Experimental study on the effect of warm acupuncture on the healing of bone nonunion in rats

Abstract

Background:

Bone nonunion is a challenging clinical issue that hinders fracture healing. Warm acupuncture, as a traditional Chinese medicine therapy, has shown potential in promoting tissue repair, but its effect on bone nonunion healing and the underlying mechanism remain unclear. This study aims to investigate the effect of warm acupuncture on the healing of bone nonunion in rats and predict its potential mechanism.

Methods:

Sprague-Dawley (SD) rats were randomly divided into a blank control group (BC group), a tibial bone nonunion model group (M group), and a warm acupuncture treatment group (WA group). The BC group was not treated. Bone nonunion modeling was performed on M and WA groups. After modeling, M group was tied up. The WA group was treated with warm acupuncture after restraint, once a day for 4 weeks. After treatment, collected blood samples, performed digital radiography (DR), micro-computed tomography (micro-CT), hematoxylin-eosin staining (HE staining), and assess the bone morphogenetic protein 2 (BMP-2) protein expression by Western blot.

Results:

After treatment, the phosphorus (P), calcium (Ca), and alkaline phosphatase (ALP) levels and the BMP-2 protein expression level were significantly higher in WA group than in BC group and M group. The difference between WA group and M group in terms of fracture scab measurement was statistically significant. The recovery of fracture structure in WA group was better than that in M group. WA group was better than M group in bending strength, bending stiffness, bending load, bending deflection, bending moment, and deflection. Bone volume fraction, trabecular thickness, and number of trabeculae were higher in WA group than in M group.

Conclusion:

Warm acupuncture can enhance the serum Ca, ALP, and P concentrations and increase the expression of BMP-2 in rats, thus improving bone scab production, promoting the healing of fractures, and increasing the biomechanical strength in rats.

Lay Summary: By establishing a model of tibial bone nonunion in rats, this study clarified that warm acupuncture could change serum calcium (Ca), alkaline phosphatase (ALP), and phosphorus (P) ion concentration, increase mechanical strength and bone morphogenetic protein 2 (BMP-2) expression in rats, thus promoting callus formation and the healing of tibial fractures in rats.

1. INTRODUCTION

Despite the rapid advances in minimally invasive surgical techniques in surgery, which have led to excellent treatment of fractures, the incidence of bone nonunion, a common and serious complication of fractures, remains high.^1,2 Bone nonunion is the failure to form bone connections after a fracture and the cessation of the bone healing process.² The current diagnostic criteria for bone nonunion still follow those established by the United States Food and Drug Administration (FDA): 9 months after fracture without healing and three consecutive months without any signs of healing.³ According to a recent survey of 4 million fracture patients, the average rate of bone nonunion is 1.9%, while the rate of bone nonunion for specific fractures (eg, tibia and clavicle) and elderly fractures is as high as 9%.⁴ The search for the etiology and mechanisms of bone nonunion has never stopped, and although great discoveries have been made, no definitive conclusions have been reached until now. The main causes of nonunion are fracture healing stagnation, inactive osteoblasts, fibrosis after the occlusion of the bone marrow cavity, fibrous tissue in contact with bone and cartilage in the fracture space, and poor angiogenesis around the fracture.² Currently, there are various treatment measures for bone nonunion, but there is no widely accepted gold standard.¹ Bone grafting is currently the main clinical treatment for bone discontinuity, but autologous bone grafting requires surgical removal of bone from the patient’s body, which can be very traumatic and may lead to wound infection, persistent pain in the donor area, and postoperative wound hematoma, causing great pain to the patient.⁵ These factors are not conducive to the widespread use of autologous bone grafting. Although allogeneic bone grafting is possible, there is a theoretical risk of transmitting infectious diseases such as hepatitis and acquired immunodeficiency syndrome (AIDS), and it may also cause immune rejection, as well as the risk of residual toxic and harmful substances during the treatment process due to different treatment methods and technical levels of manufacturers.^6,7 This shows that allogeneic bone grafts, although widely available, have severe immune rejection and poor osteogenic capacity. To overcome the shortcomings of bone grafting in the treatment of bone nonunion, many experts and scholars have started to search for alternative treatments to bone grafting.

Chinese medicine has many unique theories and experiences in the treatment of fractures, delayed healing of fractures, and bone nonunion.^8,9 Chinese medicine believes that delayed healing or nonhealing of fractures is closely related to kidney deficiency, and tonifying the liver and kidney can improve osteoblast activity, promote matrix calcification, increase trace element content, accelerate bone scab growth, and improve the quality and biomechanical properties of bone scabs.¹⁰ In addition, “if blood stasis is not removed, new blood will not be created,” and blood stasis exists in the whole process of fracture lesion, and promoting blood circulation for removing blood stasis has the function of improving microcirculation at the fracture end, thus improving the permeability of the blood vessel wall and facilitating the exchange and deposition of calcium ions.¹¹ According to the idea of three stages of identification and treatment in Chinese medicine after fracture, the method of tonifying qi, activating Blood, and tonifying liver and kidney is used to jointly control and treat bone nonunion with good effect according to the treatment principle of middle and late stages of fracture.^8,10 Acupuncture, as an effective treatment method in Chinese medicine, has remarkable effects in the treatment of orthopedic diseases.¹² Acupuncture therapy can mobilize the body to recover actively, promote blood circulation, and drain the meridians, thus enhancing the metabolic capacity of the body and promoting nutrient absorption.¹³ Warm acupuncture based on acupuncture and using methods of warming and tonifying at the same time has the effect of tonifying the liver and kidney, adjusting the balance of viscera and bowels, meridians and qi and blood, yin and yang, and promoting fracture healing and functional recovery.¹⁴ At present, there are no cases of simple acupuncture in bone nonunion and its related underlying mechanism. Therefore, in this study, a model of bone nonunion in SD rats was prepared and warm acupuncture intervention was performed using the main acupoints of Dazhu (BL11), Geshu (BL17), and Shenshu (BL23) to observe the healing of tibial bone nonunion in rats and to find its possible potential mechanisms.

2. METHODS

2.1. Experimental materials

Experimental animals and reagents: 200 clean grade 8-month-old male SD rats, weighing 332.6 ± 13.4 g, provided by the Animal Experiment Center of Southwest Medical University (Sichuan, China), Experimental Animal Use License No. SYXK (Chuan) 2013-065; Penicillin sodium for injection (Lupin Pharmaceutical Co., Ltd., Shandong, China, Lot No. B170413); BMP-2 protein assay kit (Sigma ); Ca, P, ALP assay kit (Myriad Medical, Guangdong, China); Pentobarbital (Shanghai Rinne Technology Development Co. Ltd., Shanghai, China). Main experimental instruments: AndyBio micro centrifuge; M-centrifuge electronic universal mechanics experiment machine - Shimadzu, Japan; BS biochemical analyzer (Myriad Medical); Siemens DR; Siemens Inveon micro-PET-CT.

2.2. Experimental methods

2.2.1. Experimental groups

Male SD rats weighing 300 to 350 g at 8 months of age were selected and randomly divided into a blank control group (BC group, 10 rats) and experimental group (140 rats). The experimental group was manufactured with reference to the standard experimental tibial bone nonunion model of rats.^15–17 After 2 months of modeling of bone nonunion in rats, gross observation, histopathology, and radiological examination showed that the fracture site did not heal, and the intramembrane ossification and intrachondral ossification near the fracture had stopped, which could be considered as successful modeling of the rat bone nonunion model.^18,19 After 2 months of modeling, the rats in the experimental group were examined by micro-computed tomography (CT) to confirm the diagnosis of tibial nonunion, and those rats that did not meet the diagnosis of bone nonunion were excluded, and those rats that met the diagnostic criteria of bone nonunion were randomly divided into M group and WA group. The rats in M group were not treated with warm acupuncture, but were immobilized on a rat immobilizer for half an hour, just like the rats in WA group, to reduce the differences caused by different environments. In WA group, warm acupuncture was given on the first day after the determination of bone nonunion. The procedure was as follows: the rat was fixed on the operating table with a homemade rat suit, as shown in Fig. 1, and “Dazhu (BL11), Geshu (BL17), and Shenshu (BL23)” were selected as specific points, together with “Quze (PC3), Chize (LU5), Shaohai (HT3), and Tianfu (LU3)” (refer to Experimental Acupuncture). A 0.5-inch milli-needle was selected and pricked directly for 0.5 cm. Small homemade moxa strips were placed over the needles and lit, and the needles were left in place for 20 minutes and treated once daily between 9:00 am and 11:00 am for 4 weeks. No acupuncture intervention was performed on the same day of animal sampling.

Fig. 1.

Experimental flowchart. ALP = alkaline phosphatase; BMP-2 = bone morphogenetic protein 2; Ca = calcium; P = phosphorus; SD = Sprague-Dawley.

2.2.2. Bone nonunion modeling method

Rats were anesthetized with pentobarbital 4% by volume at a dose of 2 mL/kg by intraperitoneal injection. After anesthesia took effect, the rats were placed in the supine position, the limbs were fixed on the operating table, and the hind limbs were routinely sterilized with sterile towels. An incision of about 1.5 cm in length was made along the tibia on the ventral side of the calf, and the calf muscles were bluntly dissected with a stripper to expose the tibia. A saw osteotomy was performed near the middle and lower tibia to expose both ends of the fracture. The dental cement filling device was heated by alcohol lamp, and the internal and external periosteum at both ends of the fracture was burned after the filling device turned red. The fracture was well aligned to the line with no rotation under the naked eye, thoroughly hemostatic, and sutured to each layer in turn. Each rat was kept in separate cages after surgery and injected with 40 000 units of penicillin intramuscularly daily for seven consecutive days.^16,20

2.2.3. Detection of serum Ca, P, and ALP contents

The rats in WA group underwent tail vein blood sampling 7 and 14 days after the warm acupuncture treatment, and the control and M groups were treated by drawing blood from the tail vein at the same time. Ten microliters of rat serum was placed in the measuring wells of Mindray Ca, P and ALP test kit, and the enzyme standard 610 and 520 nm were selected to test the OD value, and then the Ca, P, and ALP contents in the serum were calculated according to the formula given in the test kit instruction.

2.2.4. Imaging tests

At 7 and 14 days of warm acupuncture treatment, both hind limbs of each rat in M group and WA group were photographed in the same abduction position with orthogonal and lateral radiographs, and measurements were performed using the Picture Archiving and Communication Systems (PACS) for DR reading, and radiographs and measurements were taken by two physicians according to the double-blind principle. The scab volume was measured using the Perkins method, that is, V = 2πr1(r² − r¹) L, where r¹ is the bone radius, r² is the scab radius, and L is the scab length.

2.2.5. Pathological histological examination

At 7, 14, and 28 days of warm acupuncture treatment, five rats in M group and five rats in the WA group were executed. In each group, five tibial specimens were taken out, and the epiphyseal bone scab and the bone scab attached to the fracture end were immediately dissected (the same part was taken from the non-fracture group), trimmed into 1 cm × 1 cm size specimens, fixed in 4% neutral formalin solution, embedded in paraffin, and subjected to HE, and the pathological changes were observed under the light microscope.

2.2.6. Biomechanical test

Ten rats in M group and 10 rats in WA group were executed at 14 and 28 days of warm acupuncture treatment. In each group, 10 tibial specimens were taken out. The tibial specimens were placed on two fulcrum points of the universal material testing machine with a span of 15 mm for a three-point bending test. The loading speed was 5 mm/min, and the loading load was 500 N. The displacement produced after each loading is automatically recorded by the instrument, and the computer automatically processes the measured values of the tibia of each group of rats.

2.2.7. Micro-CT

Ten rats in M group and 10 rats in WA group were executed at 7, 14, and 28 days, in each group, 10 tibial bone specimens were removed and micro-CT was performed on the tibial fracture segment. The bone specimens were placed horizontally in the sample holder, and the voltage was set to 80 kV, and the current was set to 500 μA. Five millimeters above and below the fracture line were selected for scanning, and the analysis indices included bone trabecular thickness, bone volume fraction, bone surface area volume ratio, bone trabecular number, and bone trabecular spacing, among which bone trabecular thickness, bone volume fraction, and bone trabecular number were positive osteogenic indices, and bone trabecular spacing and bone surface area volume ratio were negative indices.

2.2.8. Western blot detection of bone morphogenetic protein 2 protein expression

Five rats in M group and five rats in WA group were executed at days 7, 14, and 28, and bone tissues 5 mm above and below the fracture line were taken for bone protein extraction. Specific steps: take 120 to 180 mg of bone tissue specimens into 10 mL centrifuge tubes, cut up in ice bath, add 1 mL of cell lysis solution, grind bone tissue by internal homogenizer, place on ice for 30 minutes, then centrifuge at 12 000 r/min for 20 minutes at 4°C, take the supernatant, divide into 0.5 mL centrifuge tubes, 20 µL per tube. The target protein concentration was measured by bicinchoninic acid (BCA) protein assay and bone morphogenetic protein 2 (BMP-2) expression in the fractured end bone tissue was measured by Western blot. The grayscale values of each band of Western blot results were scanned by gel system analysis software, and the ratio of each group of bands to the group of internal reference proteins, that is, the relative expression, was calculated and compared using β-actin as the internal reference protein.

2.3. Statistical analysis

Experimental data were processed with SPSS 20.0 statistical software and expressed as mean ± SD (x ± S), and one-way analysis of variance (ANOVA) or t test was used to compare the significance of differences between groups, with p < 0.05 indicating statistical significance.

3. RESULTS

3.1. Serum Ca, P, and ALP contents

There were no significant differences in Ca, P, and ALP contents between groups two months after surgery. Seven days after warm acupuncture treatment, serum P and ALP contents in WA group were significantly higher than those in BC group and M group, and there was no significant difference between Ca contents in WA group and BC group and M group. After 14 and 28 days of acupuncture treatment, the serum Ca, P, and ALP contents in WA group were significantly higher than those in BC group and M group (Table 1).

Table 1.

Serum Ca, P, and ALP contents (x ± S, n = 10)

Time	Group	ALP (U/L)	Ca (mmol/L)	P (mmol/L)
2 mo after modeling	Control	79.40 ± 14.90	2.34 ± 0.56	2.21 ± 0.19
	Model	72.30 ± 16.38	2.16 ± 0.84	2.05 ± 0.54
	WA	73.40 ± 7.91	2.13 ± 0.09	2.16 ± 0.04
Treatment for 7 d	Control	76.60 ± 13.20	2.24 ± 0.41	2.19 ± 0.22
	Model	69.07 ± 11.34	2.02 ± 0.35	1.96 ± 0.37
	WA	257.40 ± 24.91****	2.37 ± 0.15	2.91 ± 0.15****
Treatment for 14 d	Control	72.44 ± 13.30	2.15 ± 0.45	2.23 ± 0.21
	Model	66.3 ± 12.29	2.12 ± 0.67	2.17 ± 0.24
	WA	342.4 ± 15.31****	2.85 ± 0.13*	2.96 ± 0.21****
Treatment for 28 d	Control	69.30 ± 10.61	2.08 ± 0.39	2.34 ± 0.26
	Model	63.20 ± 11.20	2.17 ± 0.56	2.03 ± 0.20
	WA	388.1 ± 20.69****	2.98 ± 0.16**	3.05 ± 0.23****

WA group compared with model group,*p < 0.05,**p < 0.01,****p < 0.0001.

ALP = alkaline phosphatase; Ca = calcium; P = phosphorus; WA = warm acupuncture.

3.2. Calculation results of DR bone scab volume

DR detected bone scab volume in rats 2 months after surgery of bone nonunion, clear fracture lines were visible in M group and WA group, and no scab growth was seen; 14 days after warm acupuncture treatment, fracture lines in WA group were blurred, and a small amount of low-density scab was visible around them, and there was no significant change in M group; 28 days after warm acupuncture treatment, fracture lines in WA group were not obvious, and a small amount of high-density. The fracture line of M group was blurred and no obvious bone scab growth was seen (Table 2 and Fig. 2).

Table 2.

DR callus volume calculation results (mm³, x ± S, n = 10)

Group	2 mo after modeling	Treatment for 14 d	Treatment for 28 d
Model	0	0	0
WA	0	3.18 ± 2.43	13.21 ± 4.47

DR = digital radiography; WA = warm acupuncture.

Fig. 2.

Digital radiography. WA = warm acupuncture.

3.3. Pathological histological results

Two months after surgery, a large amount of fibrous connective tissue proliferation around the fracture end, sclerosis and atrophy of the fracture end, and closure of the medullary cavity could be seen; after 7 days of warm acupuncture treatment, an increase in local new capillaries could be seen in WA group; after 14 days of treatment, a small amount of osteoblast aggregation, a significant decrease in new capillaries, and a small amount of new bone trabeculae could be seen; after 28 days of treatment, a small amount of mature bone trabeculae could be seen (Fig. 3).

Fig. 3.

Histopathological pictures. WA = warm acupuncture.

3.4. Three-point bending test results

The three-point bending test was performed on the tibia of rats in M and WA groups on 7 and 14 days of acupuncture treatment. The results showed that the WA group was better than M group in bending strength, bending rigidity, bending load, bending deflection, bending moment, and contrast flexibility, which were statistically significant (Table 3).

Table 3.

Three-point tibial bending mechanical index (x ± S, n = 10)

Group	Bending load (N)			Bending moment (N·mm)			Bending deflection (mm)
	7 d	14 d	28 d	7 d	14 d	28 d	7 d	14 d	28 d
WA	34.26 ± 11.21***	78.24 ± 12.17****	103.40 ± 13.07****	192.4 ± 19.36****	479.2 ± 28.39****	616.9 ± 49.25****	0.22 ± 0.06***	0.34 ± 0.03****	0.51 ± 0.06****
Model	14.28 ± 4.18	32.21 ± 8.61	56.11 ± 7.22	98.24 ± 11.25	218.4 ± 21.81	328.2 ± 13.19	0.12 ± 0.05	0.18 ± 0.05	0.31 ± 0.07

Group	Contrast flexibility (×10−2)			Bending strength (MPa)			Bending rigidity (104, N·mm/°)
	7 d	14 d	28 d	7 d	14 d	28 d	7 d	14 d	28 d
WA	0.65 ± 0.05****	0.79 ± 0.07****	0.93 ± 0.11****	64.14 ± 8.25***	118.64 ± 15.65****	163.4 ± 12.61****	1.29 ± 0.21**	1.94 ± 0.36****	2.46 ± 0.21****
Model	0.31 ± 0.06	0.49 ± 0.12	0.65 ± 0.08	42.62 ± 5.46	62.54 ± 11.34	80.14 ± 9.34	0.93 ± 0.04	1.29 ± 0.18	1.57 ± 0.21

The indices of WA group were compared with model group, **p < 0.01, ***p < 0.001, ****p < 0.0001.

WA = warm acupuncture.

3.5. Micro-CT osteogenic index results

Two months after surgery, no bone scab formation was seen at the fracture end; after 7 days of warm acupuncture treatment, a small amount of new bone trabeculae was visible in the WA group, and local bone scab formation was visible; on day 14, bone scab formation from the distal to the proximal end of the fracture line increased significantly in the WA group, and there were significantly more new bone trabeculae; on day 28, bridge callus formation was visible, and bone scab had completely wrapped the fracture end; there was no significant bone scab formation in M group. On days 7, 14, and 28, bone volume fraction, bone trabecular thickness, and number of bone trabecular were higher in the WA group than in M group; bone trabecular gap and bone area volume ratio were larger in M group than in the WA group (Table 4 and Fig. 4).

Table 4.

Comparison of bone index in the composition of model and WA treatment group (x ± S, n = 10)

Group	Bone volume fraction (%)
	2 mo after modeling	Treatment for 7 d	Treatment for 14 d	Treatment for 28 d
WA	6.35 ± 1.25	10.18 ± 2.74*	14.36 ± 3.85****	19.12 ± 2.64****
Model	6.33 ± 0.86	6.82 ± 1.15	7.37 ± 1.46	8.26 ± 4.54

Group	Bone area volume ratio (1/mm)
	2 mo after modeling	Treatment for 7 d	Treatment for 14 d	Treatment for 28 d
WA	43.24 ± 4.85	40.54 ± 6.61	38.36 ± 8.93	36.15 ± 8.17
Model	42.98 ± 5.53	43.98 ± 7.35	45.78 ± 7.73	44.26 ± 9.28

Group	Number of trabecular bone (1/mm)
	2 mo after modeling	Treatment for 7 d	Treatment for 14 d	Treatment for 28 d
WA	1.84 ± 0.15	2.14 ± 0.36	3.98 ± 0.38****	4.77 ± 0.69****
Model	1.79 ± 0.46	1.80 ± 0.55	1.96 ± 0.21	2.24 ± 0.44

Group	Bone trabecular thickness (1/μm)
	2 mo after modeling	Treatment for 7 d	Treatment for 14 d	Treatment for 28 d
WA	28.34 ± 2.16	31.64 ± 6.18	43.76 ± 9.86**	71.97 ± 11.61****
Model	27.74 ± 1.45	28.68 ± 5.48	30.84 ± 8.35	35.87 ± 8.92

Group	Bone trabecular gap (μm)
	2 mo after modeling	Treatment for 7 d	Treatment for 14 d	Treatment for 28 d
WA	548.4 ± 87.93	504.2 ± 73.29	437.38 ± 78.22	319.8 ± 69.37***
Model	551.5 ± 92.15	548.4 ± 66.69	537.4 ± 112.4	487.7 ± 114.7

Except for 2 mo after the operation, the above indices of group WA and group model at other time points were compared *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

WA = warm acupuncture.

Fig. 4.

Micro-computed tomography. WA = warm acupuncture.

3.6. Tissue BMP-2 protein expression level

BMP-2 protein expression was detected in the fracture area of rats in each group after surgery of bone nonunion, and the expression level was low and there was no significant difference. The BMP-2 protein expression level started to increase on day 7 after treatment in the WA group and started to decrease after reaching the peak on day 14. The BMP-2 protein expression levels in the WA group increased significantly compared with M group and BC group on days 7, 14, and 28, with statistical differences, and the results are shown in Fig. 5.

Fig. 5.

Western blot. BMP-2 = bone morphogenetic protein 2; WA = warm acupuncture. **p < 0.01,****p < 0.0001.

4. DISCUSSION

Ultimately, our research found that warm acupuncture can enhance the serum Ca, ALP, and P concentrations and increase the expression of BMP-2 in rats, thus improving bone scab production, promoting the healing of fractures and increasing the biomechanical strength in rats. This suggests that warm acupuncture is effective in promoting the healing of bone nonunion.

There is no uniform definition of bone nonunion, also known as nonunion or delayed bone healing. The current clinical diagnosis still follows the FDA definition of nonunion of a fracture: a fracture that has not healed for 9 months and has shown no signs of healing for 3 consecutive months. Both conditions are indispensable. However, it is impossible to prove that the fracture has stopped growing by precise mechanical tests and histological means. Although the rapid development of technical concepts and internal fixation materials in orthopedics has opened up many new ideas and areas for fracture healing, bone nonunion is still a challenge for orthopedic surgeons, especially because the treatment of bone nonunion requires a combination of surgical and non-surgical treatments and cannot rely on the use of the same method to solve all problems.²¹

The underlying causes of bone nonunion are mainly stalled fracture healing, inactive osteoblasts, occluded fibrosis of the bone marrow cavity, fibrous tissue in contact with the bone and cartilaginous and fibrous ossification in the fracture gap, and poor vascular regeneration around the fracture.²² Western medicine still follows the Weber-Cech classification of bone nonunion, which is classified according to tissue reaction as: dystrophic bone nonunion, infected bone nonunion, hypertrophic bone nonunion, atrophic bone nonunion, and synovial pseudojoint, and each type has a corresponding treatment.^23–25 In contrast, traditional medicine does not have the names of delayed fracture healing and bone nonunion, and TCM does not have the concept of bone nonunion due to surgical infection in the treatment of fractures, so the bone nonunion described in this study does not include infected bone nonunion. In Chinese medicine, bone nonunion is classified as bone deficiency and bone wilting, which is considered to be caused by “Essence deficiency cannot be irrigated, Blood deficiency cannot be nourished, Qi deficiency cannot be filled, Blood stasis does not flow smoothly,” and the treatment is holistic, aiming to mobilize all the potential of the body to promote fracture growth.⁹ In this study, Dazhu (BL11), Geshu (BL17), and Shenshu (BL23) are used as the main points. Dazhu (BL11) is an acupoint belonging to the bladder meridian and is also a crossing point of the gallbladder meridian, triple energizer meridian, small intestine meridian, and bladder meridian. It can promote the flow of qi through the greater yang meridians and lesser yang meridians, and is clinically useful for clearing heat and dispersing wind, and for strengthening tendons and bones. In the book of “Nanjing Ben Yi,” Si Ming Chen said, “The intersection of bones is the Dazhu (BL11), bone is nourished by marrow, marrow starts at the lower part of the brain, perfuses into the Dazhu (BL11), and the Dazhu (BL11) penetrates into the spine and runs down through the bone nodes, so the essence of the bone, all converge in this.” This shows that the so-called Bone-Intersection must have the effect of strengthening the tendons and bones, and can be used to treat all bone diseases. Geshu (BL17) is the Blood-Intersection and has the function of regulating blood and activating blood, removing stasis, and creating new blood. The book of “Nanjing” says, “The intersection of blood is the Geshu (BL17). The spokesman said: this is used to treat blood diseases. The upper part of the Geshu (BL17) is the Xinshu (BL15), which is the master of blood, and the lower part of the Geshu (BL17) is the Ganshu (BL18), which collects blood. So Geshu (BL17) is the intersection of blood. Its treatment is mainly for blood disorders, and its effect of activating blood and resolving stasis is particularly prominent. According to Chinese medicine, stagnant blood accumulates inside the body, causing the qi and blood to flow poorly, resulting in the blockage of qi and blood in a certain part of the body. Stagnation of qi and blood may bring about pain. The accumulated blood stasis will clot if it does not disperse, and once it forms stasis, the normal blood will be reduced, and the generation of new blood will be affected. The Shenshu (BL23) is one of the Back-Shu acupoints, the Back-Shu acupoints are the acupoints where qi of the five viscera and six bowels is infused on the back, and “the kidney is the master of bone and marrow.” If kidney essence is sufficient and bone marrow genesis and transformation has a source, the bones are nourished and strong by the bone marrow. If kidney essence is deficient, the bone marrow is not enough to nourish the bones, and fractures and bone diseases will occur. Therefore, acupuncture Shenshu (BL23) is beneficial to kidney qi and strengthens lumbar spine. So, these three points are chosen as the main acupoints for treatment. In addition, we refer to Experimental Acupuncture to select “Quze, Chize, Tianfu, and Shaohai” as combination points. Quze is an acupoint of the pericardium meridian, with the effect of activating blood and resolving stasis; Chize and Tianfu acupoints belong to the lung meridian, which have the function of activate blood to relieve pain and tonify qi and replenish blood; Shao Hai point is a acupoint on the Heart meridian, which is the place where the heart blood converge, so it can tonify qi and replenish blood. These four acupoints are combined with the main acupoints to achieve the effect of tonifying qi and replenishing blood and activating blood and resolving stasis.

Acupuncture has made a breakthrough in fracture healing. Modern medicine believes that acupuncture therapy can mobilize the body, promote blood circulation, unblock the meridians, thus enhancing the metabolic capacity of the body, promoting nutrient absorption, and adjusting the balance of the viscera and bowels, meridians and qi and blood, yin and yang to facilitate fracture healing and functional recovery.^13,26 In this experiment, the trend of bone scab growth appeared in the rats in WA group 7 days after receiving acupuncture treatment, while the fracture line was blurred after 14 days and obvious bone scab growth was visible. Micro-CT, on the other hand, verified the growth of bone scab more precisely from the volume and quality level of bone scab. The three-point bending test showed that the strength of the scab formed after acupuncture was significantly better than that of BC group, demonstrating that acupuncture stimulates scab growth and that the formed scab gradually mineralizes to the strength required by the bone.

The concept of “Therapeutic angiogenesis” was proposed by ISNER J M in the 1990s, and its theory revealed the mechanism of in vivo revascularization. He believed that the stimulation of the ischemic periphery through intervention to induce microangiogenesis in the ischemic area could establish collateral circulation and promote fracture healing.^26,27 The results of the present experiment showed that the WA group showed an increase in local neovascularization capillaries after 7 days of warm acupuncture treatment, a small number of osteoblasts aggregation and a small number of new bone trabeculae after 14 days, and fibrous osteogenesis and intrachondral osteogenesis and a small number of mature bone trabeculae after 28 days, which also confirmed that acupuncture promoted the neovascularization and thus the formation of new bone. Trace elements are also necessary for the formation of bone scabs. Lü et al suggested that acupuncture can promote the absorption of trace elements and increase the serum and bone scab trace elements in animals, improve their bioavailability, increase the levels of various hormones in the body after fracture, and create an internal environment conducive to fracture healing.^28,29 In this experiment, P and ALP in the rats in WA group increased rapidly after 1 week of warm acupuncture treatment, especially ALP, which increased exponentially after 2 weeks of treatment. In contrast, ALP is the most important raw material for initial bone scab formation.

Bone healing is a complex process of histological and biochemical changes involving multiple cells and factors in the formation of new bone instead of injured bone tissue.³⁰ Among them, osteoblasts and osteoclasts play an important role in the bone regeneration process, and the balance between the two receives a large degree of influence from the local blood supply.³¹ It is generally believed that the role of bone marrow mesenchymal stem cells (BMSCs) as seed cells to repair bone tissue damage is mainly by using their ability to differentiate toward osteoblasts in a directed manner, with enhanced osteoblast function or reduced osteoclast function leading to increased bone mass; and vice versa, causing decreased bone mass, but some scholars believe that bone repair is enhanced by targeting crosstalk between BMSCs and the immune system.³² In contrast, BMP-2 is the factor in the BMP family that can independently induce the strongest osteogenic effect, and it plays an important role in the proliferation, differentiation, apoptosis, and internal environment homeostasis of osteoblasts.^32,33 Therefore, the results of the present experiment were consistent with the hypothesis that BMP-2 was significantly elevated after acupuncture treatment, confirming from the protein side that acupuncture can promote the body to re-recruit osteogenesis-related factors and restart fracture healing. However, this experiment did not investigate the mechanism through which warm acupuncture treats bone nonunion at a deeper level, such as molecular genes and signaling pathways, which is the future research direction of this study.

In conclusion, warm acupuncture can alter serum ALP and P ion concentrations, increase mechanical strength and BMP-2 expression in rats, which in turn initiate the conditions for bone scab production and promote the healing of tibial fractures in rats. These findings provide a new potential mechanism and treatment strategy for the treatment of bone nonunion with acupuncture. However, this study is only an animal experiment and clinical practice is needed to further validate its specific efficacy in humans.

ACKNOWLEDGMENTS

This research was supported by Luzhou’s major scientific and technology research and development project (no. 2022-SYF-42), Luzhou Municipal People’s Government - Southwest Medical University Science and Technology Strategic Cooperation Climbing Plan Project (no. 2021LZXNYD-D02), Joint Innovation Special of the Sichuan Provincial Science and Technology Plan (no. 2022YFS0609/2022YFS0609-B3), the Sichuan Science and Technology Department Project Development Project (no. 22ZDYF512), and the Program for Special project of Traditional Chinese Medicine Scientific Research of Sichuan Science and Traditional Chinese Medicine Administration (no. 2023MS019).