# Effects of Early Weight-Bearing Treadmill Training Combined with Pre-Emptive Analgesia on Femoral Fracture Recovery

Authors: Yunqiang Chen; Jiemiao Ouyang; Hong Chen
Journal: Evidence-Based Complementary and Alternative Medicine
 (2023)

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## Abstract

Background. The effect of pre-emptive analgesia plus early weight-bearing treadmill training (EWBTT) on healing and motor function recovery of femoral shaft fracture is not clear. Methods. A total of 60 SD male rats were randomly allocated into 4 groups: group A (pre-emptive analgesia with EWBTT), group B (pre-emptive analgesia with delayed weight-bearing treadmill training, DWBTT), group C (pre-emptive analgesia with no weight-bearing), and group D (EWBTT with no pre-emptive analgesia). All rats were molded by internal fixation with Kirschner wire after right femoral shaft fracture. In groups A, B, and C, tramadol was intramuscularly injected 15 minutes before surgery. EWBTT was performed at day 1 postoperatively in groups A and D, and DWBTT was performed at day 14 postoperatively in group B. Oblique plate test was accomplished to assess hindlimb motor function recovery of rats in each group. Status of fracture healing was assessed through digital radiography (DR). Hematoxylin-eosin (HE) staining and immunohistochemistry of bone morphogenetic protein-2 (MBP-2) and vascular endothelial growth factor (VEGF) in callus were performed to explore fracture healing. The expression of BMP-2 and VEGF protein in quadriceps femoris muscle was detected by Western blot technique and mRNA expression of BMP-2 and VEGF in callus ascertained via reverse transcription-polymerase chain reaction (RT-PCR) technique. Results. For oblique plate test, rats in group A outperformed those in groups B and C at all time points after operation. DR image revealed that large numbers of callus growth, blurred fracture line, and obvious continuous callus passing through the fracture line can be found in group A at day 28 postoperatively, which is the best healing status among all groups. HE staining of callus confirmed the optimal effect of healing for rats in group A. VEGF and BMP-2 expression by immunohistochemistry showed a significantly higher positive score for callus in group A while those in group C being the lowest at all time points postoperatively. Significantly higher expression level of VEGF and BMP-2 protein was detected in quadriceps femoris muscle from group A, which exceeded those in all other groups at all time points. RT-PCR testing proved the highest expression of BMP-2 and VEGF mRNA in callus of rats from group A, significantly higher than those of other groups. Conclusions. Both pre-emptive analgesia and EWBTT can effectively invoke the expression of VEGF and BMP-2 and promote recovery of hindlimb locomotor function in rats with femoral fracture, and the combination of them leads to more superior results.


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## Body

## 1. Introduction

Although it has been proven that we are able to ease intra- and postoperative pain in virtue of opioids, local anesthetics, and cyclo-oxygenase inhibitors, persistent pain following surgical healing remains as a considerable challenge in clinical practice [1]. As early as the beginning of the twentieth century, Crile [2] first proposed the concept of “pre-emptive analgesia (PA),” pointing out that local anesthesia given before general anesthesia may block the generation and delivery of noxious stimulations, thereby significantly reducing intraoperative pain and preventing postoperative pain [3]. Since then, Wall [4] presented the neural regulation mechanism involved in PA and pointed out that whether analgesic therapy can block the transmission of noxious information and inhibit the over-excitement of central neurons before incision may be the key to the effectiveness of PA. In 1993, Woolf and Chong [5] further put forward the “perioperative analgesia” concept; that is, analgesic or sedative agents are given before, during, and after the operation to achieve sufficient and effective prevention of postoperative pain, thus forming a broad sense of PA concept.Tramadol is a synthetic analogue of codeine, which can cause minimal respiratory depression and gastrointestinal disorders, and is less likely to be opioid dependent than morphine [6–8]. Guillen et al. found that a more extended time to rescue medication and lesser analgesics usage postoperatively in total were achieved in patients with pre-emptive analgesia intervention of tramadol, thereby proving the favorable role of it serving as a pre-emptive analgesia [9]. Shah et al. applied tramadol to patients undergoing third molar surgery preoperatively and found that compared with patients in control group, those in tramadol group reported lower postoperative pain scores, lower total amount of anesthetics taken after surgery, and no obvious side effects [10].The famousWolff’s law was brought forward by Julius Wolff in 1892, unveiling that mechanical stress stimulation can be momentous on the morphology and structure of bone tissue. According to Wolf’s law, applied pressure can promote fracture healing, and mechanical stress is capable of changing the electrical and electrochemical environment of bone interstitial cells, activating cellular osteogenic capability, improving cell function, increasing blood supply, and enhancing osteoblastic activity [11, 12]. More bone marrow-derived mesenchymal stem cells proliferate and differentiate into osteoblasts and participate in bone formation under mechanical pressure stimulation [13]. During bone formation, mechanical stress enhances bone strength through affecting the arrangement of collagen, at the same time upregulates the gene and protein expression of osteogenesis factors like osteocalcin, Runx2, osterix, ALP, BMP-2, and type I collagen in osteoblasts, and improves osteoblastic activity, thereby promoting the proliferation and differentiation of osteoblasts [14, 15]. Studies have shown that in the initial stage of fracture healing, capillaries are very sensitive to mechanical stress stimulation [16]. Appropriate pressure stimulation can promote capillary growth and tissue vascularization, transport nutrients, and improve local oxygen concentration to promote fracture repair. Vascular endothelial growth factor (VEGF) matters greatly in bone regeneration. VEGF not only begets neovascularization, but also directly impacts osteoblasts via endothelial cell-mediated BMP yield [17].Lower limb fracture is one of the most familiar circumstances faced by orthopedic surgeons [18]. It matters significantly to specify early weight-bearing treadmill training (EWBTT) protocol after lower extremity fractures. For femoral fractures, EWBTT aids in improving function of the affected limb, accelerating recovery, and enabling patients to return to work as soon as possible, thereby reducing patients’ pain to the greatest extent and minimizing their economic loss [18–21]. EWBTT training may, however, lead to fracture displacement or failure of internal fixation, which affects the prognosis and potentially induces secondary surgery.However, there is a lack of evidence of EWBTT plus pre-emotive analgesia on fracture healing and motor function recovery in vivo. This study aims to explore the impact of postoperative EWBTT combined with the intervention of tramadol intervention pre-emptively on the recovery of femoral shaft fracture with internal fixation, hoping to guide clinical practice.


## 2. Materials and Methods

### 2.1. Animals

60 male SPF Sprague-Dawley rats in total with 8 weeks old and mean body weight 210 g ± 10 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. Raise in plastic cages coated with sawdust in a room with a 12-h light and 12-h dark cycle under temperature of 22 ± 1°C, all animals were adaptively reared before grouping with free moving and access to food and water. Animal experimental protocol was approved by the Committee of Experimental Animal Ethics of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).


### 2.2. Femoral Shaft Fracture Model

The protocol of our study is presented in Figure1(a). The animals were divided into 4 groups randomly with 15 of them in each as follows: animals in group A were dealt with pre-emptive analgesia and EWBTT starting at day 1 postoperatively, animals in group B were treated with pre-emptive analgesia and DWBTT starting at day 7 after operation, those in group C received only pre-emptive analgesia, and those in group D experienced only EWBTT. All surgeries were carried out under pentobarbital anesthesia. Briefly [22, 23], skin and subcutaneous tissue of lateral thigh of the right hindlimb in flexion position were incised and the femoral shaft was exposed by blunt dissection along the vastus lateralis septum. Wire saws were adopted to cut the femur transversely, and 1.0-mm diameter Kirschner wires were inserted into the proximal end of the fracture retrogradely, passing through the greater trochanter of the femur. Then, Kirschner wires were inserted anterogradely into the distal end of the fracture to confirm that the fixation is reliable. 10% penicillin was injected intramuscularly right after operation for three consecutive days. The modeling process is shown in Figure 1(b). Immediately after modeling, the anteroposterior DR film of the femur of the affected limb was taken under anesthesia to confirm the type of fracture and degree of displacement. The standard of the qualified model is that the lateral displacement of the transverse fracture does not exceed 1/3 of the diameter of the femoral shaft.Figure 1
Experimental protocols, modeling process, and treadmill training facility. (a) All rats with internal fixation for femoral shaft fracture were assigned to group A, B, C, and D with 15 animals in each group. Animals in group A received PA and EWBTT, animals in group B experienced PA and DWBTT, those in group C received only PA without any treadmill training, and those in group D experienced only EWBTT without PA. Three rats out of each group were euthanized at day 7, 14, 21, 28, and 35 after operation, respectively, and assessed with oblique plate test, DR image analysis, histological and immunohistochemical analysis, Western blot, and RT-PCR. SD, Sprague-Dawley; FSF, femoral shaft fracture; EWBTT, early weight-bearing treadmill training; DWBTT, delayed weight-bearing treadmill training; PA, pre-emptive analgesia; DR, digital radiography; RT-PCR, reverse transcription-polymerase chain reaction. (b) Modeling process of internal fixation for femoral shaft fracture. (c) Treadmill training facility.
(a)(b)(c)


### 2.3. Pre-Emptive Analgesia Intervention

All rats from groups A, B, and C were infused intramuscularly with 5% tramadol (2.5 mg/kg) 15 minutes before surgery, and rats of group D were given the same amount of normal saline in the same manner.


### 2.4. EWBTT Protocol

Animals in groups A and D were arranged for treadmill training. Training protocols are 30 minutes every day with the speed of 12 meters/minute, which lasts for consecutive 6 days starting at day 1 postoperatively [24]. Animals in group B experienced treadmill training at a speed of 12 meters/minute for 30 minutes per day, which lasts 6 days consecutively starting at day 7 postoperatively. The treadmill training facility is shown in Figure 1(c). Three rats out of each group were euthanized at days 7, 14, 21, 28, and 35 after operation, respectively.


### 2.5. Evaluation of Functional Recovery

Animal’s ability to grip and maintain posture was evaluated by Rivlin’s oblique plate test [25]. For this test, all rats were placed in the middle of a plate with a rubber surface. The maximum degree in which each rat was able to maintain stable for 5 seconds was observed starting from 0° and raised at an angle of 5° each time. Animals were tested at least three times, and take the average value as the score.


### 2.6. DR Image Analysis of Fracture Healing

DR film of the affected femur of rats from the four groups was taken on the 28th day after surgery.


### 2.7. Histological and Immunohistochemical Evaluation

The rats were euthanized with 2% pentobarbital sodium at each given time. Callus tissue segments containing the lesion were embedded with paraffin after decalcification and sliced into 5-μm sections.HE staining was adopted for histopathological observation. Dewaxing of the callus paraffin sections was accomplished by xylene, and graded ethanol was used to dehydrate the slides. Then, sequentially, the slides were immersed in hematoxylin for 15 minutes, 1% aqueous hydrochloric acid solution for 10 seconds, and 1% liquid ammonia for 30 seconds. Thereafter, immerse the slides in eosin for 3 minutes and dehydrate them via graded ethanol [26].Immunohistochemical staining came after deparaffinization. Incubation in PBS solution containing 5% H2O2 for 30 minutes was applied to block the activity of endogenous peroxidase. Slides were then incubated in PBS solution containing 5% BSA for 30 minutes. After that, slides were incubated with anti-mouse primary antibody of VEGF (Abcam, ab1316, 1 : 500) and anti-mouse primary antibody of BMP-2 (R&D, AF355, 1 : 500) at 4°C overnight. Slides were then incubated with conjugated anti-mouse secondary antibody (Cell Signaling Technology, 1 : 100). Secondary antibody incubation lasted for 1 hour at room temperature. Number of positive cells and staining intensity were worked out through randomly choosing three fields containing positive expression for each sample. Immunohistochemical score (IHS) was obtained by multiplying number of positive cells and staining intensity [25].


### 2.8. Western Blot

At each given time point, a set number of rats from each group were euthanized and quadriceps femoris muscle of every affected limb was harvested for protein extraction via lysis buffer and ultrasonication. Samples were then separated and electrically transferred onto polyvinylidene fluoride membrane (Boster). After being blocked by TBST containing 5% skimmed powdered milk for 1 hour, the membranes were incubated in the following primary antibodies diluted in Western blot-specific antibody diluents (Boster, AR1017) at 4°C overnight: anti-rabbit primary antibody against VEGF (Abcam, ab32152, 1 : 1000, 151 kDa) and anti-rabbit primary antibody against BMP-2 (Abcam, ab214821, 1 : 100, 44 kDa). After washing with PBS, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (Cell Signaling Technology) diluted at 1 : 1000 in Western blot-specific antibody diluents (Boster, AR1017) for 1 hour at room temperature and then visualized through a chemiluminescence automatic gel imaging analysis system (Bio-Rad). Image Lab software (Bio-Rad) was utilized to quantify the bands with target protein levels normalized according to the internal reference protein expression.


### 2.9. RT-PCR

At each given time point, a set number of rats from each group were euthanized and bony tissue adjacent to fracture site of every affected femur was harvested for total RNA extraction via TRIzol method after decalcification. Absorbance ratio at 260 and 280 nm (OD260/OD280) and RNA concentration of samples were then detected. cDNA was synthesized from 1μg RNA for each sample. Finally, in line with common directions, step-by-step amplification was accomplished based on the primer sequences illustrated in Table 1 with reaction product undergoing RT-PCR analysis.Table 1
RT-PCR primer sequences of VEGF, BMP-2, andβ-actin mRNA.
Gene namePrimer sequenceVEGFForward: GGCTCACTTCCAGAAACACGReverse: GTGCTCTTGCAGAATCTAGTGGBMP-2Forward: GAGGAGAAGCCAGGTGTCTReverse: GTCCACATACAAAGGGTGCβ-actinForward: GAAGTACCCCATTGAACACGGReverse: TGGGTCATCTTTTCACGGTTG


### 2.10. Statistical Analysis

SPSS 18.0 software was adopted to analyze data, which was stated as mean ± standard error of mean (mean ± SEM). For comparison between two groups,t-test was utilized. For comparison among multiple groups, one-way analysis of variance (ANOVA) was employed. P<0.05 was defined as being statistically significant on the basis of repeating three times for all experiments.


## 2.1. Animals

60 male SPF Sprague-Dawley rats in total with 8 weeks old and mean body weight 210 g ± 10 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. Raise in plastic cages coated with sawdust in a room with a 12-h light and 12-h dark cycle under temperature of 22 ± 1°C, all animals were adaptively reared before grouping with free moving and access to food and water. Animal experimental protocol was approved by the Committee of Experimental Animal Ethics of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).


## 2.2. Femoral Shaft Fracture Model

The protocol of our study is presented in Figure1(a). The animals were divided into 4 groups randomly with 15 of them in each as follows: animals in group A were dealt with pre-emptive analgesia and EWBTT starting at day 1 postoperatively, animals in group B were treated with pre-emptive analgesia and DWBTT starting at day 7 after operation, those in group C received only pre-emptive analgesia, and those in group D experienced only EWBTT. All surgeries were carried out under pentobarbital anesthesia. Briefly [22, 23], skin and subcutaneous tissue of lateral thigh of the right hindlimb in flexion position were incised and the femoral shaft was exposed by blunt dissection along the vastus lateralis septum. Wire saws were adopted to cut the femur transversely, and 1.0-mm diameter Kirschner wires were inserted into the proximal end of the fracture retrogradely, passing through the greater trochanter of the femur. Then, Kirschner wires were inserted anterogradely into the distal end of the fracture to confirm that the fixation is reliable. 10% penicillin was injected intramuscularly right after operation for three consecutive days. The modeling process is shown in Figure 1(b). Immediately after modeling, the anteroposterior DR film of the femur of the affected limb was taken under anesthesia to confirm the type of fracture and degree of displacement. The standard of the qualified model is that the lateral displacement of the transverse fracture does not exceed 1/3 of the diameter of the femoral shaft.Figure 1
Experimental protocols, modeling process, and treadmill training facility. (a) All rats with internal fixation for femoral shaft fracture were assigned to group A, B, C, and D with 15 animals in each group. Animals in group A received PA and EWBTT, animals in group B experienced PA and DWBTT, those in group C received only PA without any treadmill training, and those in group D experienced only EWBTT without PA. Three rats out of each group were euthanized at day 7, 14, 21, 28, and 35 after operation, respectively, and assessed with oblique plate test, DR image analysis, histological and immunohistochemical analysis, Western blot, and RT-PCR. SD, Sprague-Dawley; FSF, femoral shaft fracture; EWBTT, early weight-bearing treadmill training; DWBTT, delayed weight-bearing treadmill training; PA, pre-emptive analgesia; DR, digital radiography; RT-PCR, reverse transcription-polymerase chain reaction. (b) Modeling process of internal fixation for femoral shaft fracture. (c) Treadmill training facility.
(a)(b)(c)


## 2.3. Pre-Emptive Analgesia Intervention

All rats from groups A, B, and C were infused intramuscularly with 5% tramadol (2.5 mg/kg) 15 minutes before surgery, and rats of group D were given the same amount of normal saline in the same manner.


## 2.4. EWBTT Protocol

Animals in groups A and D were arranged for treadmill training. Training protocols are 30 minutes every day with the speed of 12 meters/minute, which lasts for consecutive 6 days starting at day 1 postoperatively [24]. Animals in group B experienced treadmill training at a speed of 12 meters/minute for 30 minutes per day, which lasts 6 days consecutively starting at day 7 postoperatively. The treadmill training facility is shown in Figure 1(c). Three rats out of each group were euthanized at days 7, 14, 21, 28, and 35 after operation, respectively.


## 2.5. Evaluation of Functional Recovery

Animal’s ability to grip and maintain posture was evaluated by Rivlin’s oblique plate test [25]. For this test, all rats were placed in the middle of a plate with a rubber surface. The maximum degree in which each rat was able to maintain stable for 5 seconds was observed starting from 0° and raised at an angle of 5° each time. Animals were tested at least three times, and take the average value as the score.


## 2.6. DR Image Analysis of Fracture Healing

DR film of the affected femur of rats from the four groups was taken on the 28th day after surgery.


## 2.7. Histological and Immunohistochemical Evaluation

The rats were euthanized with 2% pentobarbital sodium at each given time. Callus tissue segments containing the lesion were embedded with paraffin after decalcification and sliced into 5-μm sections.HE staining was adopted for histopathological observation. Dewaxing of the callus paraffin sections was accomplished by xylene, and graded ethanol was used to dehydrate the slides. Then, sequentially, the slides were immersed in hematoxylin for 15 minutes, 1% aqueous hydrochloric acid solution for 10 seconds, and 1% liquid ammonia for 30 seconds. Thereafter, immerse the slides in eosin for 3 minutes and dehydrate them via graded ethanol [26].Immunohistochemical staining came after deparaffinization. Incubation in PBS solution containing 5% H2O2 for 30 minutes was applied to block the activity of endogenous peroxidase. Slides were then incubated in PBS solution containing 5% BSA for 30 minutes. After that, slides were incubated with anti-mouse primary antibody of VEGF (Abcam, ab1316, 1 : 500) and anti-mouse primary antibody of BMP-2 (R&D, AF355, 1 : 500) at 4°C overnight. Slides were then incubated with conjugated anti-mouse secondary antibody (Cell Signaling Technology, 1 : 100). Secondary antibody incubation lasted for 1 hour at room temperature. Number of positive cells and staining intensity were worked out through randomly choosing three fields containing positive expression for each sample. Immunohistochemical score (IHS) was obtained by multiplying number of positive cells and staining intensity [25].


## 2.8. Western Blot

At each given time point, a set number of rats from each group were euthanized and quadriceps femoris muscle of every affected limb was harvested for protein extraction via lysis buffer and ultrasonication. Samples were then separated and electrically transferred onto polyvinylidene fluoride membrane (Boster). After being blocked by TBST containing 5% skimmed powdered milk for 1 hour, the membranes were incubated in the following primary antibodies diluted in Western blot-specific antibody diluents (Boster, AR1017) at 4°C overnight: anti-rabbit primary antibody against VEGF (Abcam, ab32152, 1 : 1000, 151 kDa) and anti-rabbit primary antibody against BMP-2 (Abcam, ab214821, 1 : 100, 44 kDa). After washing with PBS, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (Cell Signaling Technology) diluted at 1 : 1000 in Western blot-specific antibody diluents (Boster, AR1017) for 1 hour at room temperature and then visualized through a chemiluminescence automatic gel imaging analysis system (Bio-Rad). Image Lab software (Bio-Rad) was utilized to quantify the bands with target protein levels normalized according to the internal reference protein expression.


## 2.9. RT-PCR

At each given time point, a set number of rats from each group were euthanized and bony tissue adjacent to fracture site of every affected femur was harvested for total RNA extraction via TRIzol method after decalcification. Absorbance ratio at 260 and 280 nm (OD260/OD280) and RNA concentration of samples were then detected. cDNA was synthesized from 1μg RNA for each sample. Finally, in line with common directions, step-by-step amplification was accomplished based on the primer sequences illustrated in Table 1 with reaction product undergoing RT-PCR analysis.Table 1
RT-PCR primer sequences of VEGF, BMP-2, andβ-actin mRNA.
Gene namePrimer sequenceVEGFForward: GGCTCACTTCCAGAAACACGReverse: GTGCTCTTGCAGAATCTAGTGGBMP-2Forward: GAGGAGAAGCCAGGTGTCTReverse: GTCCACATACAAAGGGTGCβ-actinForward: GAAGTACCCCATTGAACACGGReverse: TGGGTCATCTTTTCACGGTTG


## 2.10. Statistical Analysis

SPSS 18.0 software was adopted to analyze data, which was stated as mean ± standard error of mean (mean ± SEM). For comparison between two groups,t-test was utilized. For comparison among multiple groups, one-way analysis of variance (ANOVA) was employed. P<0.05 was defined as being statistically significant on the basis of repeating three times for all experiments.


## 3. Results

### 3.1. EWBTT and Tramadol PA Improve Functional Recovery

The results of oblique plate test in groups A, B, ,C and D were scored at 1 d before surgery, and 4, 7, 9, 14, 19, 24, 29, and 34 d after surgery, respectively, and results are shown in Table2. One day before surgery, all 60 rats were subjected to oblique plate test and differences between the four groups were not statistically significant (P>0.05). On the 4th day after surgery, angle of incline in group A was higher than those in groups B, C, and D with all the differences being statistically significant (P<0.05). At all subsequent time points, animals in group A scored the highest of the four groups. Besides, from the 19th day after the operation on, the score of group B became significantly higher than that of group C, indicating that compared to sedentary animals, delayed treadmill exercise aids in motor function recovery of femoral fracture to some extent. The variation trend of the incline angles of each group is shown in Figure 2.Table 2
Results of oblique plate test.
Groups1-d Pre4-day post9-day post14-day post19-day post24-day post29-day post34-day postA71.75 ± 1.2464.94 ± 2.39bcd65.83 ± 1.85bc68.21 ± 1.86bc70.10 ± 2.19bc71.30 ± 1.21c73.07 ± 0.31c73.03 ± 0.45cB71.37 ± 1.8357.28 ± 1.7959.28 ± 1.4162.68 ± 1.2367.56 ± 1.23c69.90 ± 1.91c71.93 ± 1.51c72.80 ± 1.08cC71.81 ± 1.2457.55 ± 1.9460.08 ± 1.2162.93 ± 2.5063.99 ± 1.6468.45 ± 0.6769.63 ± 0.9070.60 ± 0.96D71.41 ± 1.0357.55 ± 1.9466.03 ± 1.9267.58 ± 2.1470.53 ± 1.3771.10 ± 0.9573.03 ± 0.8774.20 ± 0.44In comparison with group B,bP<0.05; in comparison with group C, cP<0.05; in comparison with group D, dP<0.05.Figure 2
Functional recovery assessment. Functional recovery assessed by angle of incline analysis. In each group at every time point,n = 3 and each experiment was conducted in triplicate. ∗P<0.05 vs. group B; #P<0.05 vs. group C.


### 3.2. EWBTT and Tramadol PA Boost Fracture Healing by DR Evaluation

In each of the four groups, one rat showed fracture malunion, and the lateral displacement of the fracture exceeded 1/3 of the diameter of the femoral shaft. Except for the above four, the fracture reduction of all other animals was not lost until 35 days after operation. On the 28th day after operation, typical fracture healing of the four groups of rats shown by DR films is shown in Figure3. As can be seen from the figure, signs of good fracture healing like massive callus growth, blurred fracture line, good fracture alignment, and continuous callus passing through the fracture line can be captured in group A, while in group C there is only small amount of callus. In general, at this time point, group A healed the best, followed by group D and group B, respectively, with group C being the worst in healing.Figure 3
DR imaging evaluation result. Fracture healing assessed by DR imaging at day 28 postoperatively. Red arrow indicates the fracture site.
(a)(b)(c)(d)


### 3.3. EWBTT and Tramadol PA Promote Histopathological Features

For histopathological observation, sections of bone tissue segments containing the lesion were submitted to HE staining to evaluate histopathological features of the four groups at the fracture site. HE staining results are illustrated in Figure4.Figure 4
HE staining results. (a) Representative images of bone tissue around the fracture site from four groups at 7 d after surgery by HE staining (scale bars, 50μm). Black arrows indicate inflammatory cell infiltration, fibroplasia, or new capillaries; red arrows indicate osteoclasts; white arrow indicates osteonecrosis; blue arrow indicates abscesses. (b) Representative images of bone tissue around the fracture site from four groups at 14 d after surgery by HE staining (scale bars, 50 μm). Black arrows indicate inflammatory cell infiltration; red arrow indicates osteoclasts; purple arrow indicates fibrous callus. (c) Representative images of bone tissue around the fracture site from four groups at 21 d after surgery by HE staining (scale bars, 50 μm). Black arrow indicates inflammatory cell infiltration; green arrows indicate cartilage callus; yellow arrow indicates trabecular bone; purple arrow indicates fibrous callus. (d) Representative images of bone tissue around the fracture site from four groups at 28 d after surgery by HE staining (scale bars, 50 μm). Yellow arrow indicates trabecular bone; green arrows indicate cartilage callus. (e) Representative images of bone tissue around the fracture site from four groups at 35 d after surgery by HE staining (scale bars, 50 μm). Green arrow indicates cartilage callus; yellow arrows indicate trabecular bone; purple arrow indicates fibrous callus.
(a)(b)(c)(d)(e)What can be found through HE staining on days 7, 14, 21, 28, and 35 postoperatively in each group is elaborated in Tables3, 4, 5, 6 and 7 respectively.Table 3
HE staining results on the 7th day after surgery.
GroupsWhat can be seenAGranulation tissue consisting of a large number of inflammatory cells, proliferating fibers, and new blood vessels can be seen.BSmall abscesses, suppuration, necrotic cells, and sclerotin can be seen.CSimilar to that observed in group B.DMore necrotic bone and cells can be seen, and the inner core of the bone lacuna is lightly stained or dissolved and disappeared.Table 4
HE staining results on the 14th day after surgery.
GroupsWhat can be seenAThe fibers in the affected area proliferated obviously, forming a large range of fibrous callus and a small amount of cartilage callus.BA large number of inflammatory cell infiltration and fibrous hyperplasia can be seen.CSimilar to that observed in group B.DOsteoclasts can be seen in the affected area, and there is still a small amount of necrotic bone and fibrous callus.Table 5
HE staining results on the 21st day after surgery.
GroupsWhat can be seenAA large area of cartilage callus can be found in the affected area, some cartilage callus began to ossify, bone trabecula was formed locally, and a small area of fibrous callus was still visible.BCartilage callus, fibrous callus, and only a small amount of granulation tissue can be seen.CGranulation tissue can be seen in the affected area, and cartilage callus and fibrous callus can be seen locally.DMany fibrous callus and cartilage callus can be seen in the affected area.Table 6
HE staining results on the 28th day after surgery.
GroupsWhat can be seenAFibrous callus disappeared in the affected area, cartilage callus gradually decreased, a large number of cartilage callus began to ossify, and bone trabeculae and bone remodeling were visible.BA large range of fibrous callus and cartilage callus can be seen in the affected area, and a small part of cartilage callus begins to ossify.CMany fibrous callus and cartilage callus can be found in the affected area.DA large range of fibrous callus and cartilage callus can be seen in the affected area, and many cartilage calluses begin to ossify.Generally speaking, at every timing, optimum healing goes to group A, followed by group D which is closely behind, and group B lags slightly behind, while group C behaves the worst in healing all through. The fact that the healing progress of group A is always superior to that of group B reveals that EWBTT promotes fracture healing better than DWBTT does. The difference of healing process between group A and group D manifests that PA does have a certain role in promoting postoperative healing of fractures. Besides, the difference of healing process between group B and group C indicates that postoperative pedal walking is indeed necessary and beneficial for fracture recovery, even at a late stage.


### 3.4. EWBTT and Tramadol PA Accelerate Immunohistochemical Outcomes

To evaluate the effect of EWBTT plus tramadol PA on fracture site healing, sections of bone tissue segments containing the lesion were subjected to immunohistochemical (IHC) staining of VEGF and BMP-2 to observe expression of the two factors important for fracture healing. VEGF is the most powerful angiogenesis regulator, which accelerates proliferation of vascular endothelial cell specifically and angiogenesis by binding to receptors on cell membrane [27]. Angiogenesis serves an indispensable role in supplying oxygen to fracture segments, providing nutrients, and transporting metabolic waste, providing a favorable microenvironment for local bone regeneration and metabolism [28]. Results of IHC staining of VEGF are illustrated in Figure 5. As shown in Figure 5(a), at day 7, the double intervention group exhibited significantly higher expression level of VEGF than other three groups, indicating the best healing status with this group. Group A’s lead in IHS continued until day 35, which denotes EWBTT plus tramadol PA significantly boosted VEGF expression in bone tissue around the fracture site, thus promoting angiogenesis. Similar to what has been found and concluded in histological evaluation, EWBTT defeats DWBTT in motivating VEGF expression in bone tissue and tramadol PA, as a single factor, has been proved to facilitate angiogenesis.Figure 5
IHC staining results of VEGF. (a) Representative images of bone tissue around the fracture site from four groups at 7 d after surgery by IHC staining of VEGF (scale bars, 50μm). White arrows indicate positive cells with brown stain. (b) Representative images of bone tissue around the fracture site from four groups at 14 d after surgery by IHC staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (c) Representative images of bone tissue around the fracture site from four groups at 21 d after surgery by HE staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (d) Representative images of bone tissue around the fracture site from four groups at 28 d after surgery by HE staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (e) Representative images of bone tissue around the fracture site from four groups at 35 d after surgery by HE staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (f) Immunohistochemical score of VEGF for each group at every time points (n = 3). For quantification of each sample, 3 optic fields were chosen randomly. ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)(c)(d)(e)(f)Bone morphogenetic proteins (BMPs) are a group of multifunctional cytokines in the transforming growth factor-β (TGF-β) superfamily, which can lead to the formation of cartilage and bone. The osteogenic effect of BMP-2 has been reported the most, and studies have proved that BMP-2 has a strong ability to promote osteoblast differentiation and induce osteogenesis in vitro [29, 30]. Results of IHC staining of BMP-2 are exemplified in Figure 6. As demonstrated in Figure 6(a), at day 7, group A scored the most in BMP-2 IHS, significantly higher than all other groups, denoting the outstanding osteogenic impact of dual intervention. Over the entire time frame, BMP-2 expression of group A is the highest among all, peaking at day 28. The phenomenon that the scores of group B are all the way lower than those of group A indicates the superiority of EWBTT in inducing osteogenesis to DWBTT. Apart from that, from day 21, group B started to outscore group C, continued to lead, and maintained the significant difference until the end, which confirmed better late than never as far as weight-bearing treadmill training after fractures is concerned.Figure 6
IHC staining results of BMP-2. (a) Representative images of bone tissue around the fracture site from four groups at 7 d after surgery by IHC staining of BMP-2 (scale bars, 50μm). White arrows indicate positive cells with brown stain. (b) Representative images of bone tissue around the fracture site from four groups at 14 d after surgery by IHC staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (c) Representative images of bone tissue around the fracture site from four groups at 21 d after surgery by HE staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (d) Representative images of bone tissue around the fracture site from four groups at 28 d after surgery by HE staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (e) Representative images of bone tissue around the fracture site from four groups at 35 d after surgery by HE staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (f) Immunohistochemical score of BMP-2 for each group at every time points (n = 3). For quantification of each sample, 3 optic fields were chosen randomly. ∗P<0.05 vs. group B; #P<0.05 vs. group C.
(a)(b)(c)(d)(e)(f)Table 7
HE staining results on the 35th day after surgery.
GroupsWhat can be seenACartilage callus in the affected area is obviously reduced, and a large number of bone trabeculae and bone remodeling can be seen.BFibrous callus in the affected area is obviously reduced, ossification of cartilage callus can be found, and bone trabeculae can be seen locally.CA large range of cartilage callus can be seen in the affected area, and some cartilage callus began to ossify, but there were still many fibrous calluses.DA small amount of fibrous callus can still be seen in the affected area, and cartilage ossification and bone trabecula can be seen locally.


### 3.5. Western Blot Result Confirms the Osteogenic Effect of EWBTT and Tramadol PA

To evaluate the effect of EWBTT plus PA on VEGF and BMP-2 protein expression of skeletal muscle adjacent to the fracture site, we performed Western blot test on quadriceps femoris of the affected limb.Western blot results of VEGF expression are shown in Figure7. Our findings revealed that EWBTT and tramadol PA can both effectively induce VEGF expression in skeletal muscles supporting femur and the combined application of the two interventions acquired even better outcomes.Figure 7
Western blot results of VEGF. (a) Representative images of VEGF expression in quadriceps femoris of rats in each group at all time point by WB. (b) Quantification of VEGF protein expression level in quadriceps femoris (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)As for the expression of BMP-2, the trend of expression changes is consistent with that of VEGF, which again confirmed positive impact on fracture healing of EWBTT and tramadol PA when applied alone and jointly (see Figure8(a) and 8(b)).Figure 8
Western blot results of BMP-2. (a) Representative images of BMP-2 expression in quadriceps femoris of rats in each group at all time point by WB. (b) Quantification of BMP-2 protein expression level in quadriceps femoris (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)


### 3.6. Fracture Healing-Promoting Effect of EWBTT and Tramadol PA Is Verified by RT-PCR Consequences

To verify the effects of EWBTT and tramadol PA on VEGF and BMP-2 expression by IHC, RT-PCR was employed to see how EWBTT and tramadol PA influence mRNA expression of VEGF and BMP-2 in bony tissues.As shown in Figure9(a), at all time points, VEGF mRNA expression levels of group A were significantly higher than those of groups B and C. From the 21st day after operation on, VEGF expression levels of group A were significantly higher than those of group D indicating the superiority of EWBTT. Besides, the expression level of VEGF mMRA in group B became higher than that in group C from the 21st day after operation, and the difference between the two groups was significant on the 28th and 35th day. As shown in Figure 9(b), the trend of BMP-2 mRNA expression is highly consistent with that of VEGF. What can be concluded in RT-PCR results is consistent with the outcomes drawn by IHC, which states that EWBTT defeats DWBTT in motivating VEGF and BMP-2 expression in bone tissue and tramadol PA, as a single factor, has been proved to facilitate angiogenesis. All in all, osteogenic effect comes the best when DWBTT and tramadol PA are combined.Figure 9
RT-PCR results of VEF and BMP-2 mRNA. (a) Quantification of VEGF mRNA expression level in bone tissue around the fracture site of rats in each group at all time point by RT-PCR (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C. (b) Quantification of BMP-2 mRNA expression level in bone tissue around the fracture site of rats in each group at all time point by RT-PCR (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)


## 3.1. EWBTT and Tramadol PA Improve Functional Recovery

The results of oblique plate test in groups A, B, ,C and D were scored at 1 d before surgery, and 4, 7, 9, 14, 19, 24, 29, and 34 d after surgery, respectively, and results are shown in Table2. One day before surgery, all 60 rats were subjected to oblique plate test and differences between the four groups were not statistically significant (P>0.05). On the 4th day after surgery, angle of incline in group A was higher than those in groups B, C, and D with all the differences being statistically significant (P<0.05). At all subsequent time points, animals in group A scored the highest of the four groups. Besides, from the 19th day after the operation on, the score of group B became significantly higher than that of group C, indicating that compared to sedentary animals, delayed treadmill exercise aids in motor function recovery of femoral fracture to some extent. The variation trend of the incline angles of each group is shown in Figure 2.Table 2
Results of oblique plate test.
Groups1-d Pre4-day post9-day post14-day post19-day post24-day post29-day post34-day postA71.75 ± 1.2464.94 ± 2.39bcd65.83 ± 1.85bc68.21 ± 1.86bc70.10 ± 2.19bc71.30 ± 1.21c73.07 ± 0.31c73.03 ± 0.45cB71.37 ± 1.8357.28 ± 1.7959.28 ± 1.4162.68 ± 1.2367.56 ± 1.23c69.90 ± 1.91c71.93 ± 1.51c72.80 ± 1.08cC71.81 ± 1.2457.55 ± 1.9460.08 ± 1.2162.93 ± 2.5063.99 ± 1.6468.45 ± 0.6769.63 ± 0.9070.60 ± 0.96D71.41 ± 1.0357.55 ± 1.9466.03 ± 1.9267.58 ± 2.1470.53 ± 1.3771.10 ± 0.9573.03 ± 0.8774.20 ± 0.44In comparison with group B,bP<0.05; in comparison with group C, cP<0.05; in comparison with group D, dP<0.05.Figure 2
Functional recovery assessment. Functional recovery assessed by angle of incline analysis. In each group at every time point,n = 3 and each experiment was conducted in triplicate. ∗P<0.05 vs. group B; #P<0.05 vs. group C.


## 3.2. EWBTT and Tramadol PA Boost Fracture Healing by DR Evaluation

In each of the four groups, one rat showed fracture malunion, and the lateral displacement of the fracture exceeded 1/3 of the diameter of the femoral shaft. Except for the above four, the fracture reduction of all other animals was not lost until 35 days after operation. On the 28th day after operation, typical fracture healing of the four groups of rats shown by DR films is shown in Figure3. As can be seen from the figure, signs of good fracture healing like massive callus growth, blurred fracture line, good fracture alignment, and continuous callus passing through the fracture line can be captured in group A, while in group C there is only small amount of callus. In general, at this time point, group A healed the best, followed by group D and group B, respectively, with group C being the worst in healing.Figure 3
DR imaging evaluation result. Fracture healing assessed by DR imaging at day 28 postoperatively. Red arrow indicates the fracture site.
(a)(b)(c)(d)


## 3.3. EWBTT and Tramadol PA Promote Histopathological Features

For histopathological observation, sections of bone tissue segments containing the lesion were submitted to HE staining to evaluate histopathological features of the four groups at the fracture site. HE staining results are illustrated in Figure4.Figure 4
HE staining results. (a) Representative images of bone tissue around the fracture site from four groups at 7 d after surgery by HE staining (scale bars, 50μm). Black arrows indicate inflammatory cell infiltration, fibroplasia, or new capillaries; red arrows indicate osteoclasts; white arrow indicates osteonecrosis; blue arrow indicates abscesses. (b) Representative images of bone tissue around the fracture site from four groups at 14 d after surgery by HE staining (scale bars, 50 μm). Black arrows indicate inflammatory cell infiltration; red arrow indicates osteoclasts; purple arrow indicates fibrous callus. (c) Representative images of bone tissue around the fracture site from four groups at 21 d after surgery by HE staining (scale bars, 50 μm). Black arrow indicates inflammatory cell infiltration; green arrows indicate cartilage callus; yellow arrow indicates trabecular bone; purple arrow indicates fibrous callus. (d) Representative images of bone tissue around the fracture site from four groups at 28 d after surgery by HE staining (scale bars, 50 μm). Yellow arrow indicates trabecular bone; green arrows indicate cartilage callus. (e) Representative images of bone tissue around the fracture site from four groups at 35 d after surgery by HE staining (scale bars, 50 μm). Green arrow indicates cartilage callus; yellow arrows indicate trabecular bone; purple arrow indicates fibrous callus.
(a)(b)(c)(d)(e)What can be found through HE staining on days 7, 14, 21, 28, and 35 postoperatively in each group is elaborated in Tables3, 4, 5, 6 and 7 respectively.Table 3
HE staining results on the 7th day after surgery.
GroupsWhat can be seenAGranulation tissue consisting of a large number of inflammatory cells, proliferating fibers, and new blood vessels can be seen.BSmall abscesses, suppuration, necrotic cells, and sclerotin can be seen.CSimilar to that observed in group B.DMore necrotic bone and cells can be seen, and the inner core of the bone lacuna is lightly stained or dissolved and disappeared.Table 4
HE staining results on the 14th day after surgery.
GroupsWhat can be seenAThe fibers in the affected area proliferated obviously, forming a large range of fibrous callus and a small amount of cartilage callus.BA large number of inflammatory cell infiltration and fibrous hyperplasia can be seen.CSimilar to that observed in group B.DOsteoclasts can be seen in the affected area, and there is still a small amount of necrotic bone and fibrous callus.Table 5
HE staining results on the 21st day after surgery.
GroupsWhat can be seenAA large area of cartilage callus can be found in the affected area, some cartilage callus began to ossify, bone trabecula was formed locally, and a small area of fibrous callus was still visible.BCartilage callus, fibrous callus, and only a small amount of granulation tissue can be seen.CGranulation tissue can be seen in the affected area, and cartilage callus and fibrous callus can be seen locally.DMany fibrous callus and cartilage callus can be seen in the affected area.Table 6
HE staining results on the 28th day after surgery.
GroupsWhat can be seenAFibrous callus disappeared in the affected area, cartilage callus gradually decreased, a large number of cartilage callus began to ossify, and bone trabeculae and bone remodeling were visible.BA large range of fibrous callus and cartilage callus can be seen in the affected area, and a small part of cartilage callus begins to ossify.CMany fibrous callus and cartilage callus can be found in the affected area.DA large range of fibrous callus and cartilage callus can be seen in the affected area, and many cartilage calluses begin to ossify.Generally speaking, at every timing, optimum healing goes to group A, followed by group D which is closely behind, and group B lags slightly behind, while group C behaves the worst in healing all through. The fact that the healing progress of group A is always superior to that of group B reveals that EWBTT promotes fracture healing better than DWBTT does. The difference of healing process between group A and group D manifests that PA does have a certain role in promoting postoperative healing of fractures. Besides, the difference of healing process between group B and group C indicates that postoperative pedal walking is indeed necessary and beneficial for fracture recovery, even at a late stage.


## 3.4. EWBTT and Tramadol PA Accelerate Immunohistochemical Outcomes

To evaluate the effect of EWBTT plus tramadol PA on fracture site healing, sections of bone tissue segments containing the lesion were subjected to immunohistochemical (IHC) staining of VEGF and BMP-2 to observe expression of the two factors important for fracture healing. VEGF is the most powerful angiogenesis regulator, which accelerates proliferation of vascular endothelial cell specifically and angiogenesis by binding to receptors on cell membrane [27]. Angiogenesis serves an indispensable role in supplying oxygen to fracture segments, providing nutrients, and transporting metabolic waste, providing a favorable microenvironment for local bone regeneration and metabolism [28]. Results of IHC staining of VEGF are illustrated in Figure 5. As shown in Figure 5(a), at day 7, the double intervention group exhibited significantly higher expression level of VEGF than other three groups, indicating the best healing status with this group. Group A’s lead in IHS continued until day 35, which denotes EWBTT plus tramadol PA significantly boosted VEGF expression in bone tissue around the fracture site, thus promoting angiogenesis. Similar to what has been found and concluded in histological evaluation, EWBTT defeats DWBTT in motivating VEGF expression in bone tissue and tramadol PA, as a single factor, has been proved to facilitate angiogenesis.Figure 5
IHC staining results of VEGF. (a) Representative images of bone tissue around the fracture site from four groups at 7 d after surgery by IHC staining of VEGF (scale bars, 50μm). White arrows indicate positive cells with brown stain. (b) Representative images of bone tissue around the fracture site from four groups at 14 d after surgery by IHC staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (c) Representative images of bone tissue around the fracture site from four groups at 21 d after surgery by HE staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (d) Representative images of bone tissue around the fracture site from four groups at 28 d after surgery by HE staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (e) Representative images of bone tissue around the fracture site from four groups at 35 d after surgery by HE staining of VEGF (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (f) Immunohistochemical score of VEGF for each group at every time points (n = 3). For quantification of each sample, 3 optic fields were chosen randomly. ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)(c)(d)(e)(f)Bone morphogenetic proteins (BMPs) are a group of multifunctional cytokines in the transforming growth factor-β (TGF-β) superfamily, which can lead to the formation of cartilage and bone. The osteogenic effect of BMP-2 has been reported the most, and studies have proved that BMP-2 has a strong ability to promote osteoblast differentiation and induce osteogenesis in vitro [29, 30]. Results of IHC staining of BMP-2 are exemplified in Figure 6. As demonstrated in Figure 6(a), at day 7, group A scored the most in BMP-2 IHS, significantly higher than all other groups, denoting the outstanding osteogenic impact of dual intervention. Over the entire time frame, BMP-2 expression of group A is the highest among all, peaking at day 28. The phenomenon that the scores of group B are all the way lower than those of group A indicates the superiority of EWBTT in inducing osteogenesis to DWBTT. Apart from that, from day 21, group B started to outscore group C, continued to lead, and maintained the significant difference until the end, which confirmed better late than never as far as weight-bearing treadmill training after fractures is concerned.Figure 6
IHC staining results of BMP-2. (a) Representative images of bone tissue around the fracture site from four groups at 7 d after surgery by IHC staining of BMP-2 (scale bars, 50μm). White arrows indicate positive cells with brown stain. (b) Representative images of bone tissue around the fracture site from four groups at 14 d after surgery by IHC staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (c) Representative images of bone tissue around the fracture site from four groups at 21 d after surgery by HE staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (d) Representative images of bone tissue around the fracture site from four groups at 28 d after surgery by HE staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (e) Representative images of bone tissue around the fracture site from four groups at 35 d after surgery by HE staining of BMP-2 (scale bars, 50 μm). White arrows indicate positive cells with brown stain. (f) Immunohistochemical score of BMP-2 for each group at every time points (n = 3). For quantification of each sample, 3 optic fields were chosen randomly. ∗P<0.05 vs. group B; #P<0.05 vs. group C.
(a)(b)(c)(d)(e)(f)Table 7
HE staining results on the 35th day after surgery.
GroupsWhat can be seenACartilage callus in the affected area is obviously reduced, and a large number of bone trabeculae and bone remodeling can be seen.BFibrous callus in the affected area is obviously reduced, ossification of cartilage callus can be found, and bone trabeculae can be seen locally.CA large range of cartilage callus can be seen in the affected area, and some cartilage callus began to ossify, but there were still many fibrous calluses.DA small amount of fibrous callus can still be seen in the affected area, and cartilage ossification and bone trabecula can be seen locally.


## 3.5. Western Blot Result Confirms the Osteogenic Effect of EWBTT and Tramadol PA

To evaluate the effect of EWBTT plus PA on VEGF and BMP-2 protein expression of skeletal muscle adjacent to the fracture site, we performed Western blot test on quadriceps femoris of the affected limb.Western blot results of VEGF expression are shown in Figure7. Our findings revealed that EWBTT and tramadol PA can both effectively induce VEGF expression in skeletal muscles supporting femur and the combined application of the two interventions acquired even better outcomes.Figure 7
Western blot results of VEGF. (a) Representative images of VEGF expression in quadriceps femoris of rats in each group at all time point by WB. (b) Quantification of VEGF protein expression level in quadriceps femoris (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)As for the expression of BMP-2, the trend of expression changes is consistent with that of VEGF, which again confirmed positive impact on fracture healing of EWBTT and tramadol PA when applied alone and jointly (see Figure8(a) and 8(b)).Figure 8
Western blot results of BMP-2. (a) Representative images of BMP-2 expression in quadriceps femoris of rats in each group at all time point by WB. (b) Quantification of BMP-2 protein expression level in quadriceps femoris (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)


## 3.6. Fracture Healing-Promoting Effect of EWBTT and Tramadol PA Is Verified by RT-PCR Consequences

To verify the effects of EWBTT and tramadol PA on VEGF and BMP-2 expression by IHC, RT-PCR was employed to see how EWBTT and tramadol PA influence mRNA expression of VEGF and BMP-2 in bony tissues.As shown in Figure9(a), at all time points, VEGF mRNA expression levels of group A were significantly higher than those of groups B and C. From the 21st day after operation on, VEGF expression levels of group A were significantly higher than those of group D indicating the superiority of EWBTT. Besides, the expression level of VEGF mMRA in group B became higher than that in group C from the 21st day after operation, and the difference between the two groups was significant on the 28th and 35th day. As shown in Figure 9(b), the trend of BMP-2 mRNA expression is highly consistent with that of VEGF. What can be concluded in RT-PCR results is consistent with the outcomes drawn by IHC, which states that EWBTT defeats DWBTT in motivating VEGF and BMP-2 expression in bone tissue and tramadol PA, as a single factor, has been proved to facilitate angiogenesis. All in all, osteogenic effect comes the best when DWBTT and tramadol PA are combined.Figure 9
RT-PCR results of VEF and BMP-2 mRNA. (a) Quantification of VEGF mRNA expression level in bone tissue around the fracture site of rats in each group at all time point by RT-PCR (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C. (b) Quantification of BMP-2 mRNA expression level in bone tissue around the fracture site of rats in each group at all time point by RT-PCR (n = 3). ∗P<0.05 vs. group B; ∗∗P<0.01 vs. group B; #P<0.05 vs. group C; ##P<0.01 vs. group C.
(a)(b)


## 4. Discussion

Lower extremity fractures are the most ordinary type of trauma fractures, and femoral shaft fractures are familiar in clinical practice, with an incidence of 13/105 to 19/105 [31]. Femoral shaft fractures have the highest incidence in young and middle-aged people, and are mainly seen in high-energy injuries like traffic accidents or fall [32]. Defining the weight-bearing exercise strategy after lower extremity fracture is very important for the recovery.Clinical studies have shown that for certain types of fractures, patients who feel good in their limbs can begin weight-bearing training at an earlier postoperative time point than is advocated by routine guidelines. Early weight-bearing exercise can effectively boost the function of the affected limb, accelerate the recovery, and enable the patient to return to work as soon as possible, thereby reducing the impact of the injury. Back in 1979, it has been found that early weight-bearing training group after closed reduction and cast immobilization of tibial shaft fractures healed faster than the delayed weight-bearing training group without increased rate of complications [33]. Brumback [34] et al. reported the results of early weight-bearing training in 28 cases of comminuted femoral shaft fractures dealt with reamed intramedullary nailing showing that 26 patients (93%) returned to full weight-bearing 6 weeks after surgery with all fractures healed postoperatively. In another study, 30 patients with comminuted femoral shaft fractures were subjected to tolerable weight-bearing training immediately after intramedullary nailing, which proved that 96% of patients were able to walk unassisted with full weight-bearing at 2 months postoperatively, with complete fracture healing and no complications [19]. Adam et al. reported 25 cases of tibial shaft fractures treated with minimally invasive locking bridging plate who underwent tolerable weight-bearing training immediately after surgery, in which no loss of fracture reduction was found during long-term follow-up [35]. Cao Keyong and Ye conducted a study concerning early weight-bearing training by an upright electric bed on patients with femoral shaft fractures and found that early weight-bearing with moderate intensity (288°N) can significantly promote callus growth and improve the healing [36]. Researches about early weight-bearing after lower extremity long bone fractures mainly focus on tibia and fibula, and the existing limited results are all clinical studies, which provides our research the opportunity to fill the gap to a certain extent. In our research, it was found that EWBTT starting at day 1 after surgery significantly improved motor function recovery, boosted fracture healing, and promoted VEGF and BMP-2 expression in skeletal muscle and bony tissue surrounding the fracture site. Dongquan found that early weight-bearing and walking are beneficial to fracture patients through a study of patients with femoral neck fractures who began to perform weight-bearing and walking training on the first day after surgery, which is consistent with the results of our experiment [37].Respiration, body temperature, pulse, blood pressure, and postoperative pain, which has attracted widespread attention in recent years, have been defined by the medical community as the five vital signs that maintain the normal activities of the body [38]. Pain after fracture surgery is a problem faced by every fracture patient. At present, the routine analgesia treatment after fracture surgery is to give analgesics only when the patient complains of pain. There are two disadvantages in it: first, the onset of action requires a certain interval, and the patient needs to endure a period of pain; second, painful stimulation can cause pathological remodeling of the central nervous system (CNS), leading to hyperalgesia and ultimately reducing the analgesic effect [39, 40]. More importantly, the biggest difficulty in early postoperative rehabilitation training for fracture patients is pain intolerance, and their subjective participation and enthusiasm are not high [41]. Therefore, effective analgesia is a fundamental condition for achieving early weight-bearing training. Pre-emptive analgesia (PA) is to take measures before noxious stimuli act on the body to prevent the sensitization of peripheral and central nerves, so as to reduce or eliminate pain [42, 43]. At present, the effect of nonsteroidal analgesics in delaying fracture healing has been verified [44]. However, tramadol did not negatively affect the healing process of fractures [45]. A study comparing postoperative patient-controlled analgesia (PCA) with tramadol pre-emptive analgesia (PA) in patients undergoing laparotomy under general anesthesia found that compared with postoperative PCA, tramadol pre-emptive analgesia was effective in effectively maintaining the effect of anesthesia and analgesia, better promoting the stability of hemodynamics and reducing the occurrence of nausea and vomiting [46]. In our study, intramuscular injection of tramadol 15 minutes before the operation may reduce the afferent of nociceptive stimulation signals during the operation and the physiological and psychological effects of postoperative pain, realizing animals’ active participation in postoperative exercise training at an early time. In accordance with previous study, we proved that tramadol pre-emptive analgesia was capable of boosting motor function recovery and promoting fracture healing.As of now, it is the first time that EWBTT and tramadol PA are jointly used to exert influence on fracture healing in which it has been soundly proved that combination of the two interventions leads to much better results.There are certain limitations in this study. Firstly, there are no objective evaluation methods for analgesic effect, and thereby the specific effect of tramadol PA cannot be clearly defined, which need to be improved in future studies. Secondly, for the PA group, other drugs apart from tramadol as control were not set, as well as the dosage, mode of drug administration, and administration timing, making it unable to compare the effects of different drugs for PA. Lastly, there lack long-term observations and behavior assessment for fracture healing in the present study, which deserves to be complemented afterward to make the results more convincing.In conclusion, this study initially explored the effects of EWBTT and tramadol PA on fracture healing and motor function recovery in rats with femoral shaft fractures. It has been found that both tramadol PA and EWBTT can effectively induce the expression of BMP-2 and VEGF in bony tissues and skeletal muscle around fracture site, prolong their peak expression, and promote the fracture healing and recovery of motor function. The combined use of PA and EWBTT is even better. Besides, EWBTT did not result in loss of fracture reduction, delayed union, or nonunion. It has also been proved that delayed weight-bearing promotes fracture healing better than no weight-bearing. Our results reveal the effect of EWBTT plus tramadol PA on fracture healing and provide more inspiration for surgeons and physicians seeking enhanced recovery after surgery.



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*Source: 8498062-2023-01-31.xml*