1. Introduction
Osteoporosis is a medical condition in which the bones become brittle and fragile from a loss of tissue, typically as a result of hormonal changes or deficiency in calcium or vitamin D [
1]. This increases the likelihood of complications such as fractures, which most commonly occur in the spine, wrist and hips, but can also affect the arm or pelvis. According to the International Osteoporosis Foundation (IOF), it is estimated that there are approximately 200 million osteoporosis patients worldwide, with a particularly high prevalence in females and the elderly [
2]. In Australia, not only is there an aging population, but also relatively high rates of vitamin D deficiency. Moreover, osteoporosis heavily contributes to hospitalisation and related expenses costing approximately $1.9 billion per annum [
3]. Preventative treatment of osteoporosis and related fractures for high risk groups is important. Therefore it is necessary to develop products suitable for long-term use [
4,
5,
6]. A promising source of natural products for osteoporosis commonly uses plant-based agents such as marine plants and soybean products, which are rich in protein and calcium. Recently, the contents of animal bones have become of interest, with research into as many as 17 animals, including sika deer, red deer, pig, sheep, dog, and cow [
7]. The bone extracts from these animals may contain active minerals and proteins such as Bone Morphogenetic Proteins (BMPs) that could positively impact bone and fat metabolism. Despite this, research at this stage is primarily observational with more investigation needed for the mechanisms of action of the active ingredients of these bone products.
The wallaby is an active, lean and wild animal unique to Australia, with over sixty different species. It is estimated that the four most common species of wallaby are numerically in excess of 50 million animals [
8,
9]. At present, the wallaby industry relies heavily on producing wallaby meat [
10], while the bones are considered to be a waste product. For these reasons, we hypothesised that wallaby bone extracts may be a potentially efficacious treatment option for osteoporosis. In this study we aimed to identify and quantify the ingredients (mineral and protein) of wallaby bone extracts while also identifying the calcium to phosphorus ratio and heavy metal content. This was achieved by using inductively coupled plasma-mass spectrometry (ICP-MS) and Fourier transform infrared spectroscopy (FT-IR) spectroscopic analysis. Alongside this, liquid chromatography-mass spectrometry (LC-MS/MS) was used to perform proteomics analyses, which further clarified the wallaby bone protein composition and putative functions.
To our knowledge, this is the first scientific evaluation of Australian wallaby bone extracts using a Systems Biology approach, and may lead to the discovery of a potential alternative therapy for the treatment and prevention of osteoporosis.
2. Material and Methods
2.1. Animal and Bone Samples
Bennett’s wallaby
Macropus rufogriseus, mixed genders, were harvested from Tasmanian farmlands in Australia. The fresh femur, tibia and fibula bones were collected and supplied by Lenah Game Meats. The bones of three individual wallabies were used in the physical testing and analysis processes. In brief, after harvesting, fresh bones were trimmed free of soft tissue and washed with cold phosphate buffer saline (PBS) to remove contaminants, wrapped with gauze pre-soaked in PBS containing a protease inhibitor cocktail, then stored at −20 °C overnight. Ten grams of the parietal bone fragments (about 10 mm × 1 mm size, 1 to 2 mm thick,
n = 3) were frozen in liquid nitrogen and then ground up using a mortar and pestle for subsequent chemical analysis and protein determination [
11].
2.2. Digestion Procedures for Wallaby Bone
Wallaby bone samples which contained fibula, femur and tibia bone specimens were dried overnight and weighed (about 0.150 g). The dried samples were then placed into a 15-mL Falcon tube (Fisher Scientific, Sydney, NSW, Australia). Optima grade nitric acid (0.8 M, 3 mL) and ultrapure hydrogen peroxide (30%, 1 mL) were then added to the tubes and capped. The tubes were placed on a hot plate at 45 °C for one hour. The temperature was then increased to 80 °C for 12 h. The final solution was quantitatively transferred to 50 mL plastic centrifuge tubes (Fisher Scientific, Sydney, NSW, Australia) and filled to the mark with ultrapure deionized water (>18 MΩ·cm−1) and weighed.
2.3. Inductively Coupled Plasma-Mass Spectrometry
The ICP-MS system (Agilent Technologies 7500cx series, Santa Clara, CA, USA) was used with sample introduction via a micromist concentric nebuliser (Glass expansion, Port Melbourne, VIC, Australia) and a Scott type double pass spray chamber cooled to 2 °C. The sample solution and the spray chamber waste were carried with the aid of a peristaltic pump (Watson-Marlow, Wetherill Park, NSW, Australia). ICP-MS extraction lens conditions were selected to maximise the sensitivity of a 1% HNO3:HCl solution containing 1 ng/ml of lithium, cobalt, yttrium, cerium and thallium. Helium was added into the octopole reaction cell to reduce interferences. Calibration curves were constructed and the results analysed using Masshunter software (version 2014, Agilent Technologies, Santa Clara, CA, USA).
A certified calibration standard containing lithium (Li), beryllium (Be), boron (B), sodium (Na), magnesium (Mg), aluminium (Al), calcium (Ca), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), strontium (Sr), molybdenum (Mo), silver (Ag), cadmium (Cd), antimony (Sb), barium (Ba), lanthanum (La), europium, (Eu), holmium (Ho), ytterbium (Yb), thallium (Tl), lead (Pb), bismuth (Bi), thorium (Th), uranium (U), phosphorus (P) andmercury (Hg) standard was obtained from Choice Analytical, (Thornleigh, NSW, Australia). Baseline nitric acid (HNO3) and hydrogen peroxide (H2O2) were purchased from Choice Analytical (Thornleigh, NSW, Australia).
2.4. Fourier Transform Infrared Spectroscopy
FT-IR was carried out using a Thermo Scientific Nicolet 6700 spectrometer (Waltham, MA, USA).with a DTGS (Deuterated Tri Glycine Sulfate) (Waltham, MA, USA) detector. The powder samples were dispersed in Potassium Bromide discs and all spectra were obtained using 4 cm−1 resolution and 64 scans at room temperature.
2.5. Protein Extraction from Wallaby Bone
The protein extraction process is summarised in
Figure 1. Bone fragments (3 × 3 g) were incubated in 1.2 M HCl (10 mL for each sample) at 4 °C overnight to demineralise bone tissue. The supernatant was collected as Extract 1 (E1) after centrifugation at 4000 rpm for 15 min at 4 °C. The pelleted material was washed with water and extracted for 72 h at 4 °C in a lysis buffer containing 8 M urea, 100 mM Tris-HCl (pH 8.8) and a protease inhibitor cocktail. The supernatant was then collected as Extract 2 (E2) after centrifugation. The pelleted material was extracted further for 72 h at 4 °C in a lysis buffer containing 8 M urea, 100 mM Tris-HCl, 0.5 M tetrasodium EDTA (pH 8.8) and a protease inhibitor cocktail. The supernatant was collected as Extract 3 (E3) after centrifugation. Finally, the remaining pellet was incubated in 6 M HCl overnight at 4 °C. The solution was collected as Extract 4 (E4) after centrifugation [
12]. The acidic extracts were neutralised and the pH adjusted to ~8.
2.6. Protein Determination with Bradford Assay Kit
Disulfide bonds in the protein samples were reduced using dithiothreitol (20 mM final concentration, Bio-Rad, Hercules, CA, USA). The samples were then incubated at room temperature for 45 min, before alkylation with acrylamide monomers (20 mM final concentration, Bio-Rad), and then incubated at room temperature for 45 min. The protein was precipitated by adding 5 volumes of acetone (1:5, v/v). After centrifugation, the pellet was resuspended with a minimum volume of 8 M urea, 100 mM NH4HCO3 using a sonic probe. Protein concentrations in the four extracts of individual wallaby femur, tibia and fibula were determined using the Bradford assay kit (Bio-Rad).
2.7. Protein Concentration Assay by SDS-PAGE System
The protein concentrations of E1, E2, E3 and E4 samples were ascertained using SDS-PAGE. Samples diluted to 1:10 and 1:50 and a serial dilution of albumin (1 mg/mL) were mixed with Laemelli sample Buffer (Bio-Rad). Forty microliters of each sample was loaded onto 26-well Criterion™ TGX™ Precast Gels (Bio-Rad), and run at 160 V for 3 min. The gel was fixed and then stained with Flamingo™ Fluorescent stain (Bio-Rad). The density of bands was quantified using Quantity One software (v4.6.1, Bio-Rad) based on the BSA standard curve.
The E4 sample contained salt which was subsequently removed using Bio-spin 6 chromatography columns (Bio-Rad), and then dilutions using ratios of 1:2, 1:4 and 1:8 were carried out on the E4 sample and subjected to SDS = PAGE which showed the salt had been removed.
Approximately 100 µg of protein was diluted with 50 mM NH4HCO3 to adjust the urea concentration to 1 M. Trypsin (1 µL, MS grade, 1 µg/µL, Sigma-Aldrich, St. Louis, MO, USA) was added to each tube, and the sample tubes were incubated overnight at 37 °C. The digested samples were centrifuged at maximum speed for 5 min prior to solid phase extraction using HLB columns (Silicycle®, Quebec, QC, Canada) to recover the peptides. Samples were concentrated to approximately 15 µL (E1), or 25 µL (E2, E3 and E4).
2.8. Liquid Chromatography–Mass Spectrometry (LC-MS/MS)
Using an Eksigent AS-1 autosampler connected to a Tempo nanoLC system (Eksigent, Dublin, CA, USA) as previously described [
13], 10 µL of each combined extract sample was loaded at 20 µL/min with MS buffer A (2% Acetonitrile + 0.2% Formic Acid) onto a C8 trap column (Michrom Bioresources, Auburn, CA, USA). After washing the trap for three min, the peptides were washed off the trap at 300 nL/min onto a PicoFrit column (75 µm ID × 150 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA), then eluted from the column and into the source of a QSTAR Elite hybrid quadrupole-time-of-flight mass spectrometer (AB Sciex, Eksigent, Dublin, CA, USA) using the following program [
14,
15]: 5%–30% MS buffer B (98% acetonitrile + 0.2% formic acid) over 60 min, 30%–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80%–85% for 3 min. The eluting peptides were ionised at 2300 V. An Intelligent Data Acquisition (IDA) experiment was performed, with a mass range of 350–1500 Da continuously scanned for peptides of charge state 2+–5+ with an intensity of more than 30 counts/s. The selected peptides were fragmented and the product ion fragment masses measured over a mass range of 100–1500 Da. The mass of the precursor peptide was then excluded for 120 s.
2.9. MS/MS Data Analysis and Protein Identification
The MS/MS data files produced were searched using Mascot Daemon (version 2.4; Perkins, D.N. 1999, Boston, MA, USA) against the LudwigNR database (comprised of the UniProt, plasmoDB and Ensembl databases, vQ213) with the following parameter settings: Fixed modifications: none; Variable modifications: propionamide, oxidised methionine and deamidated asparagine; Enzyme: semitrypsin; Number of allowed missed cleavages: 3; Peptide mass tolerance: 100 ppm; MS/MS mass tolerance: 0.2 Da; Charge state: 2+, 3+ and 4+. The results of the search were then filtered by including only protein hits with at least one unique peptide (Bold Red) and excluding peptide hits with a p-value greater than 0.05. Peptides identified by the Mascot were further validated using Scaffold (v4.0, Proteome Software, Portland, OR, USA) and by manual inspection of the MS/MS spectra for the peptide to ensure the b- and y-ion series were sufficiently extensive for an accurate identification. Peptide identifications were accepted if they could be established at a probability greater than 95.0% as specified by the Peptide Prophet algorithm [
16]. Protein identifications were accepted if they could be established at probability greater than 80.0% as assigned by the Protein Prophet algorithm [
17].
2.10. In Vitro Evaluation of Wallaby Bone Extracts on Osteogenic Elements
To determine beneficial effect of wallaby bone extracts (WBE) on bone metabolism, osteogenic elements of calcium (Ca) and alkaline phosphatase (ALP) activity were measured in osteogenic cells derived from adipose tissue-derived stem cells (ADSCs) with and without WBE treatment. In brief, WBEs (1, 5 and 10 µg/mL) were added into Dulbecco's Modified Eagle Medium (DMEM) Glutmax/F12 (Gibco
®, Waltham, MA, USA) medium containing 0.1 μM dexamethasone, 50 µM ascorbate-2-phosphate, 10 mM β-glycerophosphate and 10% foetal bovine serum during 14 days of human ADSCs differentiation into osteogenic cells, as previously described [
18], and culture medium treatment as a control. At the end of the experiment, WBEs treated cells with the control cells were harvested for determination of amount of calcium and ALP activity. In brief, cell samples were completely lysed by sonication and the supernatants were collected for measuring amount of calcium and the ALP activity using the automatic biochemistry instrument (ARCHITECT, Abbot Park, IL, USA) according to manufacturer’s instruction.
2.11. Statistical Analysis
Quantitative data is expressed as the mean ± SEM. Comparisons across the four groups were done using one-way ANOVA, followed by a post hoc analysis of Tukey’s test to determine significant differences between the two groups, using Prism version 4 (Graph Pad Inc., San Diego, CA, USA). In this process, a p-value < 0.05 was considered statistically significant.
4. Discussion
From the chemical analysis of wallaby bone, the minerals analysis showed that there was not a significant difference between the femur, fibula and tibia samples (
Table 3). When these results are compared with other domestic animals’ bone, the concentrations of calcium and phosphorus in wallaby bone are significantly higher than in alpaca bone, beef bone, pork bone, lamb bone and chicken bone (
Table 6) previously reported [
21,
26]. The most interesting finding is the 2:1 ratio of calcium and phosphorus in wallaby bone, which is similar to the calcium to phosphorus ratio in human bone. This ratio may make the substance of wallaby bone readily absorbed for consumption. In addition, wallaby bone contains high levels of other essential minerals with heavy metal concentrations below the human prescribed standard.
Protein analysis has revealed that the femur contains the highest amount of protein. Extract 4 from each type of bone had the highest amount of protein among the four extracts. This result matched the protein identification results from the LC/MS/MS. A total number of 2038 spectra, 206 unique proteins and 137 clusters were identified. The analysis of Extract 4 also presented the largest number of spectra, peptides and proteins. When the proteins from the extracts were classified according to cellular component, biological process and protein role assignment, the largest component of the four extractions was also Extract 4 among groups; this was consistent with the protein assay.
These results may be attributed to a high abundance of proteins or protein fragments existing in this fraction (Extract 4) that may suppress the identification of other proteins [
12]. Additionally, among those 206 unique proteins identified, up to 65 (31.6%) were found in all four extracts, while 4 (1.9%) proteins in Extract 1, 7 (3.3%) proteins in Extract 2, 11 (5.3%) proteins in Extract 3, and 54 (26.2%) proteins in Extract 4 were uniquely observed (
Figure 3). Even the amount of overlap between proteins that had been identified was quite high in the fractions from different extraction steps. The different extraction methods (Extracts 1 to 4) still have a lot of unique proteins, particularly Extract 4.
The most interesting results found in the gene ontology and cell function groups of those identified proteins were that the major components included proteases and enzymes involved in the stimulation and proliferation of bone cells. Normally, the non-collagenous proteins only make up 10% of total bone protein content, while a majority of the biologically important proteins are naturally relatively low. However, in the proteome analysis study, we found that the cellular process and organismal process proteins of biological process classification were identified up to 69% and 54%, respectively, indicating that high amounts of bone remodelling proteins, like osteomodulin, osteopontin, osteoglycin, osteocalcin, bone sialoprotein-2 and decorin may exist in wallaby bone.
This study was designed mainly to characterize bone beneficial components from Australian Wallaby bone but the in vitro evaluation of wallaby bone extracts in the differentiation of ADSCs into osteogenic cells showed the WBEs increased deposition of calcium in osteoblasts with enhanced ALP activity at a high dose. In increased ALP activity and total calcium concentration by the nutrients have demonstrated the positive effects on the homeostasis of bone [
27]. Although more investigation is required, these data highlights a view of the future study on WBE as supplements to prevent or treat bone disorder, such as osteoporosis and bone fracture.
Overall, the results of mineral and protein analysis suggest that substance from Australian wallaby bone may have potential to be beneficial for mineral deficiency related to osteoporosis. Future studies using an animal model of osteoporosis to investigate the therapeutic effect of wallaby bone extract on bone formation, differentiation and proliferation is warranted.