阅读说明：本技术 普萘洛尔在促进成骨分化和早期种植体骨整合中的应用 (Application of propranolol in promoting osteogenic differentiation and early implant osseointegration ) 是由 王晓静 王国伟 武玉鹏 赵保东 王文雪 于 2021-01-13 设计创作，主要内容包括：本发明属生物医药技术领域,为解决目前普萘洛尔在骨代谢中的影响以及其调控机制没有研究,普萘洛尔在骨代谢中无法得到很好的应用,提供一种普萘洛尔在促进成骨分化和早期种植体骨整合中的应用。普萘洛尔在促进成骨细胞OBs增殖,抑制骨髓间充质干细胞MSCs的增殖,增强OBs和MSCs的成骨分化,增强种植体的骨整合中的应用。普萘洛尔有效促进种植体在体内的骨整合,促进OBs增殖,抑制MSCs增殖,增强OBs和MSCs的成骨分化。普萘洛尔提高BMP2、RunX2、COL-1和OCN在组织和细胞中的mRNA和蛋白表达,降低β2-AR的表达。为普萘洛尔的应用和调节机制提供了新的见解。(The invention belongs to the technical field of biological medicines, and provides application of propranolol in promoting osteogenic differentiation and early implant osseointegration, aiming at solving the problems that the influence of propranolol in bone metabolism and the regulation mechanism thereof are not researched at present and propranolol cannot be well applied in bone metabolism. The propranolol is applied to promoting the proliferation of Osteoblasts (OBs), inhibiting the proliferation of Mesenchymal Stem Cells (MSCs), enhancing the osteogenic differentiation of the OBs and the MSCs and enhancing the osseointegration of the implant. The propranolol effectively promotes the osseointegration of the implant in vivo, promotes the proliferation of OBs, inhibits the proliferation of MSCs and enhances the osteogenic differentiation of the OBs and the MSCs. Propranolol increases mRNA and protein expression of BMP2, RunX2, COL-1 and OCN in tissues and cells, and reduces expression of beta 2-AR. Provides new insight for the application and the regulation mechanism of propranolol.)

1. The application of propranolol in promoting osteogenic differentiation and early implant osseointegration is characterized in that: the propranolol is applied to promoting the proliferation of Osteoblasts (OBs), inhibiting the proliferation of Mesenchymal Stem Cells (MSCs), enhancing the osteogenic differentiation of the OBs and the MSCs and enhancing the osseointegration of the implant.

2. The use of propranolol according to claim 1 for promoting osteogenic differentiation and early implant osteointegration, wherein: the propranolol is applied to improving the mRNA and protein expression of BMP2, RunX2, COL-1 and OCN in tissues and cells and reducing the expression of beta 2-AR.

3. Use of propranolol according to claim 1 or 2 for promoting osteogenic differentiation and early implant osteointegration, wherein: the concentration of the propranolol is 1mg/kg or 10 mg/kg.

4. The use of propranolol according to claim 3 for promoting osteogenic differentiation and early implant osteointegration, wherein: the concentration of the propranolol is 1 mg/kg.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of propranolol in promoting osteogenic differentiation and early implant osseointegration.

Background

Defects and deformities in bone tissue affect the patient's daily communication and even normal function. The consequences of some severe cases can lead to mental disability in the patient. Previous treatment options include bone grafting (autologous or allogeneic bone grafting), and the use of bone substitute materials. Unfortunately, these methods also create additional problems of rejection, high surgical costs, limited bone resources, severe trauma and complicated surgery, which are difficult to popularize in clinical applications. Bone metabolic formation consists of two basic processes, bone formation and bone resorption, leading to reduced bone formation and mass, while the use of the beta blocker propranolol produces the opposite effect.

Since the sympathetic nerve's effects on bone remodeling and bone healing are complex and extensive, the mechanism is not clear. However, the effect of the sympatholytic agent propranolol on bone metabolism is mainly in the experimental phase of the animals. There are few reports indicating the effect of propranolol on Osteoblast (OB) proliferation and Osteoclast (OC) bone resorption and its mechanism.

OB from bone marrow mesenchymal stem cells (BMSCs) is essential in the bone formation process, where it generates bone matrix and releases various bioactive substances to regulate and control functions of itself and OC. Sympathetic nerve stimulation increases bone resorption and inhibits bone formation, the mechanism of which is closely related to the activity of β 2-adrenergic receptors on osteoblasts.

Documents Graewin s.j., Kiely J.M. & Lu d.et al, "Leptin modulators gapledblade genes related to gapless stone pathologenesis in Leptin-specific mice," J Am col Surg, vol.206, No.3(2008), pp.503-510 ", disclose: the presence of β 2 receptors on osteoblasts was confirmed by RT-PCR and Northern blot hybridization techniques, and no other subtypes of adrenoceptors were detected in OB.

Beta 2 blockers are common drugs for the treatment of hypertension and cardiovascular diseases. However, recent studies have shown the effect of β 2 blockers in bone tissue. In animal experiments, adrenergic receptor agonists can promote bone resorption in mice. Systemic application of beta receptor agonists reduces bone formation and mass, while propranolol, as a beta receptor blocker, produces the opposite effect. Nevertheless, the effects and regulatory mechanisms of propranolol on OB and MSC have not been fully explored.

Disclosure of Invention

The invention provides an application of propranolol in promoting osteogenic differentiation and early implant osseointegration, aiming at solving the problems that the influence of propranolol in bone metabolism and the regulation mechanism thereof are not researched at present and propranolol cannot be well applied in bone metabolism.

The invention is realized by the following technical scheme: the propranolol is applied to promoting Osteoblast (OBs) proliferation, inhibiting proliferation of Mesenchymal Stem Cells (MSCs), enhancing osteogenic differentiation of the OBs and the MSCs and enhancing osseointegration of an implant.

Further, the propranolol is applied to improving the mRNA and protein expression of BMP2, RunX2, COL-1 and OCN in tissues and cells and reducing the expression of beta 2-AR.

Further, the concentration of the propranolol is 1mg/kg, or 10 mg/kg.

Preferably, the concentration of the propranolol is 1 mg/kg.

The invention discusses the influence of the beta-AR inhibitor propranolol on the osteogenesis of a cell level animal model. The results show that propranolol can promote the osseointegration of the implant and enhance the osteogenic differentiation of the MSCs and the OBs. Propranolol can promote bone metabolism and bone formation and improve the curative effect.

The invention shows that propranolol can improve the expression of BMP2, RunX2, COL-1 and OCN in tissues and cells and simultaneously reduce the expression of beta 2-AR. This result also provides a new idea for the regulatory mechanism of propranolol.

The current studies on the effect of propranolol on osteogenesis and its mechanism of action at different concentrations are still controversial. The experiment of the invention shows that: the medium (1mg/kg) and high (10mg/kg) concentrations showed significantly higher osteogenesis promoting effects (p < 0.01) than propranolol at the low (0.1mg/kg) concentration, but the high concentration group did not show better osteogenesis effects (p > 0.05) than the medium concentration group. The method can bring guidance to the application of the medicine in clinic.

The invention shows that the beta receptor blocker propranolol can promote the osteogenic differentiation of OBs and MSCs and enhance the osseointegration of the implant by regulating the expression of osteogenic related proteins BMP2, RunX2, COL-1, OCN and beta 2-AR. Is expected to provide new insight for the application and the regulation mechanism of propranolol.

Drawings

FIG. 1 shows that propranolol promotes osteointegration of an implant. In the figure: a is the preparation of a femoral shaft fracture model of a New Zealand rabbit implant; b is X-ray examination of osseointegration of the control group and the propranolol group, which is respectively 0.1mg/kg, 1mg/kg and 10 mg/kg; c is QPCR for detecting the expression of BMP2, RunX2, COL-1 and OCN in each group; d is WB to detect the expression of BMP2, RunX2, COL-1 and OCN in each group; #, p < 0.05; #, p < 0.01;

FIG. 2 shows that propranolol inhibits the proliferation of MSCs and promotes the proliferation of OBs. In the figure: a is a fluorescence detection image after detection of different treatments in the msc; b is an edu percentage statistical chart after detecting different treatments in the msc; c is a fluorescence detection image after different treatments are detected in ob; d is a statistical graph of the edu percentage after detection of different treatments in ob; e is a diagram for flow detection of msc proliferation; f is a diagram of the proliferation condition of ob detected by flow;

FIG. 3 is a graph showing that propranolol inhibits the migration of MSCs and promotes the migration of OBs; in the figure: A. b is MSC migration efficiency detected by a cell scratch test after propranolol treatment at different concentrations and is respectively 0, 24 and 48h (200 x images are taken, and scratch areas are measured, wherein A is the 200 x images and B is a statistical graph for measuring the scratch areas, C, D is OB migration efficiency detected by the cell scratch test after propranolol treatment at different concentrations, C is the 200 x images and D is a statistical graph for measuring the scratch areas;

FIG. 4 is a graph of propranolol promoting osteogenic differentiation of MSCs and OBs; in the figure: a is alizarin red staining detection of osteogenic differentiation of MSC; b is the determination of calcium content in MSC; c is measurement of ALP activity in MSC; d is osteogenic differentiation of OB determined by alizarin red staining; e is the determination of calcium in OB; f is determination of ALP activity in OB;

FIG. 5 shows the regulation of osteogenesis related genes by propranolol; in the figure: a is the expression of BMP2 in the MSC by immunofluorescence detection; b is the expression of RunX2 in the MSC detected by immunofluorescence; c is the expression of COL-1 in the MSC by immunofluorescence detection; d is the expression of OCN in the MSC by immunofluorescence detection; panels A-D, from left to right, are protein, DAPI (stained nuclei), Merge (combined images of protein and DAPI) detected, respectively; e is the expression of BMP2 in the OB detected by immunofluorescence; f is the expression of RunX2 in the OB detected by immunofluorescence; g is the expression of COL-1 in OB detected by immunofluorescence; h is expression of OCN in OB detected by immunofluorescence; zoom in 200 ×;

in fig. 6: A-D are the expression levels of BMP2, RUNX2, COL-1 and OCN detected by immunofluorescence respectively. E-H are the mean fluorescence intensity statistical plots of each index. Each index examined the expression of control, four groups of 0.1, 1, 10. mu.M, A-D, from left to right, protein, DAPI (stained nuclei), Merge (pooled images of protein and DAPI) examined, respectively;

FIG. 7 shows qPCR and WB detection of BMP2, RunX2, COL-1, OCN and β 2-AR in MSC and OB. A, WB detects BMP2, RunX2, COL-1, OCN and beta 2-AR protein expression in MSC; b, qPCR (quantitative polymerase chain reaction) is used for detecting the mRNA expression levels of BMP2, RunX2, COL-1, OCN and beta 2-AR in the MSC; c, detecting the expression quantity of BMP2, RunX2, COL-1, OCN and beta 2-AR proteins in OB by WB; d, qPCR detection of BMP2, RunX2, COL-1, OCN and beta 2-AR mRNA expression level in OB.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

1. Establishment of implant osseointegration model

The experimental animals were carried out strictly according to the ethical committee of the affiliated hospital of Qingdao university. Healthy male New Zealand white rabbits had a body weight of about 3.5kg for 5-6 months, and were randomly divided into a control group, a low dose propranolol group (0.1mg/kg), a medium dose propranolol group (1mg/kg), and a high dose propranolol group (10 mg/kg).

The propranolol group was injected subcutaneously, the propranolol was approached to the operative site before the operation, and the control group was administered with an equivalent amount of physiological saline for 28 consecutive days. The rabbit tibia was then subjected to conventional surgical cavity preparation at the metaphysis and pure titanium implants 3.4mm in diameter were inserted using an implanter. One on the left rear leg and the other on the right rear leg, each having a length of 8 mm. From day 1 to day 3 after the operation, buprenorphine hydrochloride (0.03mg/kg) and doxycycline (3.2mg/kg) were injected subcutaneously into each group of rabbits, and the healing was good after the operation. Propranolol was injected subcutaneously for 28 days after implantation. After X-ray examination, each group of rabbits was sacrificed and the intact tibial tissue was removed for follow-up experiments.

2. Extraction of bone marrow mesenchymal stem cells BMSCs

Two experimental rabbits were decapped and placed in 75% alcohol for 15 min. The tibia and femur of the rabbits were separated under sterile conditions and placed in PBS solution. Connective tissue and periosteum were immediately removed, the medullary cavity was washed with serum-free DMEM medium, and transferred to centrifuge tubes containing 15% serum DMEM. Washing until the medullary cavity becomes white, ultrasonically shaking at 1000r/min for 5min, discarding the supernatant, and adding H-DMEM containing 15% FBS to prepare cell suspension. And (4) uniformly blowing the cell suspension, planting the cell suspension into a culture bottle, and replacing the culture solution after 24 hours. The culture medium was replaced with fresh medium every 2 to 3 days.

3. Alizarin red staining identification

Complete media for BMSCs and OBs in vitro cultures, each containing 50nM isoproterenol, were used to mimic the sympathetic effects. After culturing the extracted BMSCs in complete medium for 24h, culture was replaced with osteogenic differentiation medium containing 50nm isoproterenol and propranolol for 7 days, followed by mineralized nodule staining-alizarin red staining.

The dyeing method comprises the following specific steps: the original medium was discarded, washed three times with PBS solution, and then fixed with 4% paraformaldehyde solution for 20 min. Finally, washing the obtained product with a PBS solution, performing supplementary dyeing for 30min at 37 ℃ by using a 0.1% alizarin red dye, and then washing the obtained product with tap water for microscopic examination.

4. Alkaline phosphatase (ALP) staining

After 3 weeks, inoculating the cells into a culture dish, preparing required reagents according to the kit specification, pouring out the original culture medium, and fixing the specimen for 3min by using the fixed culture medium; the substrate solution was then added to the petri dish, covered with a hydrophobic membrane, incubated in an oven at 37 ℃ in the dark for 15min, and then rinsed clean. Finally, dye was added for 3min, the samples were rinsed with tap water and examined under a microscope.

5. Cell proliferation is affected by EDU

1×10⁷Cells were seeded in 6-well plates for culture. After overnight cell culture, EDU was added at a final concentration of 10. mu.M, and incubation was continued for 2 hours, and the medium was removed; cells were fixed with 1ml of 4% paraformaldehyde in a fixative for 15min at room temperature. Taking a fixed culture medium, adding 1ml of washing solution into each well, 3-5min each time, and washing the cells for 3 times; endogenous peroxidase was then inactivated by incubation with endogenous peroxidase blocking solution at room temperature for 20 minutes. The cells were then washed 3 times for 2 minutes each with wash solution.

6. Effect of flow cytometry on cell proliferation

Density of 1.5X 10⁵The cells were seeded in a 35mm diameter petri dish for 1 day. Most cells were allowed to enter G0 phase in medium containing 0.4% FBS for 3 days in synchrony. BrdU stock solution (1.0 mg/mL) was added to a final concentration of 0.03. mu.g/mL, and the mixture was incubated at 37 ℃ for 40min, and the medium was discarded. The cells were collected in a flow tube, centrifuged at 1000r/min for 5min and the supernatant discarded. Each tube was fixed with 1m l 4% paraformaldehyde for 15min, centrifuged at 2000r/min for 10min, and the supernatant was discarded. 2mL of glycine was added to each tube, washed with water, and then 1mL of 0.5% Triton was added and incubated for 10 min. The cells were then washed 3 times with PBS, centrifuged at 1000r/min for 5min each time, resuspended and examined by flow cytometry.

Q PCR detection

Carrying out reverse transcription reaction by using a TAKARA kit, wherein the reaction system is RNA and 2.2 mu g; oligosaccharide T, 2 μ L; dNTP, 4. mu.L; 5 Xbuffer, 4. mu.L; reverse transcriptase, 1 μ L; rnase inhibitor, 0.5 μ L; RNase double distilled water, 20. mu.L. The reaction conditions are 25 deg.C for 5min, 50 deg.C for 15min, 85 deg.C for 5min, and 4 deg.C for 10 min.

The QPCR experiment was performed according to the instructions of the QPCR kit from Qingdao Biotechnology Inc. The reaction system is a forward primer, and 0.4 mu L; reverse primer, 0.4 μ L; SYBR Green, 10 μ L; h₂O, 5.2. mu.L. The reaction conditions were 50 ℃ for 2min, 95 ℃ for 10min, 95 ℃ for 30sec, 60 ℃ for 30sec, and 40 cycles.

The primers used were as follows: BMP 2-F: 5'-TGGAAGGGCCCATTTAGAG-3' the flow of the air in the air conditioner,

BMP2-R：5’-GC TTTT CTCTGTGTGGAGC-3’；

run X2-F, 5'-TTACCTACACCCC GCCAGTC-3';

run X2-R, 5'-TGCTGGTCTGGAAGGGTCC-3';

cole-1-f, 5'-ggcttcagtggttggatg-3',

col-1-r，5’-caccaacagcaccatcgtta-3’；

OCN-f，5’-gctctgtctctctcacaca-3’；

ocn-r，5’-ccctcctgctggacatgaa-3’；

β2-AR-F，5′-GATCAAGCTTATGGGGCAACCCGGGAACGGCAGC-3′，

β2-AR-R，5‘-gatcgtcgaccagcagtagataa gggg attg-3’。

western blot detection of protein expression levels

After extraction of total protein from kidney tissue, proteins were separated on SDS-PAGE gels, and anti-BMP 2, RunX2, COL-1, OCN and β 2-AR antibodies were incubated, respectively, and corresponding secondary antibodies were incubated. Protein bands were subsequently detected by ECL luminescence. The antibodies used were as follows: BMP2 (Dilute 1: 500, BM19970, IGEE, China), RunX2 (Dilute 1: 500, BM16360, IGEE, China), COL-1 (Dilute 1: 500, BM2319, IGEE, China), OC N (Dilute 1: 500, BM18692, IGEE, China), beta 2-AR (Dilute 1: 500, BMP0265, IGEE, China) and beta-actin (Dilute 1: 2000, BMC026, IGEE, China). HRP goat anti-rabbit IgG (dilution 1: 2000, BMS014, IGEE, China).

9. Immunofluorescence detection

Paraffin sections of 4 μm thickness were baked at 60 ℃ for 2h, dewaxed, and hydrated with xylene and alcohol. The slices are placed in citrate buffer (pH6.0) for antigen retrieval, heated in a microwave oven for 20min, and naturally cooled to room temperature while being kept as it is. PBS wash 3 times, each time for 5 min. Blocking with 5% BSA for 30 min. The antibody was added once and left overnight at 4 ℃. Washing with PBS for 3 times, and rewarming for 30min, 5min each time. The corresponding secondary antibody (1 h incubation at room temperature, 3 washes with PBS, 5min each, 5min incubation in the dark with DAPI 5-10min PBS 3 washes, 1min each, sections were blocked under a fluorescence microscope, observed, photographed) was added using BMP2 (Dilute 1: 100, BM19970, IGEE, China), RunX2 (Dilute 1: 100, BM16360, IGEE, China), COL-1 (Dilute 1: 100, BM2319, IGEE, China), OC N (Dilute 1: 100, BM18692, IGEE, China), HRP goat anti-rabbit IgG (Dilute 1: 1000, BMS014, IGEE, China).

10. Statistical analysis

Statistical analysis of experimental data was performed using SPSS23.0 software. The data are expressed as mean ± standard deviation (x ± s), and paired T-test is used before and after the same group of treatments. Analysis of variance makes comparisons between groups. Where P <0.05 was considered significantly different and P <0.01 was considered very significantly different.

Results of the experiment

1. Propranolol can promote osseointegration of implant

An implant osteointegration model was established in New Zealand white rabbits and rabbits were injected with different doses of propranolol (0.1mg/kg, 1mg/kg, 10 mg/kg). After 14 days, the bone tissue was examined for the binding to the implant and bone tissue resorption by X-ray. The results are shown in fig. 1a, b, and show that the combination of implant and bone tissue is superior to the control group, and the combined effect of implant and bone tissue increases in a manner independent of propranolol dose after propranolol injection.

Bone tissue samples were collected near the implants and mRNA expression of BMP2, RunX2, COL-1, OCN and β 2-AR was examined by qPCR. The detection results are shown in fig. 1c and d, and the results show that propranolol can improve the mRNA expression of BMP2, RunX2, COL-1 and OCN and reduce the expression of beta 2-AR. The western blot was used to detect the protein expression of BMP2, RunX2, COL-1, OCN and β 2-AR, the results were consistent with qPCR.

2. Propranolol inhibits the proliferation of MSCs and promotes the proliferation of OBs

To elucidate the effect of propranolol on the proliferation of MSCs and OBs, MSCs and OBs were cultured in vitro. Cell proliferation was detected by EDU staining after 24 hours incubation with varying concentrations of propranolol in complete media containing isoproterenol. The results are shown in FIGS. 2a and b, and show that: in the MSCs, the EDU positive rate is obviously reduced along with the addition of propranolol, the EDU positive rate of a 10 mu M propranolol group is the lowest, and the propranolol is shown to inhibit the proliferation of the MSCs. As shown in FIGS. 2c and d, in OBs, the addition of propranolol can promote cell proliferation, the positive rate of EDU in OBs can be remarkably improved by 0.1 mu M propranolol, and the positive rates of EDU in 1 mu M propranolol groups and 10 mu M propranolol groups are close to each other. The above results indicate that propranolol inhibits the proliferation of MSCs and promotes the proliferation of OBs.

3. Propranolol inhibits the migration of MSCs and promotes the migration of OBs

The effect of propranolol on cell migration was then examined by the cell scratch test and scratch healing was observed at 24 hours and 48 hours, respectively. The results of the experiments show that when propranolol is added to the whole culture medium containing isoproterenol, the migration of MSCs in MSCs and OBs cultured in vitro is inhibited. In addition, the healing rate of the scratch was significantly slower than the control, and the higher the concentration, the slower the healing rate of the scratch (fig. 3A-3B). In contrast, propranolol increased the healing rate of scratches in OBs, with higher cell migration than the control (fig. 3C-3D). These results suggest that propranolol inhibits MSCs migration and promotes OBs migration.

4. Propranolol promotes osteogenic differentiation of MSCs and OBs

To elucidate the effect of propranolol on osteogenic differentiation of MSCs and OBs, different concentrations of propranolol were supplemented to the osteogenic differentiation medium containing isoproterenol in MSCs and OBs cultured in vitro. After 7 days of induction, osteoblastic differentiation of the cells was examined by alizarin red staining. Interestingly, we found that propranolol promoted osteogenic differentiation of MSCs and OBs and increased intracellular calcium nodules after induction. Calcium content and ALP activity of propranolol-treated cells were significantly higher than control (FIGS. 4A-4F).

5. Regulation effect of propranolol on osteogenesis related genes

Next, in order to elucidate the osteogenesis mechanism of propranolol in regulating MSCs and OBs, we examined the expression of osteogenesis-related genes BMP2, RunX2, COL-1, OCN and β 2-AR by immunofluorescence. The results show that propranolol can increase protein expression of BMP2, RunX2, COL-1 and OCN and decrease expression of beta 2-AR in a dose-dependent manner (FIGS. 5A-5H). The qP CR and western blot results were consistent with the immunofluorescence results, indicating that propranolol can up-regulate osteogenic gene expression, promoting osteogenic differentiation of MSCs and OB (fig. 6A-6D).

Early rapid osteointegration is considered critical for successful implantation, but it is limited by the biological inertia of the material surface itself, high elastic modulus, and limited biological effects. Therefore, how to improve the performance of titanium implants and promote and accelerate osseointegration remains the focus of attention of many researchers in recent years. Although titanium and titanium alloy implants have met with great success in the medical field, infection and poor osseointegration remain the two main causes of implant surgery failure. In addition to improving implant materials, there is great potential for improving early bone integration of implants by drug-assisted methods.

The sympathetic nervous system, a component of the autonomic nervous system of the human body, plays a crucial role in regulating life activities and body homeostasis. There is increasing evidence that the sympathetic nervous system is involved in the process of bone remodeling. The histological basis of sympathetic regulation of bone tissue is its innervation of bone tissue and bone cells. Related experiments demonstrate the presence of sympathetic nerve fibers on bone and periosteum. The prior art shows that: through histochemistry and observation of rabbit bone tissue by a fluorescence electron microscope, the surface of the blood vessel in the bone is covered with abundant adrenergic nerve fibers. Sympathetic nerves were present in bone tissues of mature rats, and presence of NE and neuropeptide Y in the sympathetic nerves was observed by immunohistochemical staining. The distribution characteristics of the sympathetic nerves are pointed out, i.e. the sympathetic nerve fibers are mostly located in the bone marrow cavity where the blood flow is abundant, while the sympathetic nerve fibers in the periosteum are less. Studies have demonstrated that leptin regulates bone formation and bone resorption, and that this effect of leptin is mediated by sympathetic nerves. When the sympathetic nerve is excited, the bone absorption can be promoted, and the bone formation can be reduced. This effect is dependent on the β 2-adrenergic receptors on the surface of osteoblasts. Immunofluorescence technology beta 2 receptors are located on the surface of human osteoblasts. In addition, β 2 receptor agonists have been found to inhibit osteoblast proliferation.

Propranolol is well known as a medicine for treating hypertension, and is one of the first-choice medicines for treating cardiovascular diseases. In recent years, the influence of propranolol on bone metabolism has attracted attention of scholars. The existing reports show that: treatment of 9 weeks of propranolol rats with surgical fractures increased mineral deposition and bone formation. The small dose of propranolol has obvious advantages in the aspect of treating osteoporosis. Several epidemiological studies have also shown that β 2-AR blockers are potential candidates for the treatment of osteoporosis and bone fractures.

The invention discusses the influence of the beta-AR inhibitor propranolol on the osteogenesis of a cell level animal model. The results show that propranolol can promote the osseointegration of the implant and enhance the osteogenic differentiation of the MSCs and the OBs. Propranolol can promote bone metabolism and bone mass and improve the curative effect.

The invention shows that propranolol can improve the expression of BMP2, RunX2, COL-1 and OCN in tissues and cells and simultaneously reduce the expression of beta 2-AR. This result also provides a new idea for the regulatory mechanism of propranolol.

The current studies on the effect of propranolol on osteogenesis and its mechanism of action at different concentrations are still controversial. The experiment of the application shows that: the medium (1mg/kg) and high (10mg/kg) concentrations showed significantly higher osteogenesis promoting effects (p < 0.01) than propranolol at the low (0.1mg/kg) concentration, but the high concentration group did not show better osteogenesis effects (p > 0.05) than the medium concentration group. The method can bring guidance to the application of the medicine in clinic.

The invention shows that the beta receptor blocker propranolol can promote the osteogenic differentiation of OBs and MSCs and enhance the osseointegration of the implant by regulating the expression of osteogenic related proteins BMP2, RunX2, COL-1, OCN and beta 2-AR. Is expected to provide new insight for the application and the regulation mechanism of propranolol.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.