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Effect of Magnesium of The Bone Strength of Rat Bones

5633 words (23 pages) Essay in Biology

08/02/20 Biology Reference this

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Introduction:

Magnesium is the fourth most abundant inorganic mineral in the body, and one of six macromolecules vital for growth. It is required in the human body for energy production, oxidative phosphorylation and glycolysis and has a major role in the structural development of bone. Magnesium can be found in green leafy vegetables such as spinach and kale, fruit, Legumes, nuts, seeds and seafood, all in which should be balanced within in the human diet in order maintain sufficient magnesium levels (National Institute of Health, 2018). A magnesium sufficient diet balances intracellular calcium levels which encourages osteoblast proliferation, which increases bone formation and replacement of damaged cells to help maintain bone mass and strength (Abed, E, Moreau, R, 2009). Magnesium has many cellular functions including the crystal formation in bone cells and is required in the metabolization and activation of Vitamin D. Deficiencies in both of these nutrients has been associated with skeletal deformities decreased bone density (Uwitonze, A.M, Razzaque, M.S, 2018). Vitamin D plays a vital role in bone strength as it promotes calcium absorption and bone resorption through osteoclast proliferation resulting in skeletal calcium and phosphate homeostasis (Reddy P, Edwards L,R, 2019).

The recommended amount of magnesium is 300mg to 500mg daily (National Institute of Health, 2018), however magnesium deficiency indirectly impacts secretion and activity of parathyroid hormone via decreased Vitamin D activation which can lead to secondary hyperparathyroidism. In turn this can result in reduced regulation of calcium level, thus reducing bone density and strength (Lips, P, van Schoor, N,M, 2011).

 

Studies show that insufficient magnesium intake can result hypomagnesemia and hypermagnesemia which may lead to an imbalance of resorption and deposition in the bone by osteoblast and osteoclast activity (Castiglioni, S, Cazzaniga, A, Albisetti, W, Maier, J.A.M, 2013). Low Magnesium intake also reduced osteocalcin synthesis due to lowered osteoblast proliferation and has a direct correlation to diminished bone volume and abnormal bone remodelling (Carpenter, T. O, Mackowiak, S.J, 1992). Furthermore, studies show that low magnesium intake can lead to damaged or deformed cartilage, matrix calcification and altered bone differentiation (Castiglioni, S, Cazzaniga, A, Albisetti, W, Maier, J.A.M, 2013). Thus, leading to bone fragility and susceptibility to fractures, both in which are associated with osteoporosis (Castiglioni, S, Cazzaniga, A, Albisetti, W, Maier, J.A.M, 2013). Studies also show that sufficient magnesium intake has a marginal correlation to bone mineral density. (Farsinejad-Marj, M, Saneei P, Esmaillzadeh, A, 2015)

Osteoporosis is common condition which impacts the bone strength through the loss of calcium, which is regulated by magnesium, and bone density, resulting in bone fragility and susceptibility to fracture. It is more common in the elderly, usually adults ages 50 and older, where bone mass is naturally deteriorating, however studies show that a lack of magnesium homeostasis can cause effects of osteoporosis in adults as young as 20 years old.

Conducting this research is important to raise awareness about the correlation of magnesium imbalance in the bone and its impact on bone strength and rigidity. This includes groups such as athletes, elderly, obese all in which could have altered magnesium levels which could increase the potential of bone fracture.

 

It is unclear, whether or not an increase in magnesium levels in the blood and bone can reverse the detrimental effects of decreased bone density and strength, which have been associated with osteoporosis, without conclusion that magnesium is a direct cause of osteoporosis. Current research shows that maintaining magnesium homeostasis is a prevention method rather than curative method (Reddy P, Edwards L,R, 2019). It is also unclear whether or not increased magnesium levels in the bone can be maintained without extracellular and intracellular processes, and magnesium’s impact on bone strength without said processes, thus making the parameters of this experiment essential to obtain valuable data regarding the chemical properties of magnesium and how they can continue to impact the bone without considering other factors such as calcium and vitamin D deficiency, hyperparathyroidism.

Human physiology is complex with many metabolic processes in order to obtain the most ethical and accurate results, rats are often used in place of humans in medical research. In comparison to humans, rat bones have a similar the anatomical structure and proportional sizes making them the most anatomically correct model for human experimental research (Annaccone, P.M, Jacob, H.J, 2009).

The research conducted will assess the tibia and femur of each rat will be used to include a variable, to determine whether or not tibia or femur is stronger, or whether the magnesium solution has more impact on one bone, over the

other. Furthermore, the left and right sides of each bone will be used to test for asymmetry. Finally, both sexes with be tested to analyse the impact of the magnesium solution on the strength of the bone on females in comparison to males. Research shows that males have larger skeletal size especially in the femur and bone mass, which will have an impact on magnesium absorption, in comparison to the small bone mass of the female bones (Nieves JW, Formica C, Ruffing J, Zion M, Garrett P, Lindsay R, Cosman F, 2009). The bones size will also impact the strength and thus normalisation of the data will be necessary.

By undertaking this research, it will be investigated whether or not an increase of magnesium levels in the bone will increase the bone strength of the left and right tibia and femur of a male and female rat.

In order to accurately undertake research regarding the effect of magnesium on bone strength, a three-point bending test on an Instron will be conducted using five rats, three treated with magnesium; two male and one female and two control rats; one female and one male. The treated bones will be dissolved in 300mg Swisse Ultiboost Magnesium” tablet, which is then dissolved 25ml of water per set of two bones (tibia and femur per side). Whereas the control rats will be soaked in demineralised water per set of two bones.

From the three-point bending test, the following biomechanical mechanical indices will be calculated; stiffness, yield load, ultimate load and ultimate deformation. The data collected from this investigation will be collated to then produce a load/deformation curve, which will allow for determination of the intended measures.

The controlled variables of the experiment are the tissue composition; either mineral or organic, as well as the size and the shape of each bone. To account for any differences in the size of the rat bones which might impact the stiffness of the bone, the data is normalised by calculating the diameter of the shaft and the cross-sectional area of the shaft to create a stress/strain curve. From this curve the yield stress, ultimate stress and elastic modules for each rat bone, both control and tested can be determined.

It is hypothesised that with an increase in the amount of magnesium in the rat bones, the ultimate load/stress needed from the 3-point bend test to break the left and right tibia and femur bones of the male and female rat will also increase. This is due to magnesium being essential for absorption and metabolism of calcium which assists in bone strength.

 

Methods:

Bone extraction

Grab three lined trays with needed equipment – small and large scissors, scapple. Place one female rat on one tray, and 2 male rat on each of the other trays. Weigh each rat individually and record in workbook. Collect 6 vials and label each with an ID tag as below.

ID22-1R & ID22-1L

Female rat

ID22-2R & ID22-2L

Male rat

ID22-3R & ID22-3L

Male rat

For each rat: Using the large scissors make window between the superficial fascia and muscle layers on the left side of the right leg of the rat and use blunt dissection to make a tunnel toward the vertebral column. Cut along the vertebral column toward the cranial end of the rat. Continue blunt dissection medially toward stomach of rat, then cut back to the window. Pull the skin over the foot of the rat so that it is detached from the body. Using the small scissors use closed to open scissor actions to snap the tendons around the calcaneus, beginning with the calcaneal tendon, and cutting all other tendons until the tibia is visible. Reflect muscles superiorly. Make incision in quadricep muscles on either side of femur to expose the head of femur. Detach head of femur from labrum of os coxa. Take detached tibia and femur and place on tray. Use smaller scissors in a rocking motion to find the joint between calcaneus and lateral malleolus to sperate the foot from the tibia. Use smaller scissors to detach the patella from the tibia. Use scalple to cut through the collateral and cruciate ligaments to separate the tibia and femur. Use small scissors and scapple to clean the remaining muscles from the bones and remove fibula from tibia. Weigh each bone individually and record in manual, Use Caliper to measure the diameter of each bone. Repeat same process for left leg. Place right and left tibia and femur of each rat into each labelled jar. Control rat bones will be extracted the using the same steps.

Treatment of bones

Dilute 2 x 300mg Swisse Ultiboost Magnesium in 50ml of water – stir until tablets dissolve and pour 25ml in each left and right tube. repeat for the other 2 rats. Leave to be treated for 1 week. Control bones with be soaked in demineralised water.

3-point bending test

Begin with ID22-1R femur and place bone in Instron, jog down the arm and begin test. Record results, Jog arm up. Record for the remaining for the 11 rat bones in order from 1-3, femur and tibia from Right to Left side. Control bones will be tested using the same methods.

Data Analysis on Excel:

Using excel spreadsheet, enter data recorded and uploaded to blackboard from Instron testing into the raw data table for all treated and control bones. Use conversions from kN to N for load data points by multiplying by 1000. Calculate the CSA by using the formula for the area of a circle =

A=πr2

. Calculate the stress and strain data sets from each sample rat by using the following formulae: Stress: =Load/CSA. Strain: = Deformation/Gauge length (21mm). Graph all load-deformation data sets to obtain Yield Load and ultimate load. Calculate Yield Stress and Ultimate stress by dividing Yield load and ultimate load by CSA. Stiffness can be calculated by using =SLOPE(Load values, deformation values) for the elastic phase obtained from load-deformation graphs in excel. Elastic Modulus can be calculated by using =SLOPE(Stress values, Strain values) for the same elastic phase range. Calculate T-tests for Bone type, sex and Side using the Data analysis tool on excel. Collect p<values use one tail if your hypothesis suggest a change (higher or lower) or two tail if no specific change was hypothesised. Pool data depending on Significant numbers (>0.05 p-values). T-test for significant data (eg. male control vs male treatment) transfer p-values to table and calculate the mean and standard deviation for treatment vs control.

Sample Size Calculation:

Use substantiative differences to calculate sample size using the mean and standard deviation.

Sample size needed to accurately analyse the effect of magnesium on male and female rats is 21 rats, 11 male, 10 female rats.

 

There were no complications with methods, however upon extraction of the tibia from ID-22 3 there were evident epiphyseal plates on the tibia’s suggesting the rat was a juvenile, this was further supported by the size of the rat which was 341.6kg.

The results from this experiment show significant differences between the male and female rat bones in accordance to p-values <0.05 as shown in table 1 below. This is further supported through figures 1 and 2 and supporting data from tables 2 and 3 that show the medians for the treatment is significantly higher than the control in both sexes shown separately and pooled in figure 3 and table 5. Specifically, the stiffness for the treated male and female rat bones in comparison to the control bones increased significantly. The rounded values gathered from table 4 provide the mean ultimate load for the treated male bones which shows an increase of 113 N from the control and a 169 N increase in females from the control. The mean stiffness for the treatment bones for males shows an increase of 137 N/mm where females show increase of 19 N/mm. After normalisation of the results, the mean ultimate stress needed to break the male treated bone showed an increase of 3.71 N/mm^2 from the control bone, however the stress needed to break the female treated bone decreased from the control bones by 1.6 N/mm^2. The mean elastic modulus of the male treated bone was significantly lower than the male control by 42.5 N/mm^2, whereas the female treated bone increased from the control by 101.5 N/mm^2.

Table 1. p-values for biomechanical indices gathered from three-point bend test

Treatment

Yield Load (N)

Yield Stress (N/mm2)

Ultimate Load (N)

Ultimate Stress (N/mm2)

Stiffness (N/mm)

Elastic Modulus (N/mm2)

Bone type (tibia vs femur)

0.660719991

0.710576168

0.452

0.912160665

0.575708777

0.630412518

Sex (male vs female)

0.000543544

0.059978956

0.001

0.04749521

0.007178711

0.077079036

Side (right vs left)

0.725649974

0.978195495

0.918

0.992802978

0.715420618

0.640141008

Control

Yield Load (N)

Yield Stress (N/mm2)

Ultimate Load (N)

Ultimate Stress (N/mm2)

Stiffness (N/mm)

Elastic Modulus (N/mm2)

Bone type (tibia vs femur)

0.47337233

0.653251739

0.111

0.194078389

0.387866185

0.351815212

Sex (male vs female)

0.430154764

0.03018707

0.249

0.104614333

0.013990346

0.018137175

Side (right vs left)

0.892371119

0.794865979

0.754

0.778186081

0.918111812

0.847243051

Table 1. shows the significant differences found by t-test and p-value evaluation of the sex of the rat bones in regard to treatment and control. Boxplots were created to further compare the female and male Variables of stiffness as seen below in figure 1 and 2 and table 2 and 3.

Figure 1. Boxplot comparing the treatment and control for the stiffness index for male rats only

Table 2. Data gathered from Figure 1

Treatment

Control

Upper whisker

746.00

292

3rd quartile

552.00

287.80

Median

295.00

218.00

1st quartile

134.50

143.46

Lower Whisker

109.00

132.28

No. Data points

8.00

4.00

Figure 1. shows an increase in median and interquartile ranges between the treatment and control bones, for stiffness which was a significant value in table 1.

Figure 2. Boxplot comparing the treatment and control for the stiffness index for female rats only

Table 3. Data gathered from Figure 2.

Treatment

Control

Upper whisker

109.22

63.84

3rd quartile

82.32

56.99

Median

54.53

43.56

1st quartile

44.98

32.91

Lower Whisker

36.33

28.81

No. Data points

4.00

4.00

Figure 2. shows an increase in median and interquartile ranges between the treatment and control bones for stiffness which was a significant figure in table 1.

 

Table 4. Biomechanical indices for pooled data for treatment and control with man, SD and P-values

Biomechanical Indices

Pooled variables

Treatment

Control

P-value

Mean

SD

Mean

SD

Yield load (N)

Male

118.8125

56.4226

27.9

3.66697

0.001337

Female

16.950

9.248243

28.55

5.983032

0.044522

Ultimate load (N)

Male

159.2625

84.73399

45.7

5.752681

0.003476

Female

19.475

14.49698

41.325

10.47931

0.029219

Stiffness (N/mm)

Male

352.3124

248.423

215.6303

83.91667

0.096908

Female

63.65081

31.57352

44.94521

15.35652

0.173338

Yield Stress (N/mm2)

Male

5.949675

3.106131

2.517697

0.426331

0.007699

Female

3.214831

2.247705

3.588494

0.776935

0.384523

Ultimate stress (N/mm2)

Male

7.802605

4.069056

4.094463

0.367546

0.018864

Female

3.595847

3.276398

5.18922

1.323657

0.209072

Elastic modulus (N/mm2)

Male

362.8977

249.3597

405.3726

153.8209

0.362474

Female

219.5801

45.5735

118.1465

39.19015

0.007474

Table 4. shows where the magnesium treated male and female rat bones increased for each biochemical index in green, where red shows the control rat bones had a higher mean, thus showing lack of effect o f magnesium on these specific indices.Highlighted yellow figures show the figures that were hypothesised to be increased with treatment and did increase. Highlighted blue figures represent figures that were hypothesised to increase with treatment but did not. Purple figures Indicate significant values as per mean for male and female for each index.

Boxplots in figure 1, 2, 3, 4 and 5 and 2,3, 4, 5 and 6 were created using table 4 and Standard deviation to analyse the significance of the increase in said parameters that were hypothesised to increase.

Figure 3. Boxplot comparing the treatment and control for the stiffness index

Table 5 Data gathered from Figure 3.

Treatment

Control

Upper whisker

746.22

291.40

3rd quartile

473.94

218.92

Median

134.68

98.06

1st quartile

82.15

43.56

Lower Whisker

36.33

28.81

No. Data points

12.00

8.00

Figure 3. shows an increase in median and interquartile ranges between the treatment and control bones which was hypothesised to increase and was shown to have significant figures in table one, thus further comparison was shown in figures 1 and 2.

Figure 4. Boxplot comparing the treatment and control for the ultimate load index

Table 6. Data gathered from Figure 4

Treatment

Control

Upper whisker

271.30

54.10

3rd quartile

195.10

50.60

Median

90.45

43.70

1st quartile

28.65

36.40

Lower Whisker

4.90

32.60

No. Data points

12.00

8.00

Figure 4. shows an increase in median and interquartile ranges between the treatment and control bones which was hypothesised to increase.

 

Figure 5. Boxplot comparing the treatment and control for the ultimate stress index

Table 7. Data gathered from Figure 5

 

Treatment

Control

Upper whisker

14.67

6.81

3rd quartile

10.15

5.13

Median

4.52

4.24

1st quartile

3.47

3.91

Lower Whisker

1.15

3.76

No. Data points

12.00

8.00

Figure 5. shows an increase in median and interquartile ranges between the treatment and control bones which was hypothesised to increase.

 

 

 

 

 

 

 

Discussion:

While analysing the data from table 4 it appears that the majority of the treated bones had increased biomechanical indices which impact bone strength. The significant values as determined by p-values <0.005 in table 4 include male and female results for yield load, ultimate load, male yield stress and female elastic modulus. When comparing Ultimate Load and stiffness between the treatment and control bones as shown in figures 3 and 4 the treated bones had much higher median and more distributed data as per tables 4 and 5. After normalising the data, Ultimate stress as shown in figure 5, had a higher interquartile range, however the median remained almost the same, although this is not indicative that the treatment didn’t increase, it shows that the results were more varied and the results for male ultimate stress may have had a large impact on the size of interquartile ranges as concluded from table 4. Table 4 also shows that the mean for all male treated bone indices except elastic modulus was higher than that of the controls, including values obtained after normalisation of data. This indicates that magnesium does increase bone strength for male bones. The only parameters that improved for the female rats were, stiffness which isn’t normalised data and may have been impacted by the weight and size of the rats. The mean Elastic modulus for the female rat was increased from the control rat, (having a p-value less than 0.05), however the overall results from the experiment still indicate that the treatment did not improve the bone strength of the female rat in comparison to the control female rat.

Therefore, with supporting evidence from figures 1 and 2, and tables 2 and 3, data shows that, overall the treatment of the male bones increased the bone strength whereas the female treated bones decreased significantly in strength. As explained in (Yang, L, Guo, P, Niu, Z, Li, F, Song, Z, Xu, C, Liu, H, Sun, W, Ren, T, 2019) a controlled magnesium increases further increases bone strength and resistance to degradation, as tested by three-point bend tests, yield strength, and ultimate load.

Research has shown that there is no direct correlation to magnesium and increased bone strength, specifically, there is so research that explains why only in female bone strength decreased which suggests experimental error. Supporting evidence shows that magnesium homeostasis is needed to maintain optimal bone strength (Castiglioni, S, Cazzaniga, A, Albisetti, W, Maier, J.A.M, 2013). As the weight of the rats and bones was not obtained until the day of the experiment, it is possible that too much magnesium was used to soak the female bones (average bone weight of 0.4725g, average CSA of 6.1mm^2) as they were significantly smaller than the male bones (average bone weight of 0.53g, average CSA 13.65mm^2) and the control female bones (average bone weight of 1.26g, average CSA 8.0mm^2), thus potentially making them brittle, and less dense and therefore they would need less stress to break, decreasing bone strength.

For the experiment there were only three treated rats, a total of 12 treated bones and 2 control rats, a total of 8 control bones. Calculation of the sample size needed for this experiment showed that a minimum of 11 male rats and 10 female rats would be needed to accurately analyse and interpret the data to be able to draw conclusion about the significance of sex when considering magnesium absorption and bone strength. Although the aim of the experiment was to analyse the effect of magnesium on bone strength, the variable of sex impacted the different indices significantly and also impacted the impact that magnesium had on the bone. While the ultimate stress needed to break the bone for males increased with treatment, it decreased for females. Whereas elastic modulus for males decreased and increased for females as displayed in table #, this could be due to a fault in experiment, however only one 1 trial was conducted, so conclusions cannot be drawn about whether this result is accurate. In order to conduct an experiment that accurately determines this significance, it is recommended that appropriate sample sizes are used.

Distribution of the rats should also be considered, one of the male rats received (ID-22 3) was a juvenile which could mean that it hadn’t had fully developed bones yet, decreasing density and therefore strength and disabling full analysis of the magnesium solution strengthening the bones. It is also important to consider that the rats may have already been deficient in minerals that impact on bone strength and density thus also impacting the parameters of the experiment results.

The data displayed in figure 4 and supplement table 5 shows that the median remained almost the same, whereas the interquartile ranges for the treatment data is larger as the data is more varied, with more data points. From the boxplot it is difficult to conclude whether or not the ultimate stress needed to break the treated bones is increased compared to the control as the treatment data is more distributed.

Conclusion:

In conclusion, the data analysed from the experiment and research from various sources support that an increase in magnesium in the bone will increase the bone strength, and thus impact its stiffness, and the amount of force needed to break the bone. Although the analysis for the female data may suggest otherwise, there is no current research that suggests that magnesium would specifically cause a decrease in bone strength for females and not males.

Bibliography

  • Farsinejad-Marj, M, Saneei P, Esmaillzadeh, A. (2015). Dietary magnesium intake, bone mineral density and risk of fracture: a systematic review and meta-analysis. doi: 10.1007/s00198-015-3400-y
  • Uwitonze, A.M, Razzaque, M.S. (2018). Role of Magnesium in Vitamin D Activation and Function doi:10.7556/jaoa.2018.037.
  • Castiglioni, S, Cazzaniga, A, Albisetti, W, Maier, J A. M. (2013). Magnesium and Osteoporosis: Current State of Knowledge and Future Research Directions. doi: 10.3390/nu5083022.
  • Lips, P, van Schoor, N,M. (2011). The effect of vitamin D on bone and osteoporosis. doi: 10.1016/j.beem.2011.05.002.
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  • Abed, E, Moreau, R. (2009). Importance of melastatin-like transient receptor potential 7 and magnesium in the stimulation of osteoblast proliferation and migration by platelet-derived growth factor. https://doi-org.ezp01.library.qut.edu.au/10.1152/ajpcell.00614.2008
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  • Nieves JW, Formica C, Ruffing J, Zion M, Garrett P, Lindsay R, Cosman F. (2009). Males have larger skeletal size and bone mass than females, despite comparable body size. Males have larger skeletal size and bone mass than females, despite comparable body size. https://doi.org/10.1359/JBMR.041005
  • Annaccone, P.M, Jacob, H.J. (2009). Rats!. doi: 10.1242/dmm.002733.
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