the nerve section staining photos of different group, the nerve morphology and fiber arrangement underwent a course from injury to recovery.
Immunohistochemical expression of NGF and p75NGFR
NGF was almost undetectable in nondistracted control tibial nerves. At Day 0 after completing distraction, it was weakly expressed around the axon and the myelin sheath (Fig. 3A,B, a-C0 group). At 7 days following distraction, the expression remarkably increased in almost all of the distracted nerve tissues (Fig. 3A,B, b-C7 group). This intensified staining persisted through 14 and 28 days after distraction (Fig. 3A,B, c- and d-C14 and C28 groups). A dramatic reduction in the expression of NGF was observed at 56 days after distraction (Fig. 3A,B, e-C56 group). This result indicates that NGF was undetectable immediately after distraction, but increased rapidly at 7 days (1 week) after distraction. This release of NGF continued for more than 3 weeks. At 56 days (8 week) after distraction, the secretion of NGF decreased to levels comparable with the control group.
No p75NGFR staining was found in the undistracted control tissues. In the C0 group (Fig. 4A,B-a), no differences were observed between the expression of p75NGFR and NGF. In the C7 group (Fig. 4A,B-b), a strong positive staining was observed in the distracted nerves. Expression of p75NGFR was found to be localized mostly in Schwann cells located in the outer layer of axons. So, the myelin sheath, the endoneurium, and the perineurium were all stained. This strong staining of p75NGFR was found to persist into 14 days post-DO (Fig. 4A,B-c). However, expression of p75NGFR was significantly reduced and nearly undetectable in the cellular components after 28 days (Fig. 4A,B-d) and showed no differences from the control nerves after 56 days (Fig. 4A,B-e). Accordingly, the C0, C28, and C56 groups had lower or undetectable p75NGFR and were similar with that observed in the control group. Only in the C7 and C14 groups was p75 activity high. The time of p75NGFR release was the same as that of NGF, but the remaining period of p75NGFR release was shorter than that of NGF.
The area and IOD of NGF and p75 expressions were measured (Tables 1 and 2). In NGF staining, there were significant differences between C7, C14, and C28 groups (three groups) compared with other two groups (C0 and C56) respectively (P < 0.01). But in p75NGFR staining, the significant differences existed between only C7 and C14 groups with C0, C28, and C56 groups, respectively (P < 0.01).
Table 1. The data summary and statistic results in NGF immunohistochemical staining
Limb lengthening is an important clinical application of DO. In this process, bone undergoes osteotomy, distraction and lengthening. At the same time, the vessels and nerves surrounding lengthened bone are also distracted. One of the desired outcomes after DO is that the resultant vessels and nerves function remain normal. Despite many experimental and clinical studies on DO of limbs that have been conducted in the last decade, the cellular events associated with distracted nerves remain poorly understood. Byun et al. (2008) observed the presence of NGF in the inferior alveolar nerve during mandible lengthening. Local application of NGF enhances recovery of inferior alveolar nerve and bone formation during mandibular DO (Wang et al., 2009a, 2009b; Du et al., 2011) Based on these findings, it can be inferred that similarities should exist in NGF secretion in the distracted tibial nerve during the limb lengthening. To elucidate whether this occurs in DO, it is helpful to have an insight as to the timing of cytokine secretion in the distracted limb nerves. The aims of our experiments were to detect the expression of NGF and its receptor at different times during the consolidation period and determine the cytokine secretion pattern. These results may potentially provide the basis for clinically effective application of NGF.
The theoretical foundation of DO is “tension stress,” a process of callus distraction leading to bone lengthening. DO is histologically similar to that of fracture healing, but with a rate of osteogenesis greater than that in embryonic development (Aronson et al., 1989). Therefore, we can deduce that the pattern of secretion of growth factors in DO may be similar or even more abundant than that of fracture healing and embryonic development.
Park et al.(Hu et al., 2001; Park et al., 2006) observed the structural changes of distracted nerve tissue including the demyelination and large myelinated destruction during 1–2 weeks after completion of distraction, nerve regeneration occurred 2 weeks after distraction. Several researchers (Farhadieh et al., 2003; Rosenstein and Krum, 2004) have described the upregulation of various neurotrophic factors and related proteins in inferior alveolar nerves after mandibular distraction. Park et al. (Heumann et al., 1987; Park et al., 2006) observed that NGF expression returned to baseline in inferior alveolar nerves at 56 days after distraction. The return of NGFR to normal levels started earlier than that of NGF, and the expression levels of NGF were elevated when compared to NGFR after peripheral nerve transection. Farhadieh et al. (2003) reported the resulting myelin sheath debris may serve as a trigger for higher expression of NGF and Brain-derived neurotrophic factor in inferior alveolar nerves, facilitating Schwann cell proliferation and remyelination of the affected segment. Byun et al. (2008) observed that p75NGFR was upregulated in inferior alveolar nerves at 7 and 14 days after distraction, and this returned to baseline at 28 days after distraction. A recent study showed the lysosomal activation is correlated with the induction of p75NGFR in demyelinating Schwann cells during Wallerian degeneration (Jung et al., 2011). Coexpression of NGF and p75NGFR in distracted inferior alveolar nerves at 7 and 14 days after distraction suggests that NGF is an autocrine growth factor in distracted nerves during the early consolidation period, and that it might contribute to the early recovery from nerve damage induced by mandibular DO.
Our study focused on the distracted tibial nerve in limb with DO. The results of the present study indicate the expression pattern of NGF and its low-affinity receptor p75NGFR vary at different time points in the consolidation period of the distracted tibial nerve during limb DO procedure. The results showed low-level expression of NGF right after distraction ended, abruptly increasing expression (strongly positive) at Days 7 and 14. Expression subsequently returned to the undetectable lower levels at Days 28 and 56. These results show that the expression level of neurotrophic factors is promoted under the stress of distraction and concurred with those of distracted inferior alveolar nerve in mandibular distraction (Heumann et al., 1987; Park et al., 2006). The regeneration of distracted nerves require a latent period, and the expression of the neurotrophic proteins matched this trend with sharply increasing expression taking place 7 days after distraction. This observation is consistent with nerve segmental regeneration (Heumann et al., 1987). Thus, the tension of mechanical distraction in DO can stimulate the expression of NGF and NGFR, and NGF may promote nerve repair during DO. Strong p75NGFR mRNA expression was observed in Schwann cells located in the outer layer of damaged axons. This result suggests that p75NGFR expression is associated with remyelination together with NGF. Therefore, the distracting force could cause subacute damage of tibial nerve and increase of NGF expression Mechanical distracting force results in detachment of the axons from Schwann cells and axonal degeneration in distracted area, afterward causing segmental degeneration.
According to the previous studies, the damaged nervous tissues are actively recovering until that period (Park et al., 2006). Other studies reported the increased NGF levels persisted for ∼2 weeks before beginning a gradual decline (Levi-Montalcini, 1987). Nerve damage caused by other reasons might be regenerated after about 2 weeks, but DO-induced nerve damage required a remodeling time of over 4 weeks, therefore indicating an increase in the expression and secretion of NGF until that time had elapsed (Park et al., 2006).
According to our experimental results, we found that NGF and its receptor were secreted in the early consolidation period and maintained at a high level for a period of at least 2 weeks. The expression and secretion of NGF and p75NGFR were found to contribute to a substantial increase after distraction, although these increases returned to normal at Day 56 (8 weeks). To protect the stimulated tibial nerve and facilitate its functional recovery, we postulate that applying NGF help shorten the recovery time of the tibial nerve and maintain function of the skeletal muscle, to ultimately lessen relative complications.