INTRODUCTION
Calcium is considered an essential mineral due to its pivotal role in bone formation and preventing bone diseases, such as osteoporosis. Osteoporosis causes weak and brittle bones, increasing the risk of fractures. The prevalence rate of osteoporosis is about 19.75%, globally.[1] Meanwhile, the death rate and the highest disability-adjusted life years (DALYs) climbed by 111.16% in 29 years from 1990 to 2019.[2] Despite being excluded from the list of countries with the highest DALYs of osteoporosis, Indonesia still reaches over 60% of the cases.[3] This is expected to be higher as the number of elderly population is rising to 71 million in 2050.[4] Therefore, higher calcium consumption is needed to alleviate these problems. However, the lack of calcium sources variety and affordability can inhibit people’s tendency to fulfill daily calcium requirements.
Sources of calcium are widely associated with dairy products such as milk and yogurt which contain approximately 100 to 180 mg per 100 g while cheese provides 1 g of calcium per 100 g.[5] However, milk has weaknesses, such as expensive prices, and having allergenic properties. These weaknesses need to be addressed by finding alternative ingredients, one of which is fish. The average growth of fish production has increased by 114.82% from 2017 to 2018.[6] The use of fish is still limited to its meat, either sold in whole (fresh) or fillet form, resulting in the accumulation of fish bone waste. As a by-product, fish bones can potentially be used as a bioavailable calcium source.[7] Fish bones have a very high calcium content (around 20%-25%) of the weight of the fish, so fish bones can be used as an alternative food source of natural calcium to meet daily needs.[8]
Calcium is generally available in micro (μ) sizes and is absorbed by the body only about 25% to 50% of the total calcium absorbed in the metabolic process, which is caused by the particle size.[9,10] The use of technology to form much smaller calcium sizes is currently being developed to increase calcium absorption in the body. Nanotechnology is one method that can form compounds that are more stable and have a high absorption rate.[11] Much smaller calcium reduction technology is currently being developed to increase the absorption of calcium in the body.[12] The synthesis of nanoparticles has attracted more attention because they perform better due to the increased surface area and absorption rate of calcium. Nano calcium which has a very small size (10-9 m) can increase its solubility in the gastrointestinal tract, which further accelerates the absorption process into the circulatory system.[13] Previous findings from Torres et al. [7] stated that by reducing particle size, a salmon fish bone has a better calcium bioavailability rate when compared to calcium carbonate.
Therefore, this study aims to observe the influence of particle size and mineral composition of various fish bone powders on bone density enhancement after oral administration to corticosteroid-induced osteoporosis rats. Types of fish are distinguished based on their place of origin, fresh water, and seawater.
METHODS
1. The making of bone flour and nanomilling process
The main material used in this study is fish. The two types of fish chosen are the most widely known fish from each region, in freshwater are catfish (Clarias gariepinus) and snakehead fish (Channa striata), in seawater are mackerel (Euthynnus affinis), and snapper fish (Lutjanus campechanus). The selection was based on the abundance, affordability, and calcium content of fish.
In general, the process of producing fish bone flour begins with washing the fresh fish, separating the meat from the bone, soaking, and softening the bone by high-pressure boiling, straining, drying, flouring, and filtering the bone flour.[11] The process of fresh fish becoming fish bone meal is explained in detail in Figure 1. There is no difference in the flouring process treatment for all types of fish, other than the length of boiling time. This was because each fishbone has a different length of time to reach the appropriate soft texture to become flour.
The milling process was carried out using the high energy milling technique by planetary ball milling (PBM; PT Nanotech Indonesia Global, Banten, Indonesia) until a size in the nanometer range was obtained. The parameters used in the process were an agitation speed of 750 rpm, a filling ratio of 1:5, and a grinding duration of 2 hr. The range of particle size is from 1 to 1,000 nm.[12] The results of milling and non-milling flour are shown in Figure 2.
2. Fish bone flour characteristic test
(1) Particle size of the flour
Particle size distribution was determined by a Horiba SZ100 particle size analyzer (PSA) instrument. Before analyzing, the sample was dispersed in distilled water and vortexed for 1 min. The fish bone meal was put into a quartz cuvette (3/4 of the volume) and then inserted into the PSA machine.
(3) Study of bone flour in experimental animals
In this study, mice were conditioned to experience osteoporosis, so the selection of mouse characteristics was also adjusted to the theory of factors that increase the risk of osteoporosis (gender and age). So the animal used was female Wistar strain rats (Rattus norvegicus) age 6 months, equivalent to the age of elderly in humans. The weight of the rats ranged from 150 to 200 g. The experimental animals were acclimatized for 7 days by feeding a basal ration and distilled water ad libitum. The composition of the basal ration was prepared according to the AIN 93 standard.
The mice were then divided into 6 groups: normal control (K0), positive control (K1) group with osteoporosis induction but no intervention, and the group induced by osteoporosis and intervened by fish bone meal. The intervention groups consist of: non-milling catfish, snapper bone (SB) meal intervention, catfish bone (CB), CB milling, SB, and SB milling. Osteoporosis induction and product intervention were carried out simultaneously for 50 days. The dose of fish bone meal given was the same in each group, equivalent to 500 mg of calcium content for the human dose, which was dissolved in water to become 1 mL of sonde solution and given daily.
To mimic osteoporosis the glucocorticoid method is used, as a cause of secondary osteoporosis.[16] Glucocorticoids are one of the drugs that are often prescribed widely for various medical conditions, such as autoimmune diseases, allergic reactions, and others.[17] In this study, the type of glucocorticoid used was dexamethasone. The dose of dexamethasone for humans was 2 times 0.75 mg per day, so when converted to mice it was 0.27 mg/head/day. The intervention was repeated 3 times per group. This research was approved by the Health Research Ethical Clearance Commission, Faculty of Dental Medicine, Universitas Airlangga (approval no. 0708/HRECC.FODM/VII/2024).
(4) Calcium absorption rate
Blood samples were obtained at every time interval after the rats were given fish bone flour products, where then 0.3 mL of blood was taken from the veins of the tail each time using a 1 mL disposable syringe. Blood sampling time started in the pre-, and post-30 min, 2 hr, 6 hr, and 24 hr. The rat blood sample taken from the heart was put into a test tube and waited for 3 hr to separate the blood and the serum.
(5) Anthropometry measurement
Body weight was measured by a digital weight scale. Body length (nose to anal) was measured by a ruler. Body mass index (BMI) was calculated by dividing body weight by the square of height.
(6) Calcium, magnesium, phosphorus serum
Blood serum was collected on the last day of the intervention. The mice were anesthetized, then their blood was taken from the heart. After the blood serum samples were collected, laboratory analysis was carried out. Calcium, magnesium, and phosphorus were assessed using the Atomic Absorption Spectrometer.
(7) Osteoblast, osteoclast, and bone density
The number of osteoblast and osteoclast cells was determined from the results of histological observations of femur bone tissue. Bone density was calculated through histological readings of mature and immature bone growth using the Masson trichrome staining method.[18] Masson’s trichrome is used in connective-muscle tissue and bone visualization using a stain.[19] The blue color is described as regenerated/immature bone (osteoblasts), collagen fibers, and osteoid. The red color is defined as the mature bone cells (osteocytes).[20]
(8) Statistical analysis
Before analyzing the differences, all data underwent an assessment to determine the normality and homogeneity of variance using the Shapiro-Wilk test. The sample size was 48 (each group included 6 primary mice and 2 spares). The assessment then continued by analyzing the differences in data using the ANOVA for variables included body length, BMI, and blood serum (calcium) in all groups. To assess the significant difference between the two groups in the variables above, the Tukey test was used. Meanwhile, the statistical analysis for body weight, blood serum (magnesium and phosphorus), osteoblast, osteoclast, and bone density used the Kruskal-Wallis test for all groups then proceeded with the Mann-Whitney test to analyze the significance. All tests used a significance level of α=0.05. Statistical analysis was performed using IBM Statistics SPSS 20 software (IBM Corp., Armonk, NY, USA).
RESULTS
1. Fish bone flour characteristics
(1) Particle size of the bone flour
Table 1 shows the results of the particle size characterization test of all bone flours from various types. The results of particle size analysis on all fish bone flour ranged from 300 to >1,000. The smallest particle size was in catfish milling bone flour (332.60 nm), while the largest was in mackerel bone flour (1,573.37 nm), thereby it cannot be defined as nanoparticles. When compared between the same type of fish, it was proven that the nanonization treatment succeeded in reducing the particle size of fish bone meal in the range of 28% to 46%.
(2) Nutritional content of bone flour
The results of the nutritional content in protein of bone flour before and after milling, are shown in Table 1. The difference in the production method of CB flour between traditional and machine methods is not very significant. The highest protein content was found in the milled snakehead fish bone meal, while the lowest was found in the milled CB meal. Although the protein content of milled CB flour in this study was the lowest, this amount was still higher than other flours. Tuna fish bones contain 28.66% [21]; CB flour contains 19.47% [22]; tilapia fish bone contains 14.81%.[23]
The highest calcium content was found in the milled CB meal (15,980.78 mg), while the lowest was found in the mackerel bone meal (6,612.98 mg). This amount is balanced with the calcium content of other fish, which showed a calcium content of 13,184.3 mg/100 g in the skeleton of tuna fish, and 15,469.3 mg/100 g in the gills of tuna.[21] As for calcium content, the highest magnesium and phosphorus content was found in milled CB meal (290.09 and 9,321.68 mg, respectively), while the lowest was found in mackerel bone meal (134.53 and 3,359.62 mg). This phosphorus content is much higher than that reported in seawater samples from other studies. Tuna fish bones contain 1,010.2 mg, and tuna fish gills contain 1,071.8 mg.[21]
(3) Calcium absorption rate of bone flour in experimental animals
The calcium absorption test in experimental animals was observed by the time intervals before intervention, 30 min, 2 hr, 6 hr, and 24 hr after intervention. Table 1 shows the results of the absorption test. There was a gradual increase over time until post 2 hr, which was the highest absorption peak, then gradually decreased until 24 hr (Fig. 3). The peak time interval at 2 hr post-administration of fish bone flour showed the maximum concentration of calcium absorption. Furthermore, there is a decrease because there is no more absorption and elimination that occurs. The calcium solubility of fish bone flour increases significantly as the particle size decreases, which is due to an increase in surface area and breakdown of the collagen matrix. Therefore, decreasing the particle size of fish bones becomes an alternative to increasing calcium absorption.[24] Therefore, CB meal showed the highest absorption which may be due to its smallest particle size.
Fishbone naturally contains calcium and phosphorus in the right ratio of approximately 2:1 from hydroxyapatite, which is considered the most bioavailable from calcium. The soluble calcium concentration correlated with total solubility, with the highest soluble calcium found at pH 3 and the lowest at pH 7.[25] At higher pH, calcium solubility was very low in all cases. In this study there was no pH adjustment, but rather temperature adjustment. This might be the drawback because the temperature did not play a significant role in the solubility profile of fishbone flour.[25]
The graph (Fig. 3) shows the highest absorption in catfish and SB meal milling. Based on this data, the two types of fish were selected to continue the analysis of experimental animals (blood serum and bone parameters).
(4) Study of experimental animal tests induced osteoporosis
The maximum body length was in the group given non-milling snapper fish bone meal (Table 2). This is in line with the results of the nutritional content test, the protein content of non-milling snapper fish bones is higher than CBs. Intake of macronutrients, especially protein, is related to increased height and z-score TB/U.[26] However, it should be remembered that this study was conducted on elderly mice, so body length did not increase optimally because the mice were not in their growth period. Measuring BMI in experimental animals such as mice is one indicator of nutritional status assessment. BMI for normal adult mice ranges from 0.457±0.02 to 0.687±0.05 g/cm2.[26] All mice at the end of the intervention period remained within the normal BMI limit.
Normal serum calcium levels in rats are 8.8 to 10.5 mg/dL,[27] or in other references 8.5 to 10.9 mg/dL.[28] When compared to the value limits, the calcium values in all groups are in the normal range, although in group K1 the value is the lowest compared to other groups. The trend of mineral levels of the three parameters (Ca, Mg, P) has similarities, namely the highest average value in group K0 (normal control), then in the second highest rank in the milled snapper fish bone meal treatment, then in sequence, namely non-milled snapper fish bone meal, milled catfish fish bone meal, and non-milled catfish fish bone meal. The results of the difference test for these three minerals also showed significant differences between groups, this indicates that the treatment of fish bone meal can increase blood serum values (calcium, magnesium, and phosphorus) in rats given osteoporosis induction. The difference in serum mineral values is suspected to be caused by protein content. It is proven that SB meal has the highest protein content (Table 1) so the results of blood serum tests also show the highest values compared to other intervention groups (Table 2).
The highest number of new bone growth and remodeling was in the normal control group, followed by snapper fish bone meal, and milling CB meal. Although there was a difference in the number of cells, statistically the difference was not significant (Table 3). This could also be due to the limited research time or the side effects of glucocorticoid induction, which may differ for each mouse. However, the interesting thing is the percentage of bone density, which is the highest in CB meal compared to other groups (although not significantly different). In terms of nutritional content, CB meal has a fairly high value and is accompanied by a phosphorus value with the right ratio, following the theory that the absorption of both minerals is optimal if at a ratio of 2:1. This finding is a recommendation that in addition to reducing particle size, the ratio of mineral content also needs to be considered to achieve maximum bone density even though it is accompanied by osteoporosis induction.
DISCUSSION
According to Greiner [29], the size of nanoparticles is in the range of 1 to 100 nm, and the size of microparticles is in the range of 1 to 1,000 µm. However, a study by Zhu et al. [30] that implemented thermoultrasonic treatment on Halibut fish bones resulted in a particle size of 605.92 nm, and he called it a nanoparticle. In addition, this product is fish bones which based on nutritional content (Table 1) contain >3,000 mg phosphorus. Based on research conducted by Li et al. [31] flour based on phosphorus, is processed by large-scale production, and has a particle size of 100 to 300 nm, referred to as nanoparticles. So from these findings, fishbone flour after milling meets the requirements for nanoparticles. But besides that, it can be agreed that the nanonization treatment succeeded in reducing the particle size of fish bone meal.
Fishbone comprises approximately 30% collagen and 60% to 70% inorganic compounds such as calcium, phosphorus, magnesium, iodine, and selenium.[32] This decrease in protein content is thought to be caused by protein denaturation caused by high heating temperatures. Through flocculation, the protein structure will be damaged. This process is the initial stage of denaturation. Analysis of the denaturation temperature range of a product and the optimal selection of thermal processing parameters are important for fish raw material because they impact the quality, stability, and functionality of the final product.[33] The difference in mineral serum levels is due to differences in species, sex, biological cycle, and environmental factors such as season. But besides that, it can be agreed that the nanonization treatment succeeded in increasing the nutritional content (protein, calcium, magnesium, and phosphorus) of fish bone meal.
Protein functions as a raw material for the formation of bones and soft tissues, which are important for increasing body length. Protein plays a role in supporting mineral absorption in the body.[28] Osteoblasts are cells responsible for bone formation, as they can synthesize collagen matrix (mineralization). Osteoblasts also have parathyroid hormone receptors, which when activated, release cytokines that will stimulate an increase in the number and activity of osteoclasts. Osteoclasts function in bone resorption and degradation, namely helping to remodel bones, reabsorb calcium, and help maintain blood calcium concentrations at optimal levels. Osteoblasts and osteoclasts work together in bone remodeling.[34,35]
Table 2 shows that the SB powder intervention obtained higher mineral serum levels (Ca, Mg, P) compared to the CB powder intervention. The difference in value is caused by the nutritional content of the product, where snapper fish bone powders (Table 1) are composed of 47.95% protein (milling) to 50% (non-milling) and accompanied by a high composition of magnesium, phosphorus, and calcium. The protein content helps minerals to be more easily absorbed by the body,[28] resulting in higher serum levels compared to CB powders which have lower protein content. However, on the contrary, the higher content of bone-forming minerals (Ca, Mg, P), with the recommended ratio (P:Ca=1:2),[36,37] and supported by the very small particle size (nano) in CB meal can cause the product to be more easily used by bone-forming cells so that the number of osteoblasts, osteoclasts, and bone density percentage is higher compared to the intervention with snapper fish bone meal. Although the difference in the number of bone-forming cells is not significant (Table 2).
It can be concluded that to increase serum minerals and form bone density optimally, in addition to reducing particle size, it is also necessary to consider the protein content which functions to help mineral absorption, and the importance of balancing the ratio of mineral content.
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