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The effect of human-like collagen calcium complex on osteoporosis mice
Materials Science & Engineering C 93 (2018) 630–639
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
The effect of human-like collagen calcium complex on osteoporosis mice a,b
a,b
Chenhui Zhu , Huan Lei , Shangshang Wang ⁎ Jianjun Denga,b, Daidi Fana,b, , Xingqiang Lva a b
a,b
, Zhiguang Duan
a,b
a,b
, Rongzhan Fu
T
,
Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi'an 710069, China Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an 710069, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Calcium supplementation Human-like collagen Osteoporosis Bone mineral density
The objective of this study was to assess the effect of a modified human-like collagen calcium complex on osteoporosis mice. BHK (Baby Hamster Kidney) cells were used to compare the cytotoxicity of different calcium reagents with the MTT test. Six-week-old male mice (n = 80) were randomly divided into eight groups: a blank group (blank), control group (control), human-like collagen calcium group (HLC-Ca), thiolated human-like collagen calcium group (SH-HLC-Ca), phosphorylated human-like collagen calcium group (Pi-HLC-Ca), gluconate group (Glc-Ca), calcium carbonate group (CaCO3) and D-cal group (B). A systematic analysis of the results available in vivo after 3 months of treatment was presented. The effects of several Ca supplements on osteoporosis mice were investigated by detecting serum calcium, alkaline phosphate activity (ALP), bone hydroxyproline (BHP) and bone mineral density (BMD). The results proved that the BMD and BHP of osteoporosis mice were significantly increased in the Pi-HLC-Ca group, while serum calcium and ALP were decreased. Therefore, Pi-HLC-Ca is likely a good calcium supplement for clinical applications. In this review, the advantage of Pi-HLCCa in preventing and delaying osteoporosis is highlighted. In addition to the current progress, further investigations are necessary to reveal the relative influences of collagen and calcium proportions on the long-term clinical effects of osteoporosis.
1. Introduction Calcium (Ca) plays a vital role in the life of human beings, as one of the most important minerals in the human body. However, calcium deficiency is also very common and can lead to osteoporosis and osteopenia, which are always related to calcium deficiency [1,2]. Calcium reagents can be used to restore the balance between resorption and formation [3]. In addition, calcium supplementation is regarded as a safe monotherapy method for osteoporosis [4]. However, even longterm supplementation with calcium only postpones bone loss and provides no favourable effect on the preservation of bone mineral density [5,6]. Moreover, collagen, the most abundant protein, plays an important role in the body. Collagen is not only an important part of bone tissue but also an essential part of bone health; for instance, type I collagen is a primary organic component that makes up the extracellular matrix of bone [7–9]. Collagen, a biocompatible material, is widely used in interventions due to its low risk of immunological reactions [10]. Human-
like collagen (HLC), which is prepared in our laboratory to be adopted as a biological material, is also widely used, including as bone materials and hydrogels that show good compatibility and that stimulate no immunological effect [11–14]. It is also known that collagen or hydrolysates in vivo can generate peptides that are useful for organic biosynthesis [15,16]. It has been shown in some studies that oral administration of hydrolysed collagen may improve bone mass content and density in rats [17]. In general, unexpected results may be achieved through combining human-like collagen with calcium. Bone, a dynamic organization, is constantly updating and remodelling to maintain its integrity [18–20]. In other words, newly generated bone is constantly replacing the old bone and is accommodating the mechanical load changes [21]. Once osteoclasts are attached to the bone surface, protons and proteolytic enzymes are secreted, which may dissolve the bone mineral and hydrolyse the matrix protein. Then, osteoblasts replace the osteoclasts to capture protons and protein to form calcium and phosphate deposits [22]. The specific process is shown in Fig. 1. In this process, the old bones
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Corresponding author at: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi'an 710069, China. E-mail addresses: [email protected] (C. Zhu), [email protected] (R. Fu), [email protected] (J. Deng), [email protected] (D. Fan), [email protected] (X. Lv). https://doi.org/10.1016/j.msec.2018.08.011 Received 8 January 2018; Received in revised form 10 July 2018; Accepted 5 August 2018 Available online 06 August 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Calcium supplementation could reduce parathyroid hormone (PTH) levels, resulting in diminished RANKL (receptor activator for nuclear factor-κB ligand) expression by osteoblasts. The content of cathepsin K (CatK) and protons that the osteoclasts captured on the bone surface were reduced. Thus, the matrix proteins (principally the type I collagen) and bone mineral were protected.
water bath, the Pi-HLC was prepared and then freeze-dried. The freezedried Pi-HLC was dissolved in a saturated KI solution at 55 °C; then, freeze-dried Pi-HLC was dissolved in MOPS buffer (10 μM, pH 7.5), and CaCl2 solution was added into the Pi-HLC solution (n (Pi-HLC): n (CaCl2) = 1:600), followed by gentle stirring at room temperature for 1–1.5 h. Finally, the reaction mixture was purified and freeze-dried. Preparation of SH-HLC-Ca: SH-HLC was prepared by adding SAMSA to HLC solutions. Then, freeze-dried SH-HLC was dissolved in ultrapure water at a concentration of 10 mg/mL, and CaCl2 solution was added into SH-HLC solution (n (SH-HLC): n (CaCl2) = 1:1000), followed by gentle stirring at room temperature for 1 h. Finally, the reaction mixture was purified and freeze-dried.
are updated and replaced to maintain skeletal balance. However, in elderly persons, the balance between formation and resorption shifts towards resorption. If the unbalanced state continues, in the long-term, calcium, bone mineral density and matrix protein will decrease, leading to osteoporosis. Osteoporosis, a common disease, is characterized by low bone mineral density and an increase in the risk of fractures of the hip, spine, and forearm [5,23]. Age is a neglected risk factor for osteoporosis. Osteoporosis may be prevented or delayed by maximizing peak bone mass during adolescence. The aim of this study was to evaluate the effect of the human-like collagen calcium complex (prepared by our laboratory) on osteoporosis mice, which are produced by oral overdoses of retinoic acid [24,25]. The primary endpoints of the study were to alleviate mouse bone disorders, such as low BMD and high serum calcium. Future studies may suggest the importance of clinical examinations as well as a way to prevent or delay osteoporosis. People suffering from calcium deficiency and osteoporosis will be treated, or the condition will be prevented, to alleviate the burden on patients and society.
2.3. Cell culture and cell viability BHK (Baby Hamster Kidney) cells (CBCAS, Shanghai, China) were cultured in RPMI 1640 medium. The cytotoxicities of HLC, HLC-Ca, SHHLC-Ca and Pi-HLC-Ca were assessed using the BHK cells. The samples were dissolved in cell culture at 20 mM (Ca2+) and sterilized with a 0.22 μm filter. The cells were cultured in a CO2 (5%) incubator at 37 °C on 96-well plates with 200 μL per well (1.0 × 104 cells·mL−1) for 24 h and then were cultured with the sample solution for 24 h, 36 h and 48 h. At the end of the incubation, 20 μL MTT (0.5%) was added to each well for another 4 h. The medium was removed, and DMSO (100 μL) was added to each well. Finally, the absorbance was measured at 490 nm. The relative cell growth (%) was calculated as follows:
2. Materials and methods 2.1. Materials Human-like collagen (HLC, China patent number: ZL01106757, Mr = 97,000) was supplied by Xi'an Giant Biogene Technology company. HLC-Ca, SH-HLC-Ca and Pi-HLC-Ca were prepared by our laboratory. Glucose calcium (Glc-Ca) was purchased from Sanchine. CaCO3 and D-cal (B) were purchased from Anshi Pharmaceutical Inc. All the reactants and solvents used were of analytical grade, and double-distilled water was used throughout the experiments.
Relative cell growth (%) = ODtest / ODcontrol × 100%
(1)
where ODtest means the absorbance value of the test sample and ODcontrol means the absorbance value of the blank sample. All tests were repeated three times, and we reported the average value of five parallel samples.
2.2. Preparation of HLC-Ca, Pi-HLC-Ca and SH-HLC-Ca
2.4. Cell fluorescence staining test
Preparation of HLC-Ca: Freeze-dried HLC was dissolved in ultrapure water at a concentration of 10 mg/mL, the pH was adjusted to 7.5, and CaCl2 solution was added into the HLC solution (n (HLC): n (CaCl2) = 1:1000), followed by gentle stirring at room temperature for 1–1.5 h. Finally, the reaction mixture was purified using dialysis and freeze-dried in a vacuum freeze drier (FD5-10, SIM, US). Preparation of Pi-HLC-Ca: Then, the freeze-dried HLC was dissolved in ultrapure water at 10 mg/mL, and G-6-P-Na2 (10 mg) was added to the HLC solution (pH 8.0–8.5), After incubating for 2–4 h in a 50 °C
A cell fluorescence staining technique was used to observe apoptosis in the cells. DAPI (2-(4-amidinophenyl)-6-indole carbinol dihydrochloride) is a kind of blue fluorescence dye that can penetrate the cell membrane and bind to DNA; then, the cells exhibit a green colour when excited in blue light. BHK cells were seeded at a density of 1.0 × 103 cells/cm2 on 48well plates and were cultured in RPMI 1640 medium in a CO2 (5%)
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conducted with 5 μL serum, and the calculation formula was as follows:
incubator at 37 °C for 24 h. The experimental groups were cultured with the HLC, HLC-Ca, SH-HLC-Ca or Pi-HLC-Ca samples, with a Ca2+ concentration of 10 μM (n = 6 per group). The control group was cultured in RPMI 1640 medium (n = 6). After 24 h, we discarded the medium and added 10 mL of 5 μg/mL DAPI into each well for 15–20 min. Finally, the reaction solution was discarded, and the cells were observed under an inverted microscope.
Serum ALP activity (king unit/100mL) = (ODtest − OD blank )/(ODstandard − OD blank ) × (0.1mg/mL) × 100mL×sample dilution ratio
(2) 2.9. Serum calcium
2.5. Animals and treatment
In an alkaline solution, serum calcium ion can be combined with methyl thymol, forming a blue complex (MTB). Colorimetric assays with standard calcium can then be used to determine the serum calcium content. We used 50 μL serum to test for calcium content. The test was performed using a calcium assay kit (Nanjing Jian Cheng Bioengineering Institute). The formula was as follows:
Male mice (n = 80), 6 weeks old and weighing approximately 20 g, were randomly divided into 8 groups of 10 animals each. The mice were housed in small cages individually under similar conditions with a 12-hour light-dark cycle at 22 ± 1 °C. All mice had free access to food and water. Body weights were recorded each week. In the blank group, the mice were fed the standard diet and given tap water freely throughout the procedure. In the control and experimental groups, mice were fed the standard diet along with a gavage of vitamin A acid 70 mg/kg each and were given tap water freely for 2 weeks. Then, the experimental groups were given by gavage the indicated amounts of HLC-Ca, SH-HLC-Ca, Pi-HLC-Ca, Glc-Ca, CaCO3 and D-cal over the 3month feeding period. At the end of the period, they were killed with thiopental sodium under deep anaesthesia. All procedures were approved by the Committee for the Use of Laboratory Animals of Northwest University, China.
Serum ALP activity (king unit/100mL) = (ODtest − OD blank )/(ODstandard − OD blank ) × standard concentration (2.5mmol/L)
(3) 2.10. BHP Right femurs were measured, cut into pieces and then treated with 1 mL 6 mol/L hydrochloric acid in a water bath for 5 h. Then, we adjusted the solution's pH to 6.0–6.8 and diluted the solution to 10 mL. We then added 20–30 mg of active carbon to the 4 mL mixture solution and centrifuged it at 3500 rpm for 10 min. The supernatant was collected, and we used 1 mL to detect the BHP content. The test was performed using a hydroxyproline assay kit (Nanjing Jian Cheng Bioengineering Institute). The calculation formula is as follows:
2.6. Body weight We weighed and recorded the mouse body weight once a week. 2.7. Blood and bone sampling
BHP (ug/g) = (ODtest − OD blank )/(ODstandard − OD blank )×standard contest × hydrolysate volume/sample wet weight
At the end of the treatment period, blood samples were collected from the abdominal aorta under ether anaesthesia and centrifuged at 3000 rpm for 10 min; the serum was stored at −70 °C until measurement of the parameters. The mice were sacrificed humanely, and the hind legs were dissected to eliminate all soft tissues. The femur and tibia were stored at −70 °C until use.
(4) 2.11. Bone mineral density Bone mineral density of the right femur was measured by dual energy X-ray absorptiometry (DXA) using a DSC-600EX-IIR bone densitometer (Aloka Co., Ltd., Tokyo, Japan) at the 10th week.
2.8. Serum alkaline phosphate activity During the early stages of osteoblast differentiation, alkaline phosphatase (ALP) expression is activated, and its activity has been traditionally regarded as a marker to determine the degree of osteoblast differentiation [26,27]. A rise in serum ALP activity is significantly elevated in diseases of the skeletal system, such as osteomalacia, fractures and rickets. The tests were performed using an alkaline phosphatase assay kit (Nanjing Jian Cheng Bioengineering Institute). The chromogenic substrate solution was dissolved in 2.5 mL of ALP reaction buffer and placed on ice. Then, 10 μL of the p-nitrophenol standard solution (10 mM) was diluted in 0.2 mL of the ALP reaction buffer so that the final concentration was 0.5 mM. The tissue was lysed, homogenized appropriately, and then centrifuged to obtain the supernatant for detection of ALP activity. Note: The lysate did not contain phosphatase inhibitors. The tissue samples were frozen at −80 °C until further use. In the test, blank control wells, standard wells, and sample wells were prepared. The amount of standard used was 4, 8, 16, 24, 32, and 40 μL, and the sample could usually be added directly to bring the total to 50 μL. If the alkaline phosphatase activity in the sample was too high, we reduced the amount of sample or made an appropriate dilution before performing the assay. After incubation at 37 °C for 5–10 min, 100 μL reaction stopper solution was added to each well to terminate the reaction. Absorbance was measured at 405 nm. Tests were
2.12. Mechanical test Mechanical testing was performed using a mechanical testing machine (Instron, Testing machine Co., model MZ-3365, USA). For the three-point bending test of the mid femur, the left femur was placed on a holding device with supports located 12 mm apart. The upper loading device was aligned to the centre of the femoral shaft on the anterior side to give an accurate span [28]. The three-point bending test was performed at a constant loading rate of 20 mm/min and a load cell of 50 kgf to measure the stiffness (N/cm). Ultimate load (N) and stiffness (N/cm) were calculated from the load-displacement curve. 2.13. SEM The left tibias of the mice were placed in a muffle furnace, burning at 800 °C for 6 h, to determine the ash composition. After they had cooled naturally, each tibia was cut into cross sections [29]. The surface morphologies of the tibia slices were examined by SEM (Hitachi S-4800, Japan) at 20 kV. We placed the tibia ash cross section into liquid nitrogen for approximately 10 min. The sections were fixed on conductive aluminium and were sputter coated with gold before scanning.
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spherical. The HLC group included more viable cells but few dead cells. The HLC-Ca group exhibited the lowest number of cells but with more dead ones, while the SH-HLC-Ca and Pi-HLC-Ca groups were similar to the HLC group; however, the numbers of dead cells were obviously increased relative to that in the HLC group. These results suggest that HLC, HLC-Ca, Pi-HLC-Ca and SH-HLC-Ca have no effect on BHK cell morphology. The dead cells were caused by nutrient limitations. Therefore, it can be concluded that HLC, HLC-Ca, SH-HLC-Ca and Pi-HLC-Ca are not only safe but can also stimulate the normal growth of cells, indicating that they are safe products for oral Ca supplementation. 3.3. Animal experiment After 2 weeks, the average serum calcium of mice fed with retinoic acid (1.75 mmol/L) was dramatically decreased in comparison with the control (2.5 mmol/L). The average ALP and bone hydroxyproline level of mice fed normal basic feedstuff were all within normal limits during the experimental period; however, in the mice gavaged with retinoic acid, they were both markedly lower than that of the control after 2 weeks. All of the above data showed that the construction of the osteoporosis mouse model was successful (Fig. 4). As shown in Fig. 4, PTH stimulates OB to synthesize RANKL, while RANKL can activate OC to synthesize H+, K and other destructive factors. These destructive factors act on bone [30], mainly K acting on bone matrix protein [31], and they decompose the bone matrix proteins so that mineral ions cannot be deposited on the bone surface. These mineral ions are mainly calcium and phosphorus. The calcium ions in the mineralized layer decrease, and the bone density also decreases due to the loss of calcium phosphate and collagen, leading to osteoporosis. Therefore, osteoporosis is mainly due to the process of bone resorption. The bone formation process is the reverse process of the bone resorption process described above; the bone matrix is synthesized using the collagen around the bone tissue, and then the mineral ions around the bone tissue are deposited on the bone matrix. Under the influence of HLC-Ca, increasing the calcium ion content promotes the reduction of PTH bound to the OB surface, which results in a decrease in the stimulation of OB by PTH, a decrease in the level of RANKL, a decrease in the synthesis and secretion of OC, a decrease in bone resorption, and thus no effect on the bone formation process. The formation process is longer than the process of bone resorption, and it effectively promotes the formation of mineralized layers of bone matrix proteins combined with calcium ions and the like so that the bone density increases. At the same time, an increase in calcium levels can also increase the amount of calcitonin, and calcitonin can also inhibit OC so that the destruction induced by H+, K and other destructive factors is reduced; thus, the bone formation process is longer than the process of bone resorption. For patients with osteoporosis, the bone formation process needs to be at a premium to maintain the bone at a healthy level. We first needed to establish a model of osteoporosis and then add the compounds to be studied to observe their effects on calcium supplementation. Many experimental studies [32] have shown that during oral administration of excessive amounts of retinoic acid, retinoic acid inhibits the activity of osteoblasts and promotes the formation of osteoclasts to stimulate the reabsorption of bone and to inhibit bone formation. This approach causes bone loss, decreased bone density, and osteoporotic fractures. We used normal mice as experimental subjects and fed the mice vitamin A acid according to a previously established experimental method, developing a model of osteoporosis. The results of the serum calcium test are shown in Fig. 4A. The serum calcium in the blank mice ranged from 1.7 mmol/L to 1.8 mmol/ L, while the serum calcium in the control mice ranged from 2.45 mmol/ L to 2.55 mmol/L. A comparison of the experimental results of the two groups showed that the serum calcium values of mice fed with retinoic acid significantly increased. The reason for this result is that retinoic acid inhibits osteoblasts when retinoic acid is used for an extended
Fig. 2. Cell viability of BHK cells with different treatments: the BHK cell viability after culturing with HLC, HLC-Ca, SH-HLC-Ca and Pi-HLC-Ca for 24 h, 36 h, and 48 h (p < 0.05). Data are presented as the mean ± SD of 6 parallel wells.
2.14. Statistical analysis The SPSS software package was used for statistical analyses. The values are shown as the means and standard deviations. The significance was analysed with a t-test, where *p < 0.05 was considered to be significant. **p < 0.01 was considered to be highly significant. 3. Results and discussion 3.1. Cell viability As we can see clearly in Fig. 2, the values of cell viability for the cells cultured with different mediums separately for 24 h, 36 h and 48 h were all higher than 100%. According to the USP toxicity classification (Table 1), the samples used for the experiment were therefore safe enough to be used as calcium supplementation reagents. We were pleased to note that the experimental groups exhibited varying degrees of growth promoting effects on BHK cells. The groups had a common growth trend with increasing incubation time, leading to increasing cell viability. The viability of cells cultured with HLC-Ca, Pi-HLC-Ca and SH-HLCCa were lower than that in the HLC group. It is possible that the HLC molecules entered the cells through a cell swallowing function or underwent enzymatic hydrolysis into small molecules that were then absorbed by the cells. The cell viability of the HLC-Ca, Pi-HLC-Ca and SHHLC-Ca groups showed a range of 105%–115%, while the HLC group had an obviously improved cell viability range of 115%–120%, which was higher than the others. Therefore, from the above analysis, all the samples were safe enough to be used as calcium reagents for application. 3.2. Cell fluorescence staining test The DAPI fluorescent staining results are shown in Fig. 3. It was observed that the viable cells were fusiform, and the dead ones were Table 1 The relationship between relative growth rate and cytotoxicity reaction gradation. RGR (%)
≥100
75–99
50–74
25–49
1–24
0
Grade
0
1
2
3
4
5
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Fig. 3. A photograph of BHK cells after staining with DAPI. HLC: HLC culture, HLC-Ca: HLC-Ca culture; SH-HLC-Ca: SH-HLC-Ca culture; Pi-HLC-Ca: Pi-HLC-Ca culture.
Fig. 4. The induction of osteoporosis in mice after feeding with retinoic acid for 2 weeks. Bone formation is a dynamic balancing process. OB: osteoblast, OC: osteoclast, PTH: parathyroid hormone, RANKL: receptor activator of nuclear factor kappa B ligand, K: proteolytic enzyme K, Form: bone formation, resorption: bone resorption. (A) The serum calcium (B) the serum ALP (C) the content of bone hydroxyproline.
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gained by the mice, and their weight rapidly dropped in the seventh week due to being long-term calcium deficient with no timely calcium supplementation [34]. The six experimental groups were compared with the blank group and the control group. The weight of the group fed with SH-HLC-Ca steadily increased, eventually by 8 g, but was smaller to that in the PiHLC-Ca group (10 g). The increased weight of the HLC-Ca group was approximately 5 g, and the weight change in the GLC-Ca group, the CaCO3 group and the B group was not significant. The main reason for the above phenomenon was that the six kinds of reagents provided the body with calcium sources that provided a balance of nutrition to maintain body weight. Different weight recoveries reflect the degree of calcium absorption in the body due to the important regulatory functions of calcium. Pi-HLC-Ca contained rich collagen, calcium and phosphorus to provide necessary nutrients. HLC not only had a trophic function but also promoted calcium absorption, but Pi-HLC-Ca had an obvious advantage in maintaining body weight. The difference in body weight between the B and Glc-Ca groups was very small, which means that the weight maintenance function of B and Glc-Ca was basically the same. These findings also revealed that Pi-HLC-Ca was a nutrient-rich calcium reagent and was better than the other three calcium reagents.
period of time. When osteoblasts are inhibited, osteoclasts predominate. The effect of osteoclasts leads to increased calcium loss from the bones that then moves into the blood, causing a significant increase in serum calcium. The increase in bone calcium in the model group also confirmed the successful establishment of the osteoporosis model. The serum alkaline phosphatase test results are shown in Fig. 4B. In the blank group, the serum alkaline phosphatase value was 0.16–0.18 U/L. In the experimental group, the serum alkaline phosphatase value was 0.27–0.29 U/L. Compared with the blank control group, the serum ALP value in the experimental group increased significantly. This result is because the serum content of alkaline phosphatase is closely related to the state of bone. When bone lesions form, alkaline phosphatase is released into the blood, and the content of alkaline phosphatase increases. The increase in alkaline phosphatase indicates that the mouse model of osteoporosis was successfully established [33]. Since serum ALP value is used as a criterion for judging skeletal diseases in clinical practice, the increase in ALP values also indicates the presence of osteoblasts. Compensatory hyperplasia results in the secretion of large amounts of alkaline phosphatase into the blood. The results of the bone hydroxyproline tests are shown in Fig. 4C. There was a significant difference between the blank group and the experimental group. The content of hydroxyproline in the blank group was 1.0–1.1 μg/mg compared with 0.45–0.55 μg/mg in the experimental group. From these results, the bone hydroxyproline content of the experimental mice was reduced by 40% to 50% compared to the blank group. Approximately 90% of the organic components in bone are type I collagen, and the special amino acid in type I collagen is hydroxyproline. The content of bone hydroxyproline can indirectly reflect the content of type I collagen in bone tissue and can indicate the state of health of the bone. In the experimental group, the content of bone hydroxyproline was profoundly decreased, which demonstrated the loss of type I collagen in the bone tissue, which is a key symptom of osteoporosis. Although the mechanism whereby retinoic acid affects bone development and metabolism is not yet clear, in the case of retinoic acid excess, bone resorption is higher than bone formation, eventually leading to osteocalcin dissolution under bone resorption and resulting in an elevated serum calcium. Alkaline phosphatase content is a common method used for clinical evaluation of skeletal diseases. When alkaline phosphatase in the serum is elevated, this is mainly due to bone and liver lesions resulting in the release of alkaline phosphatase into the serum. Based on the results of the serum calcium, serum alkaline phosphatase and bone hydroxyproline measured above, as well as the response of these results to the treatment of the mice in the experimental group, we concluded that the osteoporosis mouse model had been established. The reduction in bone hydroxyproline represents the loss of collagen. The loss of collagen protein results in a decrease in the amount of matrix proteins. The matrix proteins are also deposition sites for mineral elements such as calcium and phosphorus. If calcium phosphate cannot be deposited on the matrix proteins, it will enter the blood, causing the blood calcium concentration to increase. Establishing a model of osteoporosis is a very important process in this experiment, and it is an important means to determine the calciumsupplementing effect of human-like collagen calcium complexes.
3.5. ALP ALP is an enzyme widely distributed in tissues and fluids of the body and plays an essential role in regulating tissue mineralization. Only bone lesions, such as osteoporosis and rickets, release ALP into the bloodstream, leading to elevated levels of ALP in the blood [35]. As shown in Fig. 6A, the value of ALP in the control group, with a range of 0.16–0.17 U/L, was much higher than that of the blank group, with a range of 0.27–0.28 U/L. We presume that the higher value of ALP is due to osteoporosis, which was caused by damage to the bone cells by the excessive level of retinoic acid. Additionally, it can be found that the values of ALP in the Pi-HLC-Ca, SH-HLC-Ca, Glc-Ca, HLC-Ca, CaCO3 and B groups were much smaller than those in the control group, especially in the SH-HLC-Ca (0.19–0.21 U/L) and Pi-HLC-Ca groups (0.18–0.19 U/ L). In view of the above results and analysis, we can clearly point that Pi-HLC-Ca is a good calcium source for osteoporosis, along with calcium deposition, it enabled the bone formation process. It had an evident advantage over the other treatments, which means it may have a good effect on the treatment or prevention of osteoporosis.
3.6. Serum calcium Insufficient calcium intake and absorption causes the parathyroid hormone to increase, followed by the release of calcium deposited in bone into the blood [36]. As is known, calcium levels in the blood must be kept within a narrow range to be considered normal and functional. There is a close relationship between high blood calcium and bone health. During osteoporosis, bone resorption is increased, and bone formation is decreased, leading to high blood calcium in the control group (Fig. 6B), illustrating this point. We found that the six experimental groups and the control group all had a significantly lower serum calcium than the Ca-deficiency group, ranging from 2.35–2.45 mmol/L, combined with the values of serum calcium from the different experimental groups. We concluded that calcium supplements can reduce blood calcium levels in osteoporosis mice. The procedure of bone formation was enhanced with the accelerated calcium absorption, which resulted in the decrease in values of serum calcium in the six groups in the 12th week to varying degrees. Furthermore, the serum calcium level in the Pi-HLC-Ca group was the closest to the control. Therefore, we can conclude that Pi-HLC-Ca could not only accelerate calcium absorption but could also provide nutrition to the body.
3.4. Effects of several Ca-supplements on recovery of osteoporosis mice During the experiment, we recorded weight changes in each group of mice once a week. After 10 weeks of treatment, all the groups had significantly higher body weights than their initial body weights. The final body weights were different among the groups, especially the control group, which had a significantly lower body weight compared with the other groups (Fig. 5). The body weight of mice in the blank group showed a steady increasing trend. The trend in body weight in the control group mice was different; in the fourth week, no weight was 635
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Fig. 5. The body weight of each group throughout the experiment is presented as the mean ± SD of 10 animals (p < 0.05).
capacity of the femora was enhanced due to calcium accumulation in the bone. Through the above data analysis, the Pi-HLC-Ca group had an obvious advantage in reducing the risk of fracture. SEM was used for further analysis to confirm this conclusion.
3.7. BHP BHP is a characteristic of type I collagen, which is the primary organic component that makes up the matrix of bone. Collagen (mainly type I collagen) accounts for 90% of the bone matrix, and BHP accounts for 12.5% of the collagen [37]. The content of BHP in bone is an important indicator to evaluate the health of the bone matrix. The specificity of BHP makes it an important amino acid to judge the amount of organic material in bone. The contents of BHP in each group are shown in Fig. 6C. The control group had a lower content of BHP than did the blank group in the 12th week. These results show that the bone mass of mice with osteoporosis could not be increased without supplemental calcium reagents and instead got worse. Although the content of BHP in the six experimental groups increased, there were obvious differences, namely, SH-HLC-Ca, Pi-HLC-Ca and B had advantages in promoting increases in bone mass. The content of BHP in mice with osteoporosis increased by almost 60% after supplementing with SH-HLC-Ca or PiHLC-Ca for 12 weeks, and this high content of BHP confirmed that both SH-HLC-Ca and Pi-HLC-Ca had more of a positive impact on bone formation compared with the others.
3.9. SEM and bone mineral density The SEM micrographs of the tibia slices are shown in Fig. 8. To a certain extent, tibia cross-sections reflect the value of bone mineral density. Approximately 70% of bone composition is calcium and phosphorus minerals, and 30% is organic matter, mainly collagen I [35]. The pores generated in the tibia slice surfaces are present because they were subjected to very high temperatures and because only minerals were retained. Normal mouse images showed that the tibia pore diameters were significantly < 20 μm (Fig. 8 Blank), while the osteoporosis mice had pore diameters up to 80 μm (Fig. 8 Control). The pore diameter of the HLC-Ca group was close to the blank group as shown in Fig. 8 HLC-Ca. A smaller pore size and fewer holes means a higher bone mineral density. We were able to confirm that Pi-HLC-Ca had an excellent calcium supplementation effect on osteoporosis mice through these experiments. Their bone mineral density was improved to a high extent, which was also verified in Fig. 8. BMD is an important parameter to measure bone mass. The BMD of the Pi-HLC-Ca group was obviously higher than that of the other groups, which means that PiHLC-Ca was good at increasing bone mass. Pi-HLC-Ca has the best calcium effect, which may be due to:
3.8. Mechanical test analysis Calcium supplementation can effectively reduce the risk of fractures [35,38]. The femora of mice with osteoporosis were used to test their ultimate load, and these test results are shown in Fig. 6D. The test outcome shows that the capacity of resistance of the mice with osteoporosis was below the normal level at the beginning. However, the capacity of resistance of the bones in the experimental groups was remarkably increased after 3 months. The value of the Pi-HLC-Ca group was the highest and was closest to normal, which illustrates that PiHLC-Ca is the favourable Ca supplement for mice with osteoporosis. The schematic diagram of the mechanical test analysis is shown in Fig. 7. The fractures are largely due to calcium deficiency. While the compressive capacity of the control group changed, it did not affect the accuracy of the results. The resistance capacity of each group was improved by 43.2%, 22.7%, 10.9%, and 26.1% compared with the control group at 3 months (Fig. 6D). These results showed that the resistance
1. Pi-HLC-Ca has very good biological activity. The rate of releasing calcium into plasma is similar to that of blood calcium converted into bone calcium. Pi-HLC-Ca is safe and effective in supplying calcium to organisms. This approach solves the problem of the poor biological activity of traditional calcium supplements. 2. Pi-HLC-Ca contains collagen and phosphate, both of which are important components of bone. Pi-HLC-Ca provides supplemental collagen and phosphate while also supplementing calcium. This outcome is the primary innovation described in this article. 3. Because of the unique molecular structure of Pi-HLC-Ca and the unique binding site of calcium on collagen, calcium cannot be 636
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Fig. 6. The ultimate load on the femurs from osteoporosis mice that were supplemented with calcium for twelve weeks. (A) Serum ALP value of mice (B) Serum calcium value (C) BHP value of mice (D) ultimate load (N) of mice femora at the 12th week. Data are presented as the mean ± SD of 10 animals (p < 0.05).
Fig. 7. Three bending tests of mice femurs. Left: universal testing machine; Right: The femur in the testing machine; Down: the ultimate load on the femurs from mice.
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Fig. 8. SEM photographs of the tibia from osteoporosis mice that were supplemented with calcium for twelve weeks.
randomly combined with substances in the body to produce insoluble matter, thus avoiding excessive calcium supplementation and causing damage to the body.
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4. Conclusions Cell viability was used to evaluate the toxicity of HLC-Ca to show that it is safe enough to be a calcium supplement. HLC-Ca also had an outstanding contribution in increasing body weight. Based on the analysis of blood calcium, ALP and BHP, we drew the conclusion that Pi-HLC-Ca accelerated the absorption of calcium in the body. The compressive capacity of bones from mice with osteoporosis was enhanced after treatment, showing that the calcium deficiency situation improved after supplementation with calcium for 3 months. The BMD of the Pi-HLC-Ca group was obviously higher than that of the other groups, which means that Pi-HLC-Ca was good at improving bone mass. Both the SEM photographs of the tibia and the bone mineral density of the femora indicated that mice with osteoporosis had improved bone mass after Pi-HLC-Ca supplementation. Pi-HLC-Ca, calcium covalently linked to HLC, had an excellent effect on osteoporosis mice in regards to providing calcium for bone formation. In conclusion, it is possible that osteoporosis could be prevented or delayed with oral Pi-HLC-Ca. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (21576222, 21476184); Shaanxi Key Laboratory of Degradable Biomedical Materials Program (2014SZS07K04, 2014SZS07-P05, 15JS106, 2014SZS07-Z01, 2014SZS07-Z02, 2016SZSj-35, 2014SZS07-K03); Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program (2015HBGC-04). References [1] R.S. Adluri, L. Zhan, M. Bagchi, Comparative effects of a novel plant-based calcium supplement with two common calcium salts on proliferation and mineralization in human osteoblast cells, Mol. Cell. Biochem. 340 (2010) 73–80. [2] K.F. Moseley, D.A. Dobrosielski, K.J. Stewart, Lean mass and fat mass predict bone mineral density in middle–aged individuals with noninsulin–requiring type 2 diabetes mellitus, Clin. Endocrinol. 74 (2011) 565–571. [3] I.R. Reid, M.J. Bolland, A. Grey, Effect of calcium supplementation on hip fractures, Osteoporos. Int. 19 (2008) 1119–1123. [4] I.R. Reid, B. Mason, A. Horne, Randomized controlled trial of calcium in healthy older women, Am. J. Med. 119 (2009) 777–785. [5] S. Andersen, P. Laurberg, Age discrimination in osteoporosis screening-data from the Aalborg University Hospital Record for Osteoporosis Risk Assessment, Maturitas
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[27] S.W. Tsai, F.Y. Hsu, P.L. Chen, Beads of collagen-nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells, Acta Biomater. 4 (2008) 1332–1341. [28] N. Yahara, I. Iofani, K. Maki, Mechanical assessment of effects of grape seed proanthocyanidins extract on tibial bone diaphysis in rats, J. Musculoskelet. Neuron. 5 (2005) 162–169. [29] M. Shah, V. Simha, A. Garg, Long-term impact of bariatric surgery on body weight, comorbidities, and nutritional status, J. Clin. Endocrinol. Metab. 91 (2006) 11. [30] J. Xiong, M. Piemontese, J.D. Thostenson, Osteocyte-derived RANKL is a critical mediator of the increased bone resorption caused by dietary calcium deficiency, Bone V66146 (2014) 54. [31] Y. Zhuo, J.Y. Gauthier, W.C. Black, Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible, Bone V67269 (2014) 80. [32] A. Wang, X. Ding, S. Sheng, Retinoic acid inhibits osteogenic differentiation of rat bone marrow stromal cells, Biochem. Biophys. Res. Commun. V375 (3) (2008) 435–439.
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
The effect of human-like collagen calcium complex on osteoporosis mice a,b
a,b
Chenhui Zhu , Huan Lei , Shangshang Wang ⁎ Jianjun Denga,b, Daidi Fana,b, , Xingqiang Lva a b
a,b
, Zhiguang Duan
a,b
a,b
, Rongzhan Fu
T
,
Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi'an 710069, China Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an 710069, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Calcium supplementation Human-like collagen Osteoporosis Bone mineral density
The objective of this study was to assess the effect of a modified human-like collagen calcium complex on osteoporosis mice. BHK (Baby Hamster Kidney) cells were used to compare the cytotoxicity of different calcium reagents with the MTT test. Six-week-old male mice (n = 80) were randomly divided into eight groups: a blank group (blank), control group (control), human-like collagen calcium group (HLC-Ca), thiolated human-like collagen calcium group (SH-HLC-Ca), phosphorylated human-like collagen calcium group (Pi-HLC-Ca), gluconate group (Glc-Ca), calcium carbonate group (CaCO3) and D-cal group (B). A systematic analysis of the results available in vivo after 3 months of treatment was presented. The effects of several Ca supplements on osteoporosis mice were investigated by detecting serum calcium, alkaline phosphate activity (ALP), bone hydroxyproline (BHP) and bone mineral density (BMD). The results proved that the BMD and BHP of osteoporosis mice were significantly increased in the Pi-HLC-Ca group, while serum calcium and ALP were decreased. Therefore, Pi-HLC-Ca is likely a good calcium supplement for clinical applications. In this review, the advantage of Pi-HLCCa in preventing and delaying osteoporosis is highlighted. In addition to the current progress, further investigations are necessary to reveal the relative influences of collagen and calcium proportions on the long-term clinical effects of osteoporosis.
1. Introduction Calcium (Ca) plays a vital role in the life of human beings, as one of the most important minerals in the human body. However, calcium deficiency is also very common and can lead to osteoporosis and osteopenia, which are always related to calcium deficiency [1,2]. Calcium reagents can be used to restore the balance between resorption and formation [3]. In addition, calcium supplementation is regarded as a safe monotherapy method for osteoporosis [4]. However, even longterm supplementation with calcium only postpones bone loss and provides no favourable effect on the preservation of bone mineral density [5,6]. Moreover, collagen, the most abundant protein, plays an important role in the body. Collagen is not only an important part of bone tissue but also an essential part of bone health; for instance, type I collagen is a primary organic component that makes up the extracellular matrix of bone [7–9]. Collagen, a biocompatible material, is widely used in interventions due to its low risk of immunological reactions [10]. Human-
like collagen (HLC), which is prepared in our laboratory to be adopted as a biological material, is also widely used, including as bone materials and hydrogels that show good compatibility and that stimulate no immunological effect [11–14]. It is also known that collagen or hydrolysates in vivo can generate peptides that are useful for organic biosynthesis [15,16]. It has been shown in some studies that oral administration of hydrolysed collagen may improve bone mass content and density in rats [17]. In general, unexpected results may be achieved through combining human-like collagen with calcium. Bone, a dynamic organization, is constantly updating and remodelling to maintain its integrity [18–20]. In other words, newly generated bone is constantly replacing the old bone and is accommodating the mechanical load changes [21]. Once osteoclasts are attached to the bone surface, protons and proteolytic enzymes are secreted, which may dissolve the bone mineral and hydrolyse the matrix protein. Then, osteoblasts replace the osteoclasts to capture protons and protein to form calcium and phosphate deposits [22]. The specific process is shown in Fig. 1. In this process, the old bones
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Corresponding author at: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Xi'an 710069, China. E-mail addresses: [email protected] (C. Zhu), [email protected] (R. Fu), [email protected] (J. Deng), [email protected] (D. Fan), [email protected] (X. Lv). https://doi.org/10.1016/j.msec.2018.08.011 Received 8 January 2018; Received in revised form 10 July 2018; Accepted 5 August 2018 Available online 06 August 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Calcium supplementation could reduce parathyroid hormone (PTH) levels, resulting in diminished RANKL (receptor activator for nuclear factor-κB ligand) expression by osteoblasts. The content of cathepsin K (CatK) and protons that the osteoclasts captured on the bone surface were reduced. Thus, the matrix proteins (principally the type I collagen) and bone mineral were protected.
water bath, the Pi-HLC was prepared and then freeze-dried. The freezedried Pi-HLC was dissolved in a saturated KI solution at 55 °C; then, freeze-dried Pi-HLC was dissolved in MOPS buffer (10 μM, pH 7.5), and CaCl2 solution was added into the Pi-HLC solution (n (Pi-HLC): n (CaCl2) = 1:600), followed by gentle stirring at room temperature for 1–1.5 h. Finally, the reaction mixture was purified and freeze-dried. Preparation of SH-HLC-Ca: SH-HLC was prepared by adding SAMSA to HLC solutions. Then, freeze-dried SH-HLC was dissolved in ultrapure water at a concentration of 10 mg/mL, and CaCl2 solution was added into SH-HLC solution (n (SH-HLC): n (CaCl2) = 1:1000), followed by gentle stirring at room temperature for 1 h. Finally, the reaction mixture was purified and freeze-dried.
are updated and replaced to maintain skeletal balance. However, in elderly persons, the balance between formation and resorption shifts towards resorption. If the unbalanced state continues, in the long-term, calcium, bone mineral density and matrix protein will decrease, leading to osteoporosis. Osteoporosis, a common disease, is characterized by low bone mineral density and an increase in the risk of fractures of the hip, spine, and forearm [5,23]. Age is a neglected risk factor for osteoporosis. Osteoporosis may be prevented or delayed by maximizing peak bone mass during adolescence. The aim of this study was to evaluate the effect of the human-like collagen calcium complex (prepared by our laboratory) on osteoporosis mice, which are produced by oral overdoses of retinoic acid [24,25]. The primary endpoints of the study were to alleviate mouse bone disorders, such as low BMD and high serum calcium. Future studies may suggest the importance of clinical examinations as well as a way to prevent or delay osteoporosis. People suffering from calcium deficiency and osteoporosis will be treated, or the condition will be prevented, to alleviate the burden on patients and society.
2.3. Cell culture and cell viability BHK (Baby Hamster Kidney) cells (CBCAS, Shanghai, China) were cultured in RPMI 1640 medium. The cytotoxicities of HLC, HLC-Ca, SHHLC-Ca and Pi-HLC-Ca were assessed using the BHK cells. The samples were dissolved in cell culture at 20 mM (Ca2+) and sterilized with a 0.22 μm filter. The cells were cultured in a CO2 (5%) incubator at 37 °C on 96-well plates with 200 μL per well (1.0 × 104 cells·mL−1) for 24 h and then were cultured with the sample solution for 24 h, 36 h and 48 h. At the end of the incubation, 20 μL MTT (0.5%) was added to each well for another 4 h. The medium was removed, and DMSO (100 μL) was added to each well. Finally, the absorbance was measured at 490 nm. The relative cell growth (%) was calculated as follows:
2. Materials and methods 2.1. Materials Human-like collagen (HLC, China patent number: ZL01106757, Mr = 97,000) was supplied by Xi'an Giant Biogene Technology company. HLC-Ca, SH-HLC-Ca and Pi-HLC-Ca were prepared by our laboratory. Glucose calcium (Glc-Ca) was purchased from Sanchine. CaCO3 and D-cal (B) were purchased from Anshi Pharmaceutical Inc. All the reactants and solvents used were of analytical grade, and double-distilled water was used throughout the experiments.
Relative cell growth (%) = ODtest / ODcontrol × 100%
(1)
where ODtest means the absorbance value of the test sample and ODcontrol means the absorbance value of the blank sample. All tests were repeated three times, and we reported the average value of five parallel samples.
2.2. Preparation of HLC-Ca, Pi-HLC-Ca and SH-HLC-Ca
2.4. Cell fluorescence staining test
Preparation of HLC-Ca: Freeze-dried HLC was dissolved in ultrapure water at a concentration of 10 mg/mL, the pH was adjusted to 7.5, and CaCl2 solution was added into the HLC solution (n (HLC): n (CaCl2) = 1:1000), followed by gentle stirring at room temperature for 1–1.5 h. Finally, the reaction mixture was purified using dialysis and freeze-dried in a vacuum freeze drier (FD5-10, SIM, US). Preparation of Pi-HLC-Ca: Then, the freeze-dried HLC was dissolved in ultrapure water at 10 mg/mL, and G-6-P-Na2 (10 mg) was added to the HLC solution (pH 8.0–8.5), After incubating for 2–4 h in a 50 °C
A cell fluorescence staining technique was used to observe apoptosis in the cells. DAPI (2-(4-amidinophenyl)-6-indole carbinol dihydrochloride) is a kind of blue fluorescence dye that can penetrate the cell membrane and bind to DNA; then, the cells exhibit a green colour when excited in blue light. BHK cells were seeded at a density of 1.0 × 103 cells/cm2 on 48well plates and were cultured in RPMI 1640 medium in a CO2 (5%)
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conducted with 5 μL serum, and the calculation formula was as follows:
incubator at 37 °C for 24 h. The experimental groups were cultured with the HLC, HLC-Ca, SH-HLC-Ca or Pi-HLC-Ca samples, with a Ca2+ concentration of 10 μM (n = 6 per group). The control group was cultured in RPMI 1640 medium (n = 6). After 24 h, we discarded the medium and added 10 mL of 5 μg/mL DAPI into each well for 15–20 min. Finally, the reaction solution was discarded, and the cells were observed under an inverted microscope.
Serum ALP activity (king unit/100mL) = (ODtest − OD blank )/(ODstandard − OD blank ) × (0.1mg/mL) × 100mL×sample dilution ratio
(2) 2.9. Serum calcium
2.5. Animals and treatment
In an alkaline solution, serum calcium ion can be combined with methyl thymol, forming a blue complex (MTB). Colorimetric assays with standard calcium can then be used to determine the serum calcium content. We used 50 μL serum to test for calcium content. The test was performed using a calcium assay kit (Nanjing Jian Cheng Bioengineering Institute). The formula was as follows:
Male mice (n = 80), 6 weeks old and weighing approximately 20 g, were randomly divided into 8 groups of 10 animals each. The mice were housed in small cages individually under similar conditions with a 12-hour light-dark cycle at 22 ± 1 °C. All mice had free access to food and water. Body weights were recorded each week. In the blank group, the mice were fed the standard diet and given tap water freely throughout the procedure. In the control and experimental groups, mice were fed the standard diet along with a gavage of vitamin A acid 70 mg/kg each and were given tap water freely for 2 weeks. Then, the experimental groups were given by gavage the indicated amounts of HLC-Ca, SH-HLC-Ca, Pi-HLC-Ca, Glc-Ca, CaCO3 and D-cal over the 3month feeding period. At the end of the period, they were killed with thiopental sodium under deep anaesthesia. All procedures were approved by the Committee for the Use of Laboratory Animals of Northwest University, China.
Serum ALP activity (king unit/100mL) = (ODtest − OD blank )/(ODstandard − OD blank ) × standard concentration (2.5mmol/L)
(3) 2.10. BHP Right femurs were measured, cut into pieces and then treated with 1 mL 6 mol/L hydrochloric acid in a water bath for 5 h. Then, we adjusted the solution's pH to 6.0–6.8 and diluted the solution to 10 mL. We then added 20–30 mg of active carbon to the 4 mL mixture solution and centrifuged it at 3500 rpm for 10 min. The supernatant was collected, and we used 1 mL to detect the BHP content. The test was performed using a hydroxyproline assay kit (Nanjing Jian Cheng Bioengineering Institute). The calculation formula is as follows:
2.6. Body weight We weighed and recorded the mouse body weight once a week. 2.7. Blood and bone sampling
BHP (ug/g) = (ODtest − OD blank )/(ODstandard − OD blank )×standard contest × hydrolysate volume/sample wet weight
At the end of the treatment period, blood samples were collected from the abdominal aorta under ether anaesthesia and centrifuged at 3000 rpm for 10 min; the serum was stored at −70 °C until measurement of the parameters. The mice were sacrificed humanely, and the hind legs were dissected to eliminate all soft tissues. The femur and tibia were stored at −70 °C until use.
(4) 2.11. Bone mineral density Bone mineral density of the right femur was measured by dual energy X-ray absorptiometry (DXA) using a DSC-600EX-IIR bone densitometer (Aloka Co., Ltd., Tokyo, Japan) at the 10th week.
2.8. Serum alkaline phosphate activity During the early stages of osteoblast differentiation, alkaline phosphatase (ALP) expression is activated, and its activity has been traditionally regarded as a marker to determine the degree of osteoblast differentiation [26,27]. A rise in serum ALP activity is significantly elevated in diseases of the skeletal system, such as osteomalacia, fractures and rickets. The tests were performed using an alkaline phosphatase assay kit (Nanjing Jian Cheng Bioengineering Institute). The chromogenic substrate solution was dissolved in 2.5 mL of ALP reaction buffer and placed on ice. Then, 10 μL of the p-nitrophenol standard solution (10 mM) was diluted in 0.2 mL of the ALP reaction buffer so that the final concentration was 0.5 mM. The tissue was lysed, homogenized appropriately, and then centrifuged to obtain the supernatant for detection of ALP activity. Note: The lysate did not contain phosphatase inhibitors. The tissue samples were frozen at −80 °C until further use. In the test, blank control wells, standard wells, and sample wells were prepared. The amount of standard used was 4, 8, 16, 24, 32, and 40 μL, and the sample could usually be added directly to bring the total to 50 μL. If the alkaline phosphatase activity in the sample was too high, we reduced the amount of sample or made an appropriate dilution before performing the assay. After incubation at 37 °C for 5–10 min, 100 μL reaction stopper solution was added to each well to terminate the reaction. Absorbance was measured at 405 nm. Tests were
2.12. Mechanical test Mechanical testing was performed using a mechanical testing machine (Instron, Testing machine Co., model MZ-3365, USA). For the three-point bending test of the mid femur, the left femur was placed on a holding device with supports located 12 mm apart. The upper loading device was aligned to the centre of the femoral shaft on the anterior side to give an accurate span [28]. The three-point bending test was performed at a constant loading rate of 20 mm/min and a load cell of 50 kgf to measure the stiffness (N/cm). Ultimate load (N) and stiffness (N/cm) were calculated from the load-displacement curve. 2.13. SEM The left tibias of the mice were placed in a muffle furnace, burning at 800 °C for 6 h, to determine the ash composition. After they had cooled naturally, each tibia was cut into cross sections [29]. The surface morphologies of the tibia slices were examined by SEM (Hitachi S-4800, Japan) at 20 kV. We placed the tibia ash cross section into liquid nitrogen for approximately 10 min. The sections were fixed on conductive aluminium and were sputter coated with gold before scanning.
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spherical. The HLC group included more viable cells but few dead cells. The HLC-Ca group exhibited the lowest number of cells but with more dead ones, while the SH-HLC-Ca and Pi-HLC-Ca groups were similar to the HLC group; however, the numbers of dead cells were obviously increased relative to that in the HLC group. These results suggest that HLC, HLC-Ca, Pi-HLC-Ca and SH-HLC-Ca have no effect on BHK cell morphology. The dead cells were caused by nutrient limitations. Therefore, it can be concluded that HLC, HLC-Ca, SH-HLC-Ca and Pi-HLC-Ca are not only safe but can also stimulate the normal growth of cells, indicating that they are safe products for oral Ca supplementation. 3.3. Animal experiment After 2 weeks, the average serum calcium of mice fed with retinoic acid (1.75 mmol/L) was dramatically decreased in comparison with the control (2.5 mmol/L). The average ALP and bone hydroxyproline level of mice fed normal basic feedstuff were all within normal limits during the experimental period; however, in the mice gavaged with retinoic acid, they were both markedly lower than that of the control after 2 weeks. All of the above data showed that the construction of the osteoporosis mouse model was successful (Fig. 4). As shown in Fig. 4, PTH stimulates OB to synthesize RANKL, while RANKL can activate OC to synthesize H+, K and other destructive factors. These destructive factors act on bone [30], mainly K acting on bone matrix protein [31], and they decompose the bone matrix proteins so that mineral ions cannot be deposited on the bone surface. These mineral ions are mainly calcium and phosphorus. The calcium ions in the mineralized layer decrease, and the bone density also decreases due to the loss of calcium phosphate and collagen, leading to osteoporosis. Therefore, osteoporosis is mainly due to the process of bone resorption. The bone formation process is the reverse process of the bone resorption process described above; the bone matrix is synthesized using the collagen around the bone tissue, and then the mineral ions around the bone tissue are deposited on the bone matrix. Under the influence of HLC-Ca, increasing the calcium ion content promotes the reduction of PTH bound to the OB surface, which results in a decrease in the stimulation of OB by PTH, a decrease in the level of RANKL, a decrease in the synthesis and secretion of OC, a decrease in bone resorption, and thus no effect on the bone formation process. The formation process is longer than the process of bone resorption, and it effectively promotes the formation of mineralized layers of bone matrix proteins combined with calcium ions and the like so that the bone density increases. At the same time, an increase in calcium levels can also increase the amount of calcitonin, and calcitonin can also inhibit OC so that the destruction induced by H+, K and other destructive factors is reduced; thus, the bone formation process is longer than the process of bone resorption. For patients with osteoporosis, the bone formation process needs to be at a premium to maintain the bone at a healthy level. We first needed to establish a model of osteoporosis and then add the compounds to be studied to observe their effects on calcium supplementation. Many experimental studies [32] have shown that during oral administration of excessive amounts of retinoic acid, retinoic acid inhibits the activity of osteoblasts and promotes the formation of osteoclasts to stimulate the reabsorption of bone and to inhibit bone formation. This approach causes bone loss, decreased bone density, and osteoporotic fractures. We used normal mice as experimental subjects and fed the mice vitamin A acid according to a previously established experimental method, developing a model of osteoporosis. The results of the serum calcium test are shown in Fig. 4A. The serum calcium in the blank mice ranged from 1.7 mmol/L to 1.8 mmol/ L, while the serum calcium in the control mice ranged from 2.45 mmol/ L to 2.55 mmol/L. A comparison of the experimental results of the two groups showed that the serum calcium values of mice fed with retinoic acid significantly increased. The reason for this result is that retinoic acid inhibits osteoblasts when retinoic acid is used for an extended
Fig. 2. Cell viability of BHK cells with different treatments: the BHK cell viability after culturing with HLC, HLC-Ca, SH-HLC-Ca and Pi-HLC-Ca for 24 h, 36 h, and 48 h (p < 0.05). Data are presented as the mean ± SD of 6 parallel wells.
2.14. Statistical analysis The SPSS software package was used for statistical analyses. The values are shown as the means and standard deviations. The significance was analysed with a t-test, where *p < 0.05 was considered to be significant. **p < 0.01 was considered to be highly significant. 3. Results and discussion 3.1. Cell viability As we can see clearly in Fig. 2, the values of cell viability for the cells cultured with different mediums separately for 24 h, 36 h and 48 h were all higher than 100%. According to the USP toxicity classification (Table 1), the samples used for the experiment were therefore safe enough to be used as calcium supplementation reagents. We were pleased to note that the experimental groups exhibited varying degrees of growth promoting effects on BHK cells. The groups had a common growth trend with increasing incubation time, leading to increasing cell viability. The viability of cells cultured with HLC-Ca, Pi-HLC-Ca and SH-HLCCa were lower than that in the HLC group. It is possible that the HLC molecules entered the cells through a cell swallowing function or underwent enzymatic hydrolysis into small molecules that were then absorbed by the cells. The cell viability of the HLC-Ca, Pi-HLC-Ca and SHHLC-Ca groups showed a range of 105%–115%, while the HLC group had an obviously improved cell viability range of 115%–120%, which was higher than the others. Therefore, from the above analysis, all the samples were safe enough to be used as calcium reagents for application. 3.2. Cell fluorescence staining test The DAPI fluorescent staining results are shown in Fig. 3. It was observed that the viable cells were fusiform, and the dead ones were Table 1 The relationship between relative growth rate and cytotoxicity reaction gradation. RGR (%)
≥100
75–99
50–74
25–49
1–24
0
Grade
0
1
2
3
4
5
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Fig. 3. A photograph of BHK cells after staining with DAPI. HLC: HLC culture, HLC-Ca: HLC-Ca culture; SH-HLC-Ca: SH-HLC-Ca culture; Pi-HLC-Ca: Pi-HLC-Ca culture.
Fig. 4. The induction of osteoporosis in mice after feeding with retinoic acid for 2 weeks. Bone formation is a dynamic balancing process. OB: osteoblast, OC: osteoclast, PTH: parathyroid hormone, RANKL: receptor activator of nuclear factor kappa B ligand, K: proteolytic enzyme K, Form: bone formation, resorption: bone resorption. (A) The serum calcium (B) the serum ALP (C) the content of bone hydroxyproline.
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gained by the mice, and their weight rapidly dropped in the seventh week due to being long-term calcium deficient with no timely calcium supplementation [34]. The six experimental groups were compared with the blank group and the control group. The weight of the group fed with SH-HLC-Ca steadily increased, eventually by 8 g, but was smaller to that in the PiHLC-Ca group (10 g). The increased weight of the HLC-Ca group was approximately 5 g, and the weight change in the GLC-Ca group, the CaCO3 group and the B group was not significant. The main reason for the above phenomenon was that the six kinds of reagents provided the body with calcium sources that provided a balance of nutrition to maintain body weight. Different weight recoveries reflect the degree of calcium absorption in the body due to the important regulatory functions of calcium. Pi-HLC-Ca contained rich collagen, calcium and phosphorus to provide necessary nutrients. HLC not only had a trophic function but also promoted calcium absorption, but Pi-HLC-Ca had an obvious advantage in maintaining body weight. The difference in body weight between the B and Glc-Ca groups was very small, which means that the weight maintenance function of B and Glc-Ca was basically the same. These findings also revealed that Pi-HLC-Ca was a nutrient-rich calcium reagent and was better than the other three calcium reagents.
period of time. When osteoblasts are inhibited, osteoclasts predominate. The effect of osteoclasts leads to increased calcium loss from the bones that then moves into the blood, causing a significant increase in serum calcium. The increase in bone calcium in the model group also confirmed the successful establishment of the osteoporosis model. The serum alkaline phosphatase test results are shown in Fig. 4B. In the blank group, the serum alkaline phosphatase value was 0.16–0.18 U/L. In the experimental group, the serum alkaline phosphatase value was 0.27–0.29 U/L. Compared with the blank control group, the serum ALP value in the experimental group increased significantly. This result is because the serum content of alkaline phosphatase is closely related to the state of bone. When bone lesions form, alkaline phosphatase is released into the blood, and the content of alkaline phosphatase increases. The increase in alkaline phosphatase indicates that the mouse model of osteoporosis was successfully established [33]. Since serum ALP value is used as a criterion for judging skeletal diseases in clinical practice, the increase in ALP values also indicates the presence of osteoblasts. Compensatory hyperplasia results in the secretion of large amounts of alkaline phosphatase into the blood. The results of the bone hydroxyproline tests are shown in Fig. 4C. There was a significant difference between the blank group and the experimental group. The content of hydroxyproline in the blank group was 1.0–1.1 μg/mg compared with 0.45–0.55 μg/mg in the experimental group. From these results, the bone hydroxyproline content of the experimental mice was reduced by 40% to 50% compared to the blank group. Approximately 90% of the organic components in bone are type I collagen, and the special amino acid in type I collagen is hydroxyproline. The content of bone hydroxyproline can indirectly reflect the content of type I collagen in bone tissue and can indicate the state of health of the bone. In the experimental group, the content of bone hydroxyproline was profoundly decreased, which demonstrated the loss of type I collagen in the bone tissue, which is a key symptom of osteoporosis. Although the mechanism whereby retinoic acid affects bone development and metabolism is not yet clear, in the case of retinoic acid excess, bone resorption is higher than bone formation, eventually leading to osteocalcin dissolution under bone resorption and resulting in an elevated serum calcium. Alkaline phosphatase content is a common method used for clinical evaluation of skeletal diseases. When alkaline phosphatase in the serum is elevated, this is mainly due to bone and liver lesions resulting in the release of alkaline phosphatase into the serum. Based on the results of the serum calcium, serum alkaline phosphatase and bone hydroxyproline measured above, as well as the response of these results to the treatment of the mice in the experimental group, we concluded that the osteoporosis mouse model had been established. The reduction in bone hydroxyproline represents the loss of collagen. The loss of collagen protein results in a decrease in the amount of matrix proteins. The matrix proteins are also deposition sites for mineral elements such as calcium and phosphorus. If calcium phosphate cannot be deposited on the matrix proteins, it will enter the blood, causing the blood calcium concentration to increase. Establishing a model of osteoporosis is a very important process in this experiment, and it is an important means to determine the calciumsupplementing effect of human-like collagen calcium complexes.
3.5. ALP ALP is an enzyme widely distributed in tissues and fluids of the body and plays an essential role in regulating tissue mineralization. Only bone lesions, such as osteoporosis and rickets, release ALP into the bloodstream, leading to elevated levels of ALP in the blood [35]. As shown in Fig. 6A, the value of ALP in the control group, with a range of 0.16–0.17 U/L, was much higher than that of the blank group, with a range of 0.27–0.28 U/L. We presume that the higher value of ALP is due to osteoporosis, which was caused by damage to the bone cells by the excessive level of retinoic acid. Additionally, it can be found that the values of ALP in the Pi-HLC-Ca, SH-HLC-Ca, Glc-Ca, HLC-Ca, CaCO3 and B groups were much smaller than those in the control group, especially in the SH-HLC-Ca (0.19–0.21 U/L) and Pi-HLC-Ca groups (0.18–0.19 U/ L). In view of the above results and analysis, we can clearly point that Pi-HLC-Ca is a good calcium source for osteoporosis, along with calcium deposition, it enabled the bone formation process. It had an evident advantage over the other treatments, which means it may have a good effect on the treatment or prevention of osteoporosis.
3.6. Serum calcium Insufficient calcium intake and absorption causes the parathyroid hormone to increase, followed by the release of calcium deposited in bone into the blood [36]. As is known, calcium levels in the blood must be kept within a narrow range to be considered normal and functional. There is a close relationship between high blood calcium and bone health. During osteoporosis, bone resorption is increased, and bone formation is decreased, leading to high blood calcium in the control group (Fig. 6B), illustrating this point. We found that the six experimental groups and the control group all had a significantly lower serum calcium than the Ca-deficiency group, ranging from 2.35–2.45 mmol/L, combined with the values of serum calcium from the different experimental groups. We concluded that calcium supplements can reduce blood calcium levels in osteoporosis mice. The procedure of bone formation was enhanced with the accelerated calcium absorption, which resulted in the decrease in values of serum calcium in the six groups in the 12th week to varying degrees. Furthermore, the serum calcium level in the Pi-HLC-Ca group was the closest to the control. Therefore, we can conclude that Pi-HLC-Ca could not only accelerate calcium absorption but could also provide nutrition to the body.
3.4. Effects of several Ca-supplements on recovery of osteoporosis mice During the experiment, we recorded weight changes in each group of mice once a week. After 10 weeks of treatment, all the groups had significantly higher body weights than their initial body weights. The final body weights were different among the groups, especially the control group, which had a significantly lower body weight compared with the other groups (Fig. 5). The body weight of mice in the blank group showed a steady increasing trend. The trend in body weight in the control group mice was different; in the fourth week, no weight was 635
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Fig. 5. The body weight of each group throughout the experiment is presented as the mean ± SD of 10 animals (p < 0.05).
capacity of the femora was enhanced due to calcium accumulation in the bone. Through the above data analysis, the Pi-HLC-Ca group had an obvious advantage in reducing the risk of fracture. SEM was used for further analysis to confirm this conclusion.
3.7. BHP BHP is a characteristic of type I collagen, which is the primary organic component that makes up the matrix of bone. Collagen (mainly type I collagen) accounts for 90% of the bone matrix, and BHP accounts for 12.5% of the collagen [37]. The content of BHP in bone is an important indicator to evaluate the health of the bone matrix. The specificity of BHP makes it an important amino acid to judge the amount of organic material in bone. The contents of BHP in each group are shown in Fig. 6C. The control group had a lower content of BHP than did the blank group in the 12th week. These results show that the bone mass of mice with osteoporosis could not be increased without supplemental calcium reagents and instead got worse. Although the content of BHP in the six experimental groups increased, there were obvious differences, namely, SH-HLC-Ca, Pi-HLC-Ca and B had advantages in promoting increases in bone mass. The content of BHP in mice with osteoporosis increased by almost 60% after supplementing with SH-HLC-Ca or PiHLC-Ca for 12 weeks, and this high content of BHP confirmed that both SH-HLC-Ca and Pi-HLC-Ca had more of a positive impact on bone formation compared with the others.
3.9. SEM and bone mineral density The SEM micrographs of the tibia slices are shown in Fig. 8. To a certain extent, tibia cross-sections reflect the value of bone mineral density. Approximately 70% of bone composition is calcium and phosphorus minerals, and 30% is organic matter, mainly collagen I [35]. The pores generated in the tibia slice surfaces are present because they were subjected to very high temperatures and because only minerals were retained. Normal mouse images showed that the tibia pore diameters were significantly < 20 μm (Fig. 8 Blank), while the osteoporosis mice had pore diameters up to 80 μm (Fig. 8 Control). The pore diameter of the HLC-Ca group was close to the blank group as shown in Fig. 8 HLC-Ca. A smaller pore size and fewer holes means a higher bone mineral density. We were able to confirm that Pi-HLC-Ca had an excellent calcium supplementation effect on osteoporosis mice through these experiments. Their bone mineral density was improved to a high extent, which was also verified in Fig. 8. BMD is an important parameter to measure bone mass. The BMD of the Pi-HLC-Ca group was obviously higher than that of the other groups, which means that PiHLC-Ca was good at increasing bone mass. Pi-HLC-Ca has the best calcium effect, which may be due to:
3.8. Mechanical test analysis Calcium supplementation can effectively reduce the risk of fractures [35,38]. The femora of mice with osteoporosis were used to test their ultimate load, and these test results are shown in Fig. 6D. The test outcome shows that the capacity of resistance of the mice with osteoporosis was below the normal level at the beginning. However, the capacity of resistance of the bones in the experimental groups was remarkably increased after 3 months. The value of the Pi-HLC-Ca group was the highest and was closest to normal, which illustrates that PiHLC-Ca is the favourable Ca supplement for mice with osteoporosis. The schematic diagram of the mechanical test analysis is shown in Fig. 7. The fractures are largely due to calcium deficiency. While the compressive capacity of the control group changed, it did not affect the accuracy of the results. The resistance capacity of each group was improved by 43.2%, 22.7%, 10.9%, and 26.1% compared with the control group at 3 months (Fig. 6D). These results showed that the resistance
1. Pi-HLC-Ca has very good biological activity. The rate of releasing calcium into plasma is similar to that of blood calcium converted into bone calcium. Pi-HLC-Ca is safe and effective in supplying calcium to organisms. This approach solves the problem of the poor biological activity of traditional calcium supplements. 2. Pi-HLC-Ca contains collagen and phosphate, both of which are important components of bone. Pi-HLC-Ca provides supplemental collagen and phosphate while also supplementing calcium. This outcome is the primary innovation described in this article. 3. Because of the unique molecular structure of Pi-HLC-Ca and the unique binding site of calcium on collagen, calcium cannot be 636
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Fig. 6. The ultimate load on the femurs from osteoporosis mice that were supplemented with calcium for twelve weeks. (A) Serum ALP value of mice (B) Serum calcium value (C) BHP value of mice (D) ultimate load (N) of mice femora at the 12th week. Data are presented as the mean ± SD of 10 animals (p < 0.05).
Fig. 7. Three bending tests of mice femurs. Left: universal testing machine; Right: The femur in the testing machine; Down: the ultimate load on the femurs from mice.
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Fig. 8. SEM photographs of the tibia from osteoporosis mice that were supplemented with calcium for twelve weeks.
randomly combined with substances in the body to produce insoluble matter, thus avoiding excessive calcium supplementation and causing damage to the body.
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4. Conclusions Cell viability was used to evaluate the toxicity of HLC-Ca to show that it is safe enough to be a calcium supplement. HLC-Ca also had an outstanding contribution in increasing body weight. Based on the analysis of blood calcium, ALP and BHP, we drew the conclusion that Pi-HLC-Ca accelerated the absorption of calcium in the body. The compressive capacity of bones from mice with osteoporosis was enhanced after treatment, showing that the calcium deficiency situation improved after supplementation with calcium for 3 months. The BMD of the Pi-HLC-Ca group was obviously higher than that of the other groups, which means that Pi-HLC-Ca was good at improving bone mass. Both the SEM photographs of the tibia and the bone mineral density of the femora indicated that mice with osteoporosis had improved bone mass after Pi-HLC-Ca supplementation. Pi-HLC-Ca, calcium covalently linked to HLC, had an excellent effect on osteoporosis mice in regards to providing calcium for bone formation. In conclusion, it is possible that osteoporosis could be prevented or delayed with oral Pi-HLC-Ca. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (21576222, 21476184); Shaanxi Key Laboratory of Degradable Biomedical Materials Program (2014SZS07K04, 2014SZS07-P05, 15JS106, 2014SZS07-Z01, 2014SZS07-Z02, 2016SZSj-35, 2014SZS07-K03); Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program (2015HBGC-04). References [1] R.S. Adluri, L. Zhan, M. Bagchi, Comparative effects of a novel plant-based calcium supplement with two common calcium salts on proliferation and mineralization in human osteoblast cells, Mol. Cell. Biochem. 340 (2010) 73–80. [2] K.F. Moseley, D.A. Dobrosielski, K.J. Stewart, Lean mass and fat mass predict bone mineral density in middle–aged individuals with noninsulin–requiring type 2 diabetes mellitus, Clin. Endocrinol. 74 (2011) 565–571. [3] I.R. Reid, M.J. Bolland, A. Grey, Effect of calcium supplementation on hip fractures, Osteoporos. Int. 19 (2008) 1119–1123. [4] I.R. Reid, B. Mason, A. Horne, Randomized controlled trial of calcium in healthy older women, Am. J. Med. 119 (2009) 777–785. [5] S. Andersen, P. Laurberg, Age discrimination in osteoporosis screening-data from the Aalborg University Hospital Record for Osteoporosis Risk Assessment, Maturitas
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