Expression of nerve growth factor in the callus during fracture healing in a fracture model in aged mice

Abstract

BACKGROUND:

Impaired fracture healing results in extensive and prolonged disability and long-term pain. Previous studies reported that nerve growth factor (NGF) was expressed during fracture healing and that anti-NGF antibody improves physical activity associate with facture pain. However, NGF expression levels in delayed or non-union are not fully understood.

OBJECTIVE:

We compared chronological changes in NGF in the callus of young mice after femur fracture with those in aged mice after femur fracture as a model of bone fracture in the elderly.

METHODS:

We used young (age 8 weeks) and aged (age 10 months) male C57BL/6J mice. A fracture was generated in the femur. At 5, 7, 10, 14, 17, and 21 days after creation of a fracture, mRNA expression levels of Col2a1, Col10a1, NGF were evaluated using quantitative PCR. We examined NGF protein expression levels and localization in the callus at day 14 using ELISA and immunohistochemistry, respectively.

RESULTS:

Expression of NGF in the callus after femur fracture in aged mice was significantly greater than that in young mice at days 5, 7, 10, 17, and 21 days. NGF protein levels in the callus of aged mice were also significantly higher than that in young mice. Immunohistochemical staining showed that NGF was heavily expressed in hypertrophic chondrocytes in the callus in aged mice.

CONCLUSIONS:

It is suggested that delayed Col2a1 and Col10a1 expression reflects delayed chondrocyte formation and delayed chondrocyte maturation in aged mice and that higher NGF expression in aged mice at day 14 may be associated with the presence of remaining hypertrophic chondrocytes in callus with delaying endochondral ossification.

Keywords

Nerve growth factor fracture delayed union

1. Introduction

The physiological changes which accompany aging often result in the delayed union or nonunion of fractures [1]. Evidence indicates that elderly patients are at higher risk of delayed union or non-union after a fracture [1–3]. Additionally, fractures are associated with pain, and impaired fracture healing results in extensive and prolonged disability and long-term pain [4]. However, the mechanism of pain in delayed or non-union are not fully understood.

NGF is an important regulator of the survival, growth, proliferation, and plasticity of sensory and sympathetic neurons [5]. However, recent studies have indicated that NGF also plays an important role in the fracture healing process [6–9]. Although NGF is only found in periosteal mesenchymal osteoprogenitor cells in intact bone, it is also found in callus periosteal osteoprogenitor cells, marrow stromal cells, chondrocytes, osteoblasts, and young osteocytes during fracture healing [6]. In addition, recent studies have suggested that anti-NGF antibody relieves the pain of fractures [10,11]. However, the difference in NGF expression levels and patterns between bone undergoing normal and delayed healing has not been elucidated. We compared chronological changes in NGF in the callus of young mice after femur fracture with those in aged mice after femur fracture as a model of bone fracture in the elderly.

2. Materials and methods

2.1. Creation of a fracture model

Our previous study showed that bone union in mice aged 10 months is delayed compared with that in mice aged 8 weeks [12]. This model is suitable for examining the influence of aging on bone healing. Therefore, we used young (age 8 weeks) and aged (age 10 months) male C57BL/6J mice. A fracture was generated in the femur and fixed with a stainless-steel nail (N = 70 each). Fracture healing was monitored by an soft X-ray system (SOFTEX-CMB4; SOFTEX Corporation, Kanagawa, Japan). The calluses of the femurs in the young and aged groups were excised following treatment at 5 (N = 10 each), 7 (N = 10), 10 (N = 10), 14 (N = 20), 17 (N = 10), and 21 (N = 10) days after the creation of fractures.

2.2. Real time PCR analysis

At 5, 7, 10, 14, 17, and 21 days after creation of a fracture, total RNA were extracted from the calluses of young and aged mice (N = 10 each for each time point). Fracture callus were dissected from the femur under stereomicroscopy, homogenized in 1 ml of TRIzol reagent and purified for total RNA according to the manufacturer’s protocol. cDNA synthesis was performed using Superscript III according to the manufacturer’s protocol. PCR primer pair sequences for use in qPCR analysis (Table 1). Relative mRNA expression levels of Col2a1 (proliferative chondrocyte marker), Col10a1 (hypertrophic chondrocyte marker), and NGF (marker of sensory nerve activity) were evaluated using qPCR (CFX-96^®, Bio-Rad, Richmond CA, USA). Expression levels of Col2a1, Col10a1, and NGF mRNA were normalized to the expression of the housekeeping gene, GAPDH.

Table 1
Sequences of primers used in this study

Gene Direction Primer sequence (5^′ –3^′) Product size (bp)

Col2a1 F GGAGAGACCATGAACGGTGG 78

R CATCTGGACGTTAGCGGTGT

Col10a1 F TAGGCAGCAGCATTACGACC 85

R CACATGCACGTGGTAGGAGA

NGF F ATGGTGGAGTTTTGGCCTGT 192

R GTACGCCGATCAAAAACGCA

Gapdh F AACTTTGGCATTGTGGAAGG 223

R ACACATTGGGGGTAGGAACA

Gene	Direction	Primer sequence (5^′ –3^′)	Product size (bp)
Col2a1	F	GGAGAGACCATGAACGGTGG	78
	R	CATCTGGACGTTAGCGGTGT
Col10a1	F	TAGGCAGCAGCATTACGACC	85
	R	CACATGCACGTGGTAGGAGA
NGF	F	ATGGTGGAGTTTTGGCCTGT	192
	R	GTACGCCGATCAAAAACGCA
Gapdh	F	AACTTTGGCATTGTGGAAGG	223
	R	ACACATTGGGGGTAGGAACA

2.3. Enzyme-linked immunosorbent assay (ELISA)

NGF mRNA expression was markedly increased at day 14 in the aged group. We examined NGF protein expression levels in the callus at day 14 using ELISA. Fracture calluses of young and aged mice (N = 5 each) were dissected from the femur under stereomicroscopy, homogenized in 1 ml of RIPA buffer, centrifuged at 15,000 rpm, and the supernatants were collected as total protein. Total protein concentration in supernatant were determined by BCA assay to adjust the protein concentration of each sample. A total 250 μg of protein was applied to an NGF ELISA kit (R&D system).

2.4. Immunohistochemistry

NGF mRNA expression was markedly increased at day 14 in the aged group. We performed immunohistochemical staining and examined the local distribution of NGF protein (N = 5 each). The PFA-fixed femoral samples were embedded in paraffin and sectioned into 4-μm-thick sections, deparaffinized with xylene for 1 h, hydrated in serial dilutions of ethanol and then rinsed in distilled water. Endogenous peroxidases were blocked by incubation of the sections using 3% hydrogen peroxide in methanol for 15 min. The slides were washed in phosphate-buffered saline (PBS), incubated at room temperature with 10% goat serum (Nichirei, Tokyo, Japan), and then incubated overnight at 4 °C with anti-NGF monoclonal rabbit IgG (cat. no: ab52918, Abcam). Location of the primary antibodies was determined via the streptavidin-biotin-peroxidase method (Histofine SAB-PO Kit; Nichirei), with counterstaining using Meyer’s hematoxylin.

2.5. Statistical analysis

Changes in Col2a1 and Col10a1 expression in calluses harvested at 7, 10, 14, 17, and 21 days after operation were compared with those at 5 days in each group using the Wilcoxon signed-rank test. NGF expression at 5, 7, 10, 14, 17, and 21 days, and NGF protein levels in the callus at day 14 in both groups were compared using the Mann–Whitney U test. When the p-value was less than 0.05 (p < 0.05), the data were considered statistically significant.

3. Results

3.1. Delayed fracture healing process in aged mice

Radiographical analysis showed that young mice achieved healing at D21 (Fig. 1). However, aged mice show non-union at D21. Col2a1 expression increased at D7 and 10 compared to that at D5 in young mice (Fig. 2A, P = 0.009 and P < 0.001, respectively). Thereafter, Col2a1 expression level decreased at D14, D17 and 21, at which time it showed no significant difference compared to D5 (Fig. 2A). In aged mice, in contrast, Col2a1 expression increased at D10 compared to D5 (Fig. 2A, P < 0.001) and remained significantly elevated up to D17 (D14, P < 0.001; D17, P < 0.0001). Col10A1 expression in the young group was increased at D10 compared to D5 (Fig. 2B, P < 0.001), and was higher at D14 than that at D5 in aged mice (Fig. 2B, P < 0.001).

Fig. 1.

Representative post-fracture radiographic images of femurs in 8- and 40-week-old mice. X-ray images taken at 0, 1, 2 and 3 weeks after fracture. (A–D) 8-week-old, (E–G) 40-week-old. Radiographical analysis showed that young mice achieved healing at 3 weeks after fracture. However, aged mice showed non-union at 3 weeks after fracture.

Fig. 2.

Col2a1 and Col10a1 expression in callus of young and aged mice. Col2a1 (A) and Col0a1 (B) expression at 5, 7, 10, 14, 17, and 21 days after fracture creation in young and aged mice. Col2a1 expression increased at D7 and 10 compared to that at D5 in young mice. In aged mice, Col2a1 expression increased at D10 compared to that at D5 and remained significantly elevated up to D17. Col10A1 expression in the young group was increased at D10 compared to that at D5, and was higher at D14 than that at D5 in aged mice. ^aStatistical difference between the young and aged groups at the same time point. ^bStatistical difference in comparison to day 5 and other days.

3.2. NGF expression during fracture healing

Expression of NGF in the callus after femur fracture in aged mice was significantly higher than that in young mice at all time points (Fig. 3A, D5, p = 0.003; D7, p = 0.002; D10, p = 0.026; D14, p < 0.001; D17, p < 0.001; D21, p = 0.002). NGF protein levels in the callus of aged mice were also significantly higher than those in young mice (Fig. 3B, p < 0.001).

Fig. 3.

NGF expression in callus of young and aged mice. (A) NGF expression at 5, 7, 10, 14, 17, and 21 days after fracture creation. Expression of NGF in the callus after femur fracture in aged mice was significantly higher than that in young mice at all time points. (B) NGF protein expression at 14 days after fracture creation. NGF protein levels in the callus of aged mice were also significantly higher than those in young mice.

Fig. 4.

Localization of NGF in callus of young and aged mice. NGF expression in callus of young (A–B) and aged (C–D) mice at day 14. Scale bars indicate 500 μm. The sections were stained with anti-NGF antibody and visualized with DAB (brown color). NGF was heavily expressed in hypertrophic chondrocytes in the callus in aged mice.

3.3. NGF localization

NGF-positive cells were observed in fibroblastic cells and chondrocytic cells in both young (Fig. 4A-B) and aged mice (Fig. 4C-D). Consistent with real time PCR analysis, immunohistochemical staining showed that NGF was heavily expressed in hypertrophic chondrocytes in the callus in aged mice.

4. Discussion

Previous studies reported that aged animals showed delayed fracture healing compared to young mice [12–14]. Compared to young mice (7–9 weeks), fractures in aged mice (age 52–56 weeks) show delays in chondrogenesis, completion of endochondral ossification, and bone union. Consistent with these reports, fractures in young mice (aged 8 weeks) achieved union at D21, whereas those in aged mice (10 months) remained to non-union at D21. Our model using aged mice at 10 months might represent delayed union in the aged.

A previous study reported that aged mice had delayed chondrocyte formation and chondrocyte maturation [15]. In our study, expression of the chondrocyte marker Col2a1 and mature chondrocyte marker (hypertrophic chondrocyte) Col10A1 increased rapidly at day 10 and then decreased rapidly in young mice. In aged mice, Col2a1 expression increased rapidly at day 10 and then gradually decreased, whereas Col10a1 expression increased rapidly at day 14 and then decreased rapidly. These results suggest that delayed Col2a1 and Col10a1 expression reflects delayed chondrocyte formation and delayed chondrocyte maturation in aged mice.

A previous immunohistochemical study showed that NGF expression was observed at day 7 after fracture in a rat fracture model [6]. Immunoreactivity for NGF was observed in osteoblast-like cells, fibroblast-like cells surrounding osteoblast-like cells and chondrocytes. In our study, NGF expression was observed in the callus of both young and aged mice. However, in aged mice, NGF expression increased rapidly in the callus at day 14 but then decreased rapidly until day 17. Expression was higher in the callus of aged mice, especially in hypertrophic chondrocytes. A previous study reported that endochondral ossification was delayed in aged compared with young mice [15]. Both Col10a1 and NGF mRNA expression increased at day 14 in aged mice. Therefore, higher NGF expression in aged mice at day 14 may be associated with the presence of remaining hypertrophic chondrocytes in callus with delaying endochondral ossification. The reason why NGF expression decreased rapidly until day 17 in aged mice is unclear, however, several studies have reported that NGF contributes to fracture-related pain and physical activity in mice [10,16]. Further experiments are expected to investigate whether higher NGF expression is associated with pain in delayed union.

5. Conclusions

We compared chronological changes in NGF in the callus of young mice after femur fracture with those in aged mice after femur fracture as a model of bone fracture in the elderly. It is suggested that delayed Col2a1 and Col10a1 expression reflects delayed chondrocyte formation and delayed chondrocyte maturation in aged mice and that higher NGF expression in aged mice at day 14 may be associated with the presence of remaining hypertrophic chondrocytes in callus with delaying endochondral ossification.

Footnotes

Acknowledgements

The authors thank DMC Corp for editing drafts of this manuscript.

Conflict of interest

None to report.

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