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Introduction

Although fractures heal, bone nonunion remains one of the major difficulties for bone fracture treatment (1). There were ~10,000 patients suffering from bone fractures in the USA in 2012 (2). Bone nonunion causes significant discomfort for patients, with body pain and mental distress, and increases the burden for the patient, society and the economy (3).

The typical therapeutic methods for bone nonunion include sclerosis bone excision, bone nonunion tissue removal, medullary cavity excavation, bone grafting and fixation (4). During the treatment process, many studies have detected that the majority of hypertrophic nonunion cases achieve bony union through compressive fixation; and atrophic nonunion is generally accompanied by bone defects (5). Bone marrow mesenchymal stem cell (BMMSC) implantation is not exposed and some bone nonunion can be healed (6). However, the mechanism underlying the reason that bone nonunion may be healed without direct treatment of the fracture site remains unclear.

Studies have revealed that fracture healing is a complicated and consecutive process (7,8). At bone nonunion, hematoma and tissues between the fracture ends gradually develop into tissues between the fracture ends of bone nonunion (9). Tissues and hematoma join the fracture ends and in vitro its reaction is similar to that of bone mesenchymal stem cells. Such cells are separated from tissues between fracture ends of bone nonunion (10). BMMSCs have multiple potentialities and present positive expression to surface antigen of bone mesenchymal stem cells (8).

Extracorporeal shock wave therapy (ESWT) is an emerging treatment strategy, which is effective for muscle and joint diseases (11). It is a mechanical pulse pressure wave that is mediated by a physical mechanism. It predominantly includes four types of shockwave generator: Liquid electric, magnetic, piezoelectric and air pressure ballistic (12). The former three are types of focused shockwave and the latter is radial. The former three types to work on the area that requires treatment through focused shockwaves via X-ray or ultrasonic location (12,13). The radial shockwave is to treat larger areas of damaged tissue (14).

Purinergic receptor P2X 7 (P2X7) receptor is widely expressed in a variety of tissues and exerts diverse physiological functions (15). It mediates intracellular signal transduction and intercellular signal communication, and performs significant physiological actions (16). For example, the ion channel formed by the P2X7 receptor mediates the quick response of cells on extracellular signals (17). P2X7 receptor activation by ATP mediates intercellular communication, and causes a series of physiological functions, such as cell apoptosis and necrosis induced by macrophage fusion, induction of lymphocyte membrane bubble formation, promotion of neurotransmitter release at nerve endings, and expression and release of cytokines and chemokines (15,18). ATP activates multiple intracellular signaling pathways mediated by P2X7 (15).

Numerous endogenous cytokines induce differentiation of bone mesenchymal stem cells (BMSCs) into chondrocytes (19). A recent study demonstrated that cytokines promoting the differentiation of BMSCs into chondrocytes include cartilage-derived morphogenetic protein, insulin-like growth factor (IGF), bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) (20). Another study adopted IGF-I to successfully induce the differentiation of mesenchymal stem cells (MSCs) into chondrocytes, the majority of which used BMP-2/4 as factors to induce the differentiation of MSCs into chondrocytes (21).

For example, P2X7 receptor mediates the interleukin-1 (IL-1) maturity of monocytes and macrophages, IL-1 release and precursor protein processing (18). The detection of activated P2X7 receptor excites and conducts associated signaling pathways of phospholipase D (22). In addition, P2X7 mediates proliferation, differentiation and other reactions of extracellular signals by activating mitogen-activated protein kinases. The aim of the present study was to evaluate the effect of ESWT combined with bone marrow mesenchymal stem cell (BMMSC) transplantation to improve bone repair in a rabbit bone nonunion model.

Materials and methods

Experimental animals

A total of 24 purebred New Zealand rabbits (age, 5–6 months; weight, 2.5–3.0 kg) were randomly divided into three groups: Sham (n=8), model (n=8) and combination (n=8). Rabbits were anesthetized via an intramuscular injection of ketamine (50 mg/kg) and positioned on an operating table. The right forelimb was shaved and disinfected using iodine. Skin was sliced (2.5–3.0 cm in length) to expose the radius. In the bone nonunion model and combination group, a 15-mm length of bone was removed from the mid-radius and controlled to ≤0.1 mm using Vernier calipers. A rongeur was used for gouging the bone stump and the injury was sealed using bone wax. The wound was closed using no. 1 silk sutures and disinfected with iodine, and the rabbits were administered oral amoxicillin for 12 days. The sutures were then removed. The present study was approved by the Institute of Animal Care and Use Committee at Southern Medical University (Guangdong, China).

Study design

Bone marrow was extracted from the tibia of the New Zealand rabbits (n=2-3) and mechanically disintegrated and diluted with Sigma-Aldrich Dulbecco's modified Eagle's medium-low glucose (DMEM-LG; Merck KGaA, Darmstadt, Germany). DMEM-LG was centrifuged at 500 × g for 5 min at 4°C. Cells were resuspended using standard growth medium (GM) consisting of DMEM-LG supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin-streptomycin and 0.5% Fungizone® solution (Thermo Fisher Scientific, Inc.) and seeded on one 100-mm culture dish. Fresh GM was replaced every 2–3 days. BMMSCs was induced for 2 weeks and then was used to study. XY-K-SHOCKMASTER-500 ESW Power (Xiangyu Medical Equipment Company, Anyang, China) was used to perform the ESWT therapy (12 kW, 0.45 mJ/mm2, 1,000 shockwaves, 60 per min). BMMSCs (2×106/day) were injected at the wound site. The ESWT session (which was painless) lasted ~6 min and was performed once per day every 5 days for 15 days.

Biomechanical testing

Mechanical strength [Newton (N)] and fracture stiffness (N/mm) were measured using three-point bending with a mechanical testing machine (Zwick/Roell, 1446; Zwick GmbH & Co. KG, Ulm, Germany) and calculated with regard to the radius of the bone. The bearing distance between the two points of support was 21 mm, and three-point cantilever bending was applied at 2 mm/min with the fulcrum placed over the fracture callus.

Histological scoring

The right forelimb was fixed in 10% neutral buffered formalin for 4–5 days, decalcified for 15–20 days in 10% formic acid and embedded in paraffin. Bone was cut into 5 µm longitudinal sections and stained with hematoxylin and eosin at 37°C for 20 min. Histological scoring was performed under a light microscope (magnification, ×100) as previously described (1).

Alkaline phosphatase (ALP) miRNA expression analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from the bone tissue of three groups using Invitrogen TRIzol (Thermo Fisher Scientific, Inc.). cDNA was reverse-transcribed using a One Step SYBR RT-PCR kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed using SYBR Premix Ex Taq (Takara Bio, Inc.) with a ViiA7 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequences of primers for ALP were as follows: forward-GTT TTC TGT TCT GTA AGA CGG G and reverse-GCC GTT AAT TGA CGT TCC GA. Conditions were as follows: 5 min at 95°C (one cycle); 30 sec at 94°C; 30 sec at 60°C and 30 sec at 72°C (40 cycles); then 72°C for 5 min.

ELISA

Peripheral blood was collected from the eye socket of the rabbits and centrifuged at 4,000 × g for 10 min at 4°C to separate the serum. A BCA assay (P0009; Beyotime Institute of Biotechnology, Haimen, China) was used to measure the protein concentration according to manufacturer's protocols. Protein (10 µg) was used to measure the ALP activity, IGF-1 and vascular endothelial growth factor (VEGF) expression levels, and TGF-β contents were measured using ELISA kits (A059-2, H041, H044 and H034 respectively; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

Western blotting

Bone tissue was lysed in RIPA buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) to extract proteins. Equal quantities (50 µg) of protein extracts were loaded and separated by 6–10% SDS-PAGE using 12% acrylamide gradients (80 V for 30 min; 120 V for 60 min) and transferred electrophoretically onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Membranes were blocked with 5% nonfat dry milk in tris-buffered saline containing 0.05% Tween-20 and incubated with anti-osteopontin (OPN; sc-20788; 1:500), anti-runt related transcription factor 2 (RUNX-2; sc-10758; 1:500), anti-collagen type I a1 chain (COL1-A1; sc-28657; 1:500), anti-BMP-2 (sc-402; 1:500), anti-BMP-4 (sc-9003; 1:500), P2X7 (sc-25698; 1:500) and GAPDH (sc-25778; 1:500; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse immunoglobulin G (7074; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was incubated for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence (P0018; Beyotime Institute of Biotechnology).

Statistical analysis

Data are presented as the mean ± standard deviation. Differences between groups were calculated by one-way analysis of variance and Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

ESWT combined with BMMSC transplantation increases mechanical strength, fracture stiffness and histological scoring in a rabbit bone nonunion model

Fig. 1 demonstrated that mechanical strength, fracture stiffness and histological scoring were significantly inhibited in a rabbit bone nonunion model, when compared with the sham group (P<0.01). Furthermore, ESWT combined with BMMSC significantly decreased the inhibition of mechanical strength, fracture stiffness and histological scoring in the rabbit bone nonunion model (P<0.01; Fig. 1).

Figure 1. ESWT combined with BMMSC transplantation increased mechanical strength, fracture stiffness and histological scoring in a rabbit bone nonunion model. ESWT combined with BMMSC transplantation increased (A) fracture stiffness, (B) mechanical strength and (C) histological scoring in a rabbit bone nonunion model. Sham, sham group; model, bone nonunion model group; combination, ESWT combined with BMMSC transplantation group. ##P<0.01 vs. sham group; **P<0.01 vs. model group. ESWT, extracorporeal shock-wave therapy; BMMSC, bone marrow mesenchymal stem cell.

ESWT combined with BMMSC transplantation increases ALP activity and miRNA expression levels in a rabbit bone nonunion model

The results of ELISA and PCR demonstrated that ALP activity and miRNA expression levels were significantly decreased in a rabbit bone nonunion model group, as compared with the sham control group (P<0.01; Fig. 2). Furthermore, ESWT combined with BMMSC significantly enhanced ALP activity and miRNA expression levels in the rabbit bone nonunion model (P<0.01; Fig. 2).

Figure 2. ESWT combined with BMMSC transplantation increased ALP activity and miRNA expression levels in a rabbit bone nonunion model. ESWT combined with BMMSC transplantation increased (A) ALP activity and (B) miRNA expression levels in a rabbit bone nonunion model. Sham, sham group; model, bone nonunion model group; combination, ESWT combined with BMMSC transplantation group. ##P<0.01 vs. sham group; **P<0.01 vs. model group. ESWT, extracorporeal shock-wave therapy; BMMSC, bone marrow mesenchymal stem cell; ALP, alkaline phosphatase.

ESWT combined with BMMSC transplantation increases OPN, RUNX-2 and COL1-A1 protein expression levels in a rabbit bone nonunion model

After 15 days of treatment, OPN, RUNX-2 and COL1-A1 protein expression levels in the rabbit bone nonunion model were significantly suppressed, when compared with the sham group (P<0.01; Fig. 3). ESWT combined with BMMSC transplantation significantly induced OPN, RUNX-2 and COL1-A1 protein expression levels in the rabbit bone nonunion model (P<0.01; Fig. 3).

Figure 3. ESWT combined with BMMSC transplantation increased OPN, RUNX-2 and COL1-A1 protein expression levels in a rabbit bone nonunion model. ESWT combined with BMMSC transplantation enhanced OPN, RUNX-2 and COL1-A1 protein expression levels. (A) Western blot analysis and statistical analysis of (B) OPN, (C) RUNX-2 and (D) COL1-A1 protein expression levels in a rabbit bone nonunion model. Sham, sham group; model, bone nonunion model group; combination, ESWT combined with BMMSC transplantation group. ##P<0.01 vs. sham group; **P<0.01 vs. model group. ESWT, extracorporeal shock-wave therapy; BMMSC, bone marrow mesenchymal stem cell; OPN, osteopontin; RUNX-2, anti-runt related transcription factor 2; COL1-A1, anti-collagen type I a1 chain.

ESWT combined with BMMSC transplantation enhances IGF-1, VEGF and TGF-β contents in a rabbit bone nonunion model

To detect the underlying mechanism of ESWT combined with BMMSC in bone nonunion, IGF-1, VEGF and TGF-β contents were measured using ELISA kits. Fig. 4 indicates that the IGF-1, VEGF and TGF-β contents of the rabbit bone nonunion model group were markedly lower than those of the sham control group. ESWT combined with BMMSC transplantation significantly promoted IGF-1, VEGF and TGF-β contents in the rabbit bone nonunion model (Fig. 4).

Figure 4. ESWT combined with BMMSC transplantation enhanced IGF-1, VEGF and TGF-β contents in a rabbit bone nonunion model. ESWT combined with BMMSC transplantation enhanced (A) IGF-1, (B) VEGF and (C) TGF-β contents in a rabbit bone nonunion model. Sham, sham group; model, bone nonunion model group; combination, ESWT combined with BMMSC transplantation group. ##P<0.01 vs. sham group; **P<0.01 vs. model group. ESWT, extracorporeal shock-wave therapy; BMMSC, bone marrow mesenchymal stem cell; IGF-1, insulin-like growth factor; TGF, transforming growth factor-β; VEGF, vascular endothelial growth factor.

ESWT combined with BMMSC transplantation enhances P2X7, BMP-2 and BMP-4 protein expression levels in the rabbit bone nonunion model

In the current study, the inhibition of P2X7, BMP-2 and BMP-4 protein expression levels in a rabbit bone nonunion model were observed, compared with the sham control group (Fig. 5). After day 15, ESWT combined with BMMSC transplantation significantly elevated P2X7, BMP-2 and BMP-4 protein expression levels in the rabbit bone nonunion model (Fig. 5).

Figure 5. ESWT combined with BMMSC transplantation enhanced P2X7, BMP-2 and BMP-4 protein expression levels in a rabbit bone nonunion model. ESWT combined with BMMSC transplantation enhanced P2X7, BMP-2 and BMP-4 protein expression levels. (A) Western blot analysis and statistical analysis of (B) BMP-2, (C) BMP-4 and (D) P2X7 protein expression levels in a rabbit bone nonunion model. Sham, sham group; model, bone nonunion model group; combination, ESW combined with BMMSC group. ##P<0.01 vs. sham group; **P<0.01 vs. model group. ESWT, extracorporeal shock-wave therapy; BMMSC, bone marrow mesenchymal stem cell; BMP, bone morphogenetic protein; P2X7, purinergic receptor P2X 7l.

Discussion

Fracture and bone tissue damage are common and serious problems in the clinical setting (6). With the population aging, this type of problem is becoming increasingly prominent (23). Development of artificial bone with high biocompatibility, that is completely biodegradable with a degradation rate that matches bone cell growth rate, with high biological activity and stable mechanical properties is an effective approach for treating the above-mentioned bone issues (5).

In recent years, MSCs have been a particularly popular research topic, with the most development potential (6). MSCs differentiate into various mesodermal cell types, such as fat precursor cells, bone cells and cartilage cells, and differentiate to bone, heart and smooth muscle cells (7). Furthermore, MSCs present morphological characteristics of non-mesoblast cells, such as neuronal and hepatic cells (7). The present results indicated that ESWT combined with BMMSC transplantation effectively enhanced mechanical strength, fracture stiffness and histological scoring, and increased ALP activity in a rabbit bone nonunion model.

RUNX-2 is a key transcription factor that controls osteogenic differentiation of human MSCs (24). The cytokine regulates regulatory proteins jointly and induces the undifferentiated progenitors to differentiate into osteoblasts (24). Furthermore, Osterix (Osx) and RUNX-2 regulate the transcription factor of osteogenic differentiation (25). In particular, bone cells are essential for regulating the effects of early- and late-stage osteoblast differentiation (25). RUNX-2 and Osx combination initiates the differentiation of preosteoblast (which expresses type I collagen and bone sialoprotein) to osteoblasts. Subsequently, it induces and activates transcription factors, and induces the maturity of osteoblasts jointly with Osx and various Wnt/β-link protein signaling components (25). Collectively, the present study demonstrated that ESWT combined with BMMSC transplantation promoted ALP miRNA expression levels and activity, and induced OPN, RUNX-2 and COL1-A1 protein expression in a rabbit bone nonunion model.

It remains controversial as to whether there are P2X7 receptors in osteoblast cell lines (15). Previously, it was detected that the MG-63 osteogenesis cell strain contains P2X7 transcriptional factors (26). However, P2X7 specific receptor adjusts osteoblast differentiation (27). Subsequently, in vitro immune cell chemical analysis and membrane pore forming reaction experiments demonstrated P2X7 receptor expression in a human bone-derived stem cell subgroup (22). In addition, whether ESWT combined with BMMSC transplantation significantly increased P2X7 protein expression levels in a rabbit bone nonunion model was investigated in the present study.

Studies have also indicated that following bone fracture, TGF-β is activated with extensive expression, and this increased expression is maintained for the duration of the healing process; in addition, ectogenic TGF-β stimulates osteoblasts and accelerates fracture rehabilitation (28). Studies on bone formation and fracture recovery must not ignore the effects of cytokines and polypeptide GFs, and must focus on the effects of TGF-β (28,29).

IGF-1 exerts moderate mitosis effects on osteoblasts. It regulates cell cycle activities and exerts para-insulin effects. IGF-1 is an essential GF for bone cell secretion (30). It contributes to promoting the formation of osteoclasts, stimulating the activity of osteoclasts, adjusting bone resorption and participating in bony remodeling (31). Notably, the current study indicated that ESWT combined with BMMSC transplantation significantly promoted IGF-1, VEGF and TGF-β contents in a rabbit bone nonunion model.

BMP are important in the growth and development of bones, as well as during rehabilitation following trauma. However, the primary function is to induce the formation of bones (29). BMPs induce specific, undifferentiated and active ectomesenchymal cells in the muscles and around blood vessels to differentiate them into cartilage and osteocytes. The process is irreversible (32). The bone induction ability of BMPs primarily presents on cartilage, muscles and blood vessels (32). For liver, spleen, kidney and other organs, however, it does not exhibit an obvious bone induction ability. Currently, BMP is caused by different reactions of mesenchymal cells (33). It also indicates that the osteoinductive activity of BMP is closely associated with the surrounding environment. BMPs show a strong ability to induce bone formation at cartilage muscles and around blood vessels (34). Studies have confirmed that BMPs binding to receptors may phosphorylate and release Smad proteins, and subsequently enter into the nucleus to activate the transcription and expression of specific genes, resulting in osteogenic and chondrogenic differentiation (19,21). BMP-2/4 is vital to cartilage tissue engineering, and is considered to be associated with MSCs regulating the cell cycle and differentiation of chondrocytes; thus, BMP-2/4 facilitates the synthesis and secretion of cartilage matrix (35). In the current study, ESWT combined with BMMSC transplantation significantly induced BMP-2 and BMP-4 protein expression levels in a rabbit bone nonunion model. Pfaff et al (36) demonstrated that ESWT enhances bone healing via BMP-2, BMP-4, IGF-1, VEGF, and TGF-β expression (36).

In conclusion, the present study demonstrated that ESWT combined with BMMSC transplantation improves bone repair in a rabbit bone nonunion model via BMPs and P2X7 expression. ESWT combined with BMMSC transplantation was presented as a novel and effective method to improve bone repair in a rabbit bone nonunion model, which may be useful in clinical applications. However, this study only employed an in vivo model, which is a limitation, model study or clinical research are required for further study.

Acknowledgements

The present study was supported by Southern Medical University Scientific Research Start-up Program and Guangdong Province Science and Technology Project (grant no. 2017ZC0121).

References