Background
The process of fracture healing consists of inflammation stage, soft callus stage, hard callus stage and remodeling stage. Inflammation reaction in the initial phase of fracture which contains macrophages and inflammatory cytokines has been recognized critical for fracture healing. These inflammatory cells secrete a large number of cytokines which has been proved to play an important role in cell recruitment and angiogenesis as well as bone repair [
1].
Macrophages can be categorized into inflammatory macrophage and resident macrophage. Inflammatory macrophages can be further classified into classically activated M1 phenotype and alternatively activated M2 phenotype, which can be shifted into each other under certain conditions [
2]. In general, M1 macrophages produce inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and mediate inflammatory response, while M2 macrophages secret interleukin-10 and interleukin-1 receptor antagonist, and mediate tissue repair and resolution of inflammation [
3]. Inflammatory macrophages are central mediators of the inflammatory response, the dynamic changes and balance between macrophage M1/M2 play important roles in inflammatory regulation and tissue repair. Different from inflammatory macrophages, resident macrophages are present in all tissue with tissue-specific phenotypes and functional abilities [
4,
5]. Resident macrophages in bone tissue located on periosteal and endosteal surface are termed osteal macrophages [
6]. Studies have demonstrated that inflammatory macrophages were highly presented in the early anabolic phase; while osteal macrophages mature the bone callus, predominating in the late anabolic and remodeling phases during fracture healing [
7,
8].
Macrophages and inflammation cytokines are proved to play important roles in callus formation, cartilage deposition and callus remodeling during bone fracture healing [
7,
9‐
12]. Several studies have proved that the dysregulation of inflammatory environment during bone fracture could impair fracture healing, which was seen in diabetics, obesities and smokers [
13‐
15]. Menopause, one of the most important factors in the pathogenesis of postmenopausal osteoporosis, was considered to induce a systemic chronic inflammatory reaction with the increase of TNF-α, IL-1β, IL-6 which were the major inflammatory factors during fracture healing [
16]. Fracture healing in animal models of postmenopausal osteoporosis revealed a decrease in bone callus formation in the early stage, delay of endochondral bone formation and hard callus remodeling in the middle and late stages [
17,
18]. In addition, the pathological histology of postmenopausal osteoporotic fracture was similar to fracture with abnormal expression of macrophages or inflammatory cytokines [
17,
18].
A systematic review analyzed the differences in inflammatory responses in normal and osteoporotic fractures, and found that it was inconclusive whether OVX animals have higher or lower local inflammatory response, and declared that there was a need for further studies to better understand the role of the inflammatory response in the healing cascade for potential immunomodulation to enhance osteoporotic fracture healing [
19]. A recent study found that OVX impaired the innate immune response locally at the fracture site in rats. However, there was no study observing the dynamic changes in inflammatory response during osteoporotic fracture healing systematically, especially in the inflammation stage [
20]. To address this issue, in this study, we established tibia fracture model in ovariectomized (OVX) mice, and observed the changes in macrophages and inflammatory cytokine expressions during different stages of fracture healing.
Methods
Animal experimentation
All applicable institutional and national guidelines for the care and use of animals were followed. The experimental protocols were performed with the approval of Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM18121414). Female C57BL/6 mice were purchased from Charles River Company (Beijing, China). The mice were housed in environmentally controlled animal facilities at 22 °C and humidity of 50 to 60%, with a 12 h light/dark cycle, and were fed a commercial diet and distilled water ad libitum. The maximum caging density was five mice.
Following a 1-week acclimatization phase, 210 16-week-old mice were randomly assigned to be bilaterally ovariectomized or to undergo sham surgery. Random numbers were generated using the standard = RAND() function in Microsoft Excel. Four mice in the OVX group and 3 in the sham group died after ovariectomy or sham surgery. Twelve weeks after the surgery, mice were performed a mid-shaft transverse osteotomy fracture or sham surgery on the left tibias, and the tibias were fixed with 0.5-mm-diameter intramedullary metallic pins. Another 14 mice, including 6 mice in the sham group and 8 in the OVX group, were excluded from following observations because comminuted fractures were made. Another 3 mice were used to supplement the sample size of the OVX group. All the mice were anaesthetized by intraperitoneal injection of ketamine (67 mg/kg) and xylazine (5 mg/kg) before fracture. After fracture, mice were allowed spontaneous recovery in warmed cages and unrestricted weight bearing. Mice were given Buprenorphine (0.1 mg/kg) for 3 days to relief any pain.
Mice in each group were sacrificed by exsanguination and confirmation of death by cervical dislocation before fracture or 1, 3, 5, 7, 14, 21, 28 days post-fracture (DPF) (n = 12 at each time point). Serum was separated for estradiol (E2) detection. The left tibias (6 from each group per time point) were harvested and fixed in 10% buffered formalin for 24 h. After fixation, the specimens were washed by phosphate buffered saline, and stored in 75% ethanol at 4 °C for X-ray and micro-CT scanning. Following that, specimens were decalcified with 14% ethylene diaminetetraacetic acid disodium salt solution for 14 days, dehydrated and embedded. Four-μm thick serial sections were cut for histomorphometric study. The rest 6 tibias of each group were harvested for real-time polymerase chain reaction analysis (PCR).
Blinding
For each mouse, different investigators were involved as follows: 2 investigators (JW and XL) administered the random allocation of the mice, and 5 investigators (DZ, CX, YT, SZ and HZ) performed bilaterally ovariectomy. Another 5 investigators (LC, SC, KS, YZ and JY) performed fracture and sacrifice procedures. Each investigator was responsible for part of the surgeries, including anesthesia, skin preparation and disinfection, model establishment, suturing, and resuscitation of the mice. LC, SC and KS assessed the results of the experiment. Two investigators (JW and SL) were responsible for statistical analysis of the data.
Serum E2 detection
Blood of mice in 0 DPF group was collected, and serum was separated after centrifugation at 3000 rpm for 15 min at room temperature. Serum concentrations of E2 were measured using a commercially available kit (ml058533, MLBIO, China) according to the manufacturer’s instructions.
Three-dimensional (3D) reconstruction analyses
The fractured tibias underwent X-ray photography first. A five-point radiographic scoring system was used to quantify fracture healing status at each time point by two researchers separately, and the average scores were adopted. The intramedullary metallic pins in the fractured tibias were then removed, and underwent scanning using a micro-CT imaging system (μCT80, Bassersdorf, Switzerland) with 10 mm slice increment. The source voltage was 55 kV and the source current was 72 μA. The integration time was 300 ms. A reconstruction of the bitmap data set was used to construct the 3D images of the fracture site using the built-in software (Scanco Holding AG, Brüttisellen, Switzerland). Total volume (TV, mm3), bone volume (BV, mm3) and the ratio of bone volume to total volume (BV/TV, %) of the callus were also analyzed.
Histological examination
Alcian blue hematoxylin/orange G (ABH/OG) staining was used to evaluate the histological changes of callus at different time points. Briefly, dewaxed sections of each group were stained with alcian blue/hematoxylin solution for 30 min followed by a 15-s wash in 1% ammonia solution and another 1-min wash in 95% ethanol solution. The sections were then incubated in orange G/eosin solution with phloxine B for 1 min. After dehydration in ascending series of ethanol solution, clearing in xylene and mounting, all sections were analyzed by an Olympus VS120-S6-W slide loader system (Olympus, Japan).
Antibodies
The antibodies used for immunohistochemistry staining and immunofluorescence staining were purchased from Abcam (Shanghai, China) including anti-TNF-α antibody (ab6671, 1:200), anti-IL-1β antibody (ab9722, 1:400), anti-IL-6 antibody (ab83339, 1:2000), anti-F4/80 antibody (ab6640, for immunohistochemistry staining, 1:200), anti-F4/80 antibody (Alexa Fluor 488, ab204266, for immunofluorescence staining, 1:400), anti-inducible nitric oxide synthase (iNOS) antibody (Alexa Fluor 647, ab209027, 1:200), anti-Mannose receptor antibody (anti-CD206, Alexa Fluor 647, ab195192, 1:200), anti-Galectin 3 (Mac-2) antibody (ab53082, 1:200).
Immunohistochemistry staining
Immunohistochemistry staining was used to evaluate the expressions of TNF-α, IL-1β, IL-6, F4/80 and Mac-2 in the fracture haematoma or callus. Deparaffinized and rehydrated serial sections were successively treated with 0.1% trypsin solution and 3% H
2O
2 solution for 15 min at 37 °C. Sequentially the sections were incubated in diluted primary antibodies overnight at 4 °C. Negative control sections were incubated in corresponding IgG solution instead. Then sections were incubated with secondary antibody and horseradish peroxidase (HRP)-streptavidin as instructed by the manufacturer (Polink-2 plus polymer HRP detection system kit, PV-9001/ PV-9004, ZSGB-BIO, Beijing, China). After staining with diaminobenzidine and counterstaining with hematoxylin, the images of the sections were obtained by an Olympus VS120-S6-W slide loader system, and were analyzed with an image Pro Plus 6.0 software (Media Cybernetics, PA, USA). Quantification of osteal macrophages (F4/80
+ Mac-2
−) was performed referring to the previous study [
7]. For each section of the haematoma or callus, 6 regions were randomly selected for quantitative analysis, and the average value was calculated. For each group, 6 samples were included for statistical analyses.
Immunofluorescence staining
To identify different types of macrophages in the fracture haematoma or callus tissue, dual immunofluorescence staining was performed with deparaffinized and rehydrated sections. Immunofluorescence staining was performed as described previously [
21]. Briefly, sections were incubated with anti-F4/80 and anti-iNOS antibodies to identify M1 phenotypic macrophages; or incubated with anti-F4/80 and anti-CD206 antibodies to identify M2 phenotypic macrophages [
22]. Finally, the sections were mounted in mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Ca H-1200, Vector, USA) and the images were obtained and analyzed by an Olympus VS120-S6-W slide loader system. For quantitative analysis of each section, positive stained cells of six random fields were counted and the average number of positive stained cells was calculated.
Real-time PCR analysis
Total RNA was isolated from the tissue located 2 mm distal and proximal to the fracture site. Reverse transcription was performed using a Primescript™ RT reagent Kit (Takara Bio, Shanghai, China) and real-time PCR was performed in a total volume of 20 μl solution containing 10 μl SYBR Premix EX Taq (Takara Bio, Shanghai, China), 1 μl diluted cDNA, 10pM forward and reverse primers. Primers specific for the genes were listed in Table
1. The mRNA expression of each gene was normalized to β-actin. The experiments were repeated at least three times.
Table 1
Sequences of PCR primers for specific genes
β-actin | Forward | 5′-GGAGATTACTGCCCTGGCTCCTA-3’ |
Reverse | 5′-GACTCATCGTACTC CTGCTTGCTG-3’ |
Tnf-α | Forward | 5′-CCTGTAGCCCACGTCGTAG-3’ |
Reverse | 5′-GGGAGTAGACAAGGTACAACCC-3’ |
Il-1β | Forward | 5′-GGAGATTACTGCCCTGGCTCCTA-3’ |
Reverse | 5′-GACTCATCGTACTCCTGCTTGCTG-3’ |
Il-6 | Forward | 5′-GACAAAGCCAGAGTCCTTCAGA-3’ |
Reverse | 5′-GTCTTGGTCCTTAGCCACTCC-3’ |
Cd16 | Forward | 5′-CAGAATGCACACTCTGGAAGC-3’ |
Reverse | 5′-GGGTCCCTTCGCACATCAG-3’ |
Cd206 | Forward | 5′-GGAAACGGGAGAACCATCAC-3’ |
Reverse | 5′-GGCGAGCATCAAGAGTAAAG-3’ |
Mcsf | Forward | 5′-AACAGCTTTGCTAAGTGCTCTA-3’ |
Reverse | 5′-ACTTCCACTTGTAGAACAGGAG-3’ |
Mcp-1 | Forward | 5′-TTTTTGTCACCAAGCTCAAGAG-3’ |
Reverse | 5′-TTCTGATCTCATTTGGTTCCGA-3’ |
Il-4 | Forward | 5′-AGT GAG CTC GTC TGT AGGGC-3’ |
Reverse | 5′-CAGGCA TCG AAA AGC CCG AA-3’ |
Statistical evaluation
All data were presented as mean ± standard deviation and were analyzed by GraphPad Prism statistical software (version 6.01, California Corporation, America) as appropriate. Data sets was performed a Shapiro-Wilk test for normality and confirmed a comparison of variances test for Homogeneity. ANOVA with Turkey’s post hoc test or unpaired Student’s t tests was performed for comparison between two groups. P value < 0.05 was considered statistically significant.
Discussion
The expressions of inflammatory cytokines during fracture healing follow a biphasic pattern, with a transient peak in the initial inflammatory phase, and a second peak during chondrocyte maturation and endochondral ossification [
1,
24]. Being different from those secreted by inflammatory cells in the initial phase, the inflammatory cytokines expressed during chondrocyte maturation and endochondral ossification are mainly synthesized by osteoblasts, chondrocytes and other cells, which can result the second peak in the middle and later stage of fracture [
25,
26]. In murine fracture models, inflammatory response of TNF-α, IL-1β and IL-6 peak 24 h following the fracture injury and decline to baseline levels rapidly by 72 h. Thereafter, a second peak in TNF-α, IL-1β expression occur approximately 14 days following the fracture injury [
24,
27].
Fracture healing comprises two bone repair processes: intramembranous and endochondral bone formation. The roles of inflammatory cytokines in intramembranous bone formation can be summarized into two aspects. On one hand, inflammatory cytokines acted as chemokines which were responsible for bone marrow stromal cell (bMSC) recruitment [
28]. On the other hand, inflammatory cytokines could promote intramembranous bone formation directly by inducing bMSCs osteogenic differentiation [
29]. Endochondral bone formation includes a series of processes such as cartilage proliferation, maturation, hypertrophy and apoptosis, and inflammatory cytokines also play an important role [
30]. Clinical studies confirmed that inflammatory cytokines such as TNF-α, IL-1β and IL-6 were highly expressed in osteoarthritis, which induced chondrocyte apoptosis and destroy articular cartilage [
31,
32]. TNF-α could promote apoptosis of chondrocytes by up-regulating the apoptotic genes in chondrocytes [
33], and chondrocyte apoptosis and resorption of the mineralized cartilage during the endonchondral period were delayed in TNF-α receptor deficient mice [
34].
In our study, TNF-α expression was dysregulated during fracture healing in OVX mice. Reduced transient expression in the initial inflammation phase and endochondral ossification phase might lead to decreased abilities in bMSC recruitment and osteogenic differentiation as well as chondrocyte maturation and endochondral ossification. Furthermore, TNF-α expression in OVX mice was consistently up-regulated at other time points except for the initial inflammation phase and endochondral ossification phase of fracture healing. TNF-α has dual roles on osteogenic differentiation depended on the exposure time. Huang et al. [
29] reported that long-term treatment of TNF-α induced inhibitory effect on osteogenic differentiation in vitro while short-term promoted osteogenic differentiation. Also TNF-α was proved with strong ability to induce osteoclast formation [
35]. Therefore, consistent up-regulated TNF-α expression in OVX mice could contribute partially to decreased trabecular bone number and sparser structure of callus, which was consistent with previous studies [
17,
36].
IL-6 levels in the plasma and fracture callus peaked as early as 6 h after fracture, and progressively declined to a low level in 72 h. Compared with sham mice, plasma IL-6 expression in OVX mice was even higher at 6 h after fracture [
37], which might due to the increase of neutrophils [
38]. In this study, IL-6 expression in OVX mice was increased before fracture, and the increase was even obvious during the early stage of fracture which was consistent with the previous finding. Furthermore, there was almost no significant difference in IL-6 expression between OVX and sham mice during the middle and late stages of fracture healing. IL-6 is a potential inducer of osteoclast differentiation independent of receptor activator of nuclear factor kappa-B ligand (RANKL) [
39], and can be produced by plenty of cells under physiological and pathological conditions including immune-mediated cells, mesenchymal cells, vascular endothelial cells, fibroblasts, and so on. Unlike TNF-α, the action of IL-6 signaling is dependent on the availability of RANKL in the microenvironment [
40].
However, no change in IL-1β expression between sham mice and OVX mice was found. This was also consistent with previous animal study that during the fracture healing of IL-1β receptor (I
lr1−/−) mice, no differences in callus, cartilage or bone matrix production could be found [
41].
Previous studies have demonstrated macrophages presented in multiple stages of fracture healing [
7,
8]. Inflammatory macrophages were observed in the early anabolic events of femoral fracture healing; the phenotype changed during the first 3 days from predominantly M1 to M2, and M2 macrophages dominated the gap area at 7 days. Osteal macrophages were predominantly activated at day 14 to 21 after fracture [
7,
42]. In our study, both M1 and M2 macrophages were predominantly presented in the initial phase, and osteal macrophages presented in the middle and late stage during fracture healing in sham mice. OVX led to deceased M1 and M2 macrophages in the initial phase and rarely detected osteal macrophages in the middle and late stages of fracture healing. Moreover, OVX mice also showed fewer M2 macrophages in the endochondral ossification phase of fracture healing. M-SCF has been confirmed a significant contributor to differentiation and maintenance of macrophage multiple populations [
43], and is typically used in vitro to differentiate human monocytes into macrophages [
44]. Therefore, the dramatically decreased expression of M-SCF at 1 DPF in OVX mice may partially contributed to the loss of both M1 and M2 macrophages in the initial phase of bone fracture. IL-4 is capable of stimulating M2 polarization, while MCP-1 is an inhibitory factor for M2 polarization from bone marrow-derived macrophages [
45,
46]. Increased expression of MCP-1 and decreased expression of IL-4 could also partially explain the phenotype of M2 macrophage reduction at the early and middle phases of fracture healing.
M1 macrophages can secrete pro-inflammatory cytokines such as TNF-α, promoting angiogenesis and recruiting bMSCs. M2 macrophages participate in phagocytosing apoptotic cells, dissolving thrombus and promoting tissue repair [
47]. In addition, M2 macrophages were reported a higher angiogenic potential compared to other macrophage subsets [
48]. In vitro experiment also evidenced increased osteogenic markers expression and bone mineralization of M2 macrophage co-cultured bMSCs [
49]. Therefore, decreased inflammatory macrophages as well as lower expressions of inflammatory cytokines, especially TNF-α, disturbed the initiation inflammatory responses of fracture healing, and deceased M2 macrophages could also impaired angiogenesis, osteogenesis, and endochondral ossification in OVX mice. Osteal macrophages were predominantly presented within the maturing/remodeling hard callus during fracture; depletion of osteal macrophages during bone healing has been demonstrated impaired endochondral callus formation and osteoblast mineralization using the Mafia transgenic mouse model [
7,
8,
50]. However, the exact molecular mechanisms by which osteal macrophages affect callus remodeling have not been fully addressed.
There were still some limitations of this study. Firstly, we only examined the expressions of several critical cytokines due to limited samples obtained from mice models, including TNF-α, IL-1β and IL-6. There were plenty of inflammatory or pro-inflammatory cytokines presenting in the process of fracture healing, and more studies should be carried out to totally reveal the expression patterns of these cytokines. Secondly, activated resident macrophages including osteal macrophages, are able to shift into pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages by different cytokines [
51]. Therefore, the cell shift between inflammatory macrophages and osteal macrophages continuously occurred during the process fracture healing. Ovariectomy leads to a chronic increase in systemic inflammation, and it may probably result in dysregulation of the cell shift. However, this issue was not confirmed in the current study. Thirdly, due to the limited sample size, mechanical test which could contribute the evaluation of the quality of fracture healing was not included in this study. Lastly, we selected a small sample size at each time point since it was the first time to evaluate the changes in macrophages and inflammatory cytokine expressions during fracture healing between sham and OVX mice, which might limit greater extrapolation of our results.
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