In Vitro Effects of Biofield Energy Treated Vitamin D3 Supplementation on Bone Formation by Osteoblasts Cells

Various bone disorders including fractures, significant pain and height loss, disability to stand up, and walk occurs due to insufficient level of vitamin D and calcium in the body. Both plays an important role to improve the patients' quality of life with respect to bone disorders. The present experimental analysis designed to explore the potential of Consciousness Energy Healing based vitamin D3 and DMEM medium on various bone health parameters such as alkaline phosphatase enzyme (ALP) activity, collagen levels and bone mineralization. Both the test items (TI) i.e. vitamin D3 and DMEM medium were divided into two parts. Each sample received the Consciousness Energy Healing Treatment by Sakina Aleemah Ansari and those samples were labeled as the Biofield Energy Treated (BT) samples, while the other parts of each sample were denoted as the untreated test items (UT). Cell viability using MTT assay showed that cell viability was more than 79% with safe and nontoxic profile on MG-63 cell line. The level of ALP was significantly increased by 119% (at 50 μg/mL), 161.5% (at 1 μg/mL), and 252.3% (at 50 μg/mL) in UT-DMEM+BT-TI, BT-DMEM+UT-TI, and BT-DMEM+BT-TI groups, respectively as compared with the untreated test item and DMEM group. Collagen level was significantly increased by 81% (at 10 μg/mL), 117.9% (at 1 μg/mL), and 134.2% (at 0.1 μg/mL) in UT-DMEM+BT-TI, BT-DMEM+UT-TI, and BT-DMEM+BT-TI groups, respectively as compared with the untreated test item and DMEM group. In addition, the percent of bone mineralization was significantly increased by 235.2% at 100 μg/mL in UT-DMEM+BT-TI group, while 152.7% and 92.6% at 0.1 and 1 μg/mL, respectively in BT-DMEM+UT-TI group as compared with the untreated group. In addition, BT-DMEM+BT-TI group showed a significant increased bone mineralization by 158% at 1 μg/mL as compared with the untreated group. Overall, the experimental data suggested that the Biofield Energy Treated vitamin D3 and DMEM would play an important role in the promotion and maintenance of strong and healthy bones, which improve quality of life. Biofield Energy Treatment might also regulates the osteoblast function, improves bone mineralization, and calcium absorption in wide range of bone disorders along with wide range of adverse health conditions, comprising cancer and certain autoimmune diseases.


Introduction
Vitamin D has multiple effects which regulate the functions in different organs such as brain, lungs, liver, kidneys, and heart, immune, skeletal, and reproductive systems. Moreover, it has significant anti-inflammatory, antiarthritic, anti-osteoporosis, anti-stress, anti-aging, anti-apoptotic, wound healing, anti-cancer, anti-psychotic, and anti-fibrotic roles. Vitamin D receptors (VDRs) are widely present in most of the body organs like brain, heart, lungs, kidney, liver, pancreas, large and small intestines, muscles, reproductive, nervous system, etc. [1]. VDRs influence cellto-cell communication, normal cell growth, cell differentiation, cell cycling and proliferation, hormonal balance, neurotransmission, skin health, immune and cardiovascular functions. Bone-related health issues become a major problem among the population from village to the cities. Vitamin D plays a vital role in preserving a healthy mineralized skeleton of most of the vertebrates including humans. Cod liver oil, irradiation of other foods including plants, sunlight, etc. are found to be effective against bone related disorders, which lead to discovering the active principle-vitamin D [1]. The role of vitamin D has been well defined not only for improving the bone mineralization but also with increased bone resorption, aging, inflammation and overall quality of life. Vitamin D 3 is synthesized in the skin by sunlight and once formed it sequentially metabolized in the liver and kidney to 1,25-dihydroxyvitamin D (calcitriol, the vitamin D hormone) [2]. Calcitriol play an important role in maintaining the normal level of calcium and phosphorus, promotes bone mineralization, induce or repress the genes responsible for conserving the mineral homeostasis and skeletal integrity, and inhibit hypertension, kidney damage, cardiovascular and immune disorders (such as Lupus, Addison Disease, Graves' Disease, Hashimoto Thyroiditis, Multiple Sclerosis, Myasthenia Gravis, Anemia, Sjogren Syndrome, Systemic Lupus Erythematosus, Diabetes, Alopecia Areata, Fibromyalgia, Vitiligo, Psoriasis, Scleroderma, Chronic Fatigue Syndrome and Vasculitis), and the secondary hyperparathyroidism [3]. Vitamin D insufficiency and deficiency is the major health problem, which causes metabolic bone disease in the young and elderly populations [4]. Fortified foods have a variable amount of vitamin D and most of the foods do not contain vitamin D, which can be fulfilled using some supplements. In order to avoid the bone related disorders such as osteomalacia, exacerbate osteoporosis, hyperparathyroidism, immune disorders, etc. calcium 1000-1500 mg/day along with vitamin D supplement around 400 IU/day is very important for maintaining the good bone health [5].
Various in vitro studies have readily demonstrated the role of bone health using cell lines and its resorbing effects using three important key biomarkers, such as ALP, collagen and calcium. MG-63 cell line derived from juxtacortical osteosarcoma, which represents an immature osteoblast phenotype and undergoes temporal development in long term culture. The response of MG-63 cells to 1,25dihydroxyvitamin D 3 (1,25 (OH) 2D 3 ) administration has been studied to be similar to normal human osteoblast cells [6]. Hence, MG-63 cell line is widely used for studying the potential of any test compounds to improve the bone health [7]. The formation of new bone involves a complex series of events including the proliferation and differentiation of osteoblasts, and eventually the formation of a mineralized extracellular matrix. ALP is a phenotypic marker for the early differentiation and maturation of osteoblasts. ALP increases the local concentration of inorganic phosphate for bone mineralization and hence is an important marker for osteogenic activity [8]. Similarly, active osteoblasts synthesize and extrude collagen, which plays an important role in the formation of bone extracellular matrix by providing strength and flexibility. Collagen fibrils formed an arrays of an organic matrix known as Osteoid [9]. Likewise, calcium phosphate is deposited in the Osteoid and gets mineralized (combination of calcium phosphate and hydroxyapatite) and provides rigidity to the bone [10]. Thus, these parameters are very essential in order to study the bone health in cell lines. Authors evaluated the in vitro effect of the Biofield Energy Treated vitamin D 3 as a test item, a Complementary and Alternative Medicine (CAM) on bone health using MG-63 cell line for major biomarkers.
Within the burgeoning ground of CAM therapies, Biofield Energy Treatment or energy medicine, is emerging with significant benefits in various scientific fields. The effects of the CAM therapies have great potential, which include external qigong, Johrei, Reiki, therapeutic touch, yoga, Qi Gong, polarity therapy, Tai Chi, pranic healing, deep breathing, chiropractic/osteopathic manipulation, guided imagery, meditation, massage, homeopathy, hypnotherapy, progressive relaxation, acupressure, acupuncture, special diets, relaxation techniques, Rolfing structural integration, healing touch, movement therapy, pilates, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines in biological systems both in vitro and in vivo [11]. Biofield Energy Healing Treatment (The Trivedi Effect ® ) contain a putative bioenergy, which is channeled by a renowned practitioners from a distance. Biofield Energy Healing as a CAM showed a significant results in biological studies [12]. However, the National Center for Complementary and Alternative Medicine (NCCAM), well-defined Biofield therapies in the subcategory of Energy Therapies [13]. The Trivedi Effect ® -Consciousness Energy Healing Treatment has been reported with significant revolution in the physicochemical properties of metals, chemicals, ceramics and polymers [14][15][16], improved agricultural crop yield, productivity, and quality [17,18], transformed antimicrobial characteristics [19][20][21], biotechnology [22][23], improved bioavailability [24][25][26], skin health [27,28], nutraceuticals [29,30], cancer research [31,32], and human health and wellness.
Based on the significant outcomes of Biofield Energy Treatment and vital role of vitamin D 3 on bone health, authors sought to evaluate the impact of the Biofield Energy Treatment (The Trivedi Effect ® ) on vitamin D 3 as test sample for bone health activity with respect to the assessment of different bone health parameters like ALP, collagen content, and bone mineralization using standard in vitro assays in MG-63 cells.

Cell Culture
Human bone osteosarcoma cell line -MG-63 was used as test system in the present study. The MG-63 cell line was maintained in DMEM growth medium for routine culture supplemented with 10% FBS. Growth conditions were maintained as 37°C, 5% CO 2 and 95% humidity and subcultured by trypsinisation followed by splitting the cell suspension into fresh flasks and supplementing with fresh cell growth medium. Three days before the start of the experiment (i.e., day -3), the growth medium of nearconfluent cells was replaced with fresh phenol-free DMEM, supplemented with 10% charcoal dextran stripped FBS (CD-FBS) and 1% penicillin-streptomycin [33].

Experimental Design
The experimental groups consisted of cells in baseline control, vehicle control groups (0.05% DMSO with Biofield Energy Treated and untreated DMEM), positive control group (rutin hydrate) and experimental test groups. The experimental groups included the combination of the Biofield Energy Treated and untreated vitamin D 3 /DMEM. It consisted of four major treatment groups on specified cells with Untreated-DMEM + Untreated-Test item (UT-TI), UT-DMEM + Biofield Energy Treated test item (BT-TI), BT-DMEM + UT-TI, and BT-DMEM + BT-TI.

Consciousness Energy Healing Treatment Strategies
The test item and DMEM were divided into two parts. One part each of the test item and DMEM was treated with the Biofield Energy by a renowned Biofield Energy Healer (also known as The Trivedi Effect ® ) and coded as the Biofield Energy Treated item, while the second part did not receive any sort of treatment. This Biofield Energy Healing Treatment was provided by Sakina Aleemah Ansari remotely for ~5 minutes. Biofield Energy Healer was remotely located in the USA, while the test samples were located in the research laboratory of Dabur Research Foundation, New Delhi, India. This Biofield Energy Treatment was administered for 5 minutes through the Healer's unique Energy Transmission process remotely to the test samples under laboratory conditions. Sakina Aleemah Ansari in this study never visited the laboratory in person, nor had any contact with the test item and medium. Further, the control group was treated with a sham healer for comparative purposes. The sham healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions for experimental study.

Determination of Non-Cytotoxic Concentration
The cell viability was performed by MTT assay in human bone osteosarcoma cell line (MG-63). The cells were counted and plated in 96 well plates at the density corresponding to 5 X 10 3 to 10 X 10 3 cells/well/180 µL of cell growth medium. The above cells were incubated overnight under growth conditions and allowed the cell recovery and exponential growth, which were subjected to serum stripping or starvation. The cells were treated with the test item, DMEM, and positive control. The untreated cells were served as baseline control. The cells in the above plate (s) were incubated for a time point ranging from 24 to 72 hours in CO 2 incubator at 37°C, 5% CO 2 , and 95% humidity. Following incubation, the plates were taken out and 20 µL of 5 mg/mL of MTT solution were added to all the wells followed by additional incubation for 3 hours at 37°C. The supernatant was aspirated and 150 µL of DMSO was added to each well to dissolve formazan crystals. The absorbance of each well was read at 540 nm using Synergy HT micro plate reader, BioTek, USA [34]. The percentage cytotoxicity at each tested concentrations of the test substance were calculated using the following equation (1): Where, X = Absorbance of treated cells; R = Absorbance of untreated cells The percentage cell viability corresponding to each treatment was obtained using the following equation (2): The concentrations exhibiting ≥70% Cell viability was considered as non-cytotoxic.

Assessment of ALP Activity
The cells were counted using an hemocytometer and plated in a 24-well plate at the density corresponding 1 x 10 4 cells/well in phenol free DMEM supplemented with 10% CD-FBS. Following respective treatments, the cells in the above plate were incubated for 48 hours in CO 2 incubator at 37°C, 5% CO 2 , and 95% humidity. After 48 hours of incubation, the plate was taken out and processed for the measurement of ALP enzyme activity. The cells were washed with 1X PBS and lysed by freeze thaw method i.e., incubation at -80°C for 20 minutes followed by incubation at 37°C for 10 minutes. To the lysed cells, 50 µL of substrate solution i.e., 5 mM of p-nitrophenyl phosphate (pNPP) in 1M diethanolamine and 0.24 mM magnesium chloride (MgCl 2 ) solution (pH 10.4) was added to all the wells followed by incubation for 1 hour at 37°C. The absorbance of the above solution was read at 405 nm using Synergy HT micro plate reader (Biotek, USA). The absorbance values obtained were normalized with substrate blank (pNPP solution alone) absorbance values [33]. The percentage increase in ALP enzyme activity with respect to the untreated cells (baseline group) was calculated using equation (3): Where, X = Absorbance of cells corresponding to positive control and test groups R = Absorbance of cells corresponding to baseline group (untreated cells)

Assessment of Collagen Synthesis
The MG-63 cells were counted using an hemocytometer and plated in 24-well plate at the density corresponding to 10 x 10 3 cells/well in phenol free DMEM supplemented with 10% CD-FBS. Following respective treatments, the cells in the above plate were incubated for 48 hours in CO 2 incubator at 37°C, 5% CO 2 , and 95% humidity. After 48 hours of incubation, the plate was taken out and the amount of collagen accumulated in MG-63 cells corresponding to each treatment was measured by Direct Sirius red dye binding assay. In brief, the cell layers were washed with PBS and fixed in Bouin's solution (5% acetic acid, 9% formaldehyde and 0.9% picric acid) for 1 hours at room temperature (RT). After 1 hour of incubation, the above wells were washed with milliQ water and air dried. The cells were then stained with Sirius red dye solution for 1 hour at RT followed by washing in 0.01 N HCl to remove unbound dye. The collagen dye complex obtained in the above step was dissolved in 0.1 N NaOH and absorbance was read at 540 nm using Biotek Synergy HT micro plate reader. The level of collagen was extrapolated using standard curve obtained from purified Calf Collagen Bornstein and Traub Type I (Sigma Type III) [33]. The percentage increase in collagen level with respect to the untreated cells (baseline group) was calculated using equation (4): Where, X = Collagen levels in cells corresponding to positive control and test groups R = Collagen levels in cells corresponding to baseline group (untreated cells)

Assessment of Bone Mineralization by Alizarin Red S Staining
The MG-63 cells were counted using an hemocytometer and plated in 24-well plate at the density corresponding to 10 x 10 3 cells/well in phenol free DMEM supplemented with 10% CD-FBS. Following respective treatments, the cells in the above plate were incubated for 48 hours in CO 2 incubator at 37°C, 5% CO 2 , and 95% humidity to allow cell recovery and exponential growth. Following overnight incubation, the above cells will be subjected to serum stripping for 24 hours. The cells will be then be treated with non-cytotoxic concentrations of the test samples and positive control. After 3-7 days of incubation with the test samples and positive control, the plates were taken out cell layers and processed further for staining with Alizarin Red S dye. The cells were fixed in 70% ethanol for 1 hour, after which Alizarin Red solution (40 µm; pH 4.2) was added to the samples for 20 minutes with shaking. The cells were washed with distilled water to remove unbound dye. For quantitative analysis by absorbance evaluation, nodules were solubilized with 10% cetylpyridinium chloride for 15 minutes with shaking. Absorbance was measured at 562 nm using Biotek Synergy HT micro plate reader [33]. The percentage increase in bone mineralization with respect to the untreated cells (baseline group) was calculated using the following equation (5): Where, X = Absorbance in cells corresponding to positive control or test groups; R = Absorbance in cells corresponding to baseline (untreated) group.

Statistical Analysis
All the values were represented as percentage of respective parameters. For multiple group comparison, one-way analysis of variance (ANOVA) was used followed by posthoc analysis by Dunnett's test. Statistically significant values were set at the level of p≤0.05.  Figure 1. The percentage of cell viability in different tested groups showed significant improved cell viability. The results showed that both the test samples in combination at tested concentration ranges were found to have significant cell viability with more than 79%. MTT assay for cell viability is one of the primary method to find cell growth, proliferation, and morphological effects. These data suggests that the test item along with DMEM groups were found safe up to maximum of 100 µg/mL against the tested MG-63 cells. Thus, different concentrations i.e. safe concentrations are used to study the bone health parameters such as on the levels of ALP activity, collagen synthesis, and bone mineralization in MG-63 cells.

Study of ALP Enzyme Activity
ALP enzyme activity was evaluated for Biofield Energy Treated test item and DMEM at various concentrations on MG-63 cell line ( Figure 2). The results in terms of percentage ALP were described and compared with respect to the untreated group. The positive control, rutin showed a significant increased values of ALP by 59.53%, 60.38%, and 84.53% with respect to the untreated cells. The experimental test group's viz. untreated medium and Biofield Treated Test item (UT-DMEM+BT-TI) showed a significant increase in the ALP level by 119% at 50 µg/mL, while Biofield Treated medium and untreated Test item (BT-DMEM+UT-TI) showed a significant increased ALP level by 161.5%, 86.4%, and 69.5% at 1, 10, and 50 µg/mL, respectively as compared with the untreated test item and DMEM group. However, the Biofield Energy Treated medium and Biofield Energy Treated Test item (BT-DMEM+BT-TI) showed a significant increased ALP level by 52.5% and 252.3% at 10 and 50 µg/mL, respectively as compared with the untreated test item and DMEM group. Various reports suggested that bone diseases such as post-menopausal women, osteoporosis, bone cancers, Paget's disease of bone, healing fracture, bone growth, acromegaly, myelofibrosis, osteogenic sarcoma, or bone metastases, leukemia, and rarely myeloma were related to ALP abnormalities and can be overcome by the supplementation [35][36][37]. The experimental data suggest that Biofield Energy Healing Treatment in test samples showed a significant improved level of the ALP, which could be the best supplementation to treat various bone related diseases such as osteoporosis, in middle and old-aged, post-and menopausal women [36]. Thus, overall data concluded that The Trivedi Effect ® -Energy of Consciousness Healing based vit D 3 and DMEM could be used to improve the ALP concentration in many bone disorders.

Estimation of Collagen Synthesis
Biofield Energy Treated vit D 3 and DMEM were estimated for the level of collagen among various tested concentrations. The results of collagen are presented as % values with respect to the untreated cells in Figure 3. The rutin hydrate showed a significant increased value of collagen by 40.55%, 45.70%, and 58.59% at 0.01, 0.1, and 1 µg/mL, respectively. Besides, the experimental test groups such as UT-DMEM+BT-TI showed a significant increased collagen level by 65.8%, 70.8%, and 81.0 % at 0.1, 1, and 10 µg/mL, respectively while BT-DMEM+UT-TI group showed a significant increased collagen level by 86.1%, 117.9%, and 11.6% at 0.1, 1, and 10 µg/mL, respectively as compared with the untreated test item and DMEM group. However, BT-DMEM+BT-TI group showed a significant increased collagen level by 134.2%, 117.0% and 95.9% at 0.1, 1, and 10 µg/mL, respectively as compared with the untreated test item and DMEM group. Collagen in various forms has been reported with increased bone mass and play important role against many joint diseases. Collagen, a therapeutic agent of potential utility that is mostly utilized in the treatment of bone diseases such as osteoarthritis and osteoporosis. Besides, collagen supplements were reported to have high level of safety for long-term use in these chronic bone disorders. With increase in age, the level of collagen production decreases that can be maintained using collagen supple1mentation and with other nutritional factors [38,39].
However, the present experimentation suggested that the Biofield Energy Treated vit D 3 and DMEM groups showed a significant improved level of collagen compared with the untreated group. Biofield Energy Treated vit D 3

(The Trivedi
Effect ® ) would be used to improve the collagen level for bone health, which can be used to decrease aging process and inflammation.

Study of Bone Mineralization
Bone mineralization play an important role in the treatment of osteoporosis or other bone diseases. Loss in bone mass results due to the decreased bone mineralization capacity, and poor calcium absorption in bones, which leads to reduced bone mineral density (BMD) and various structural abnormalities [40,41]. Biofield Energy Treated vit D3 and DMEM groups showed a significant improved bone mineralization on MG-63 cell line. The results are presented in term of percentage change of bone mineralization among different experimental groups in Figure 4. The positive control, rutin group showed a significant increased value of bone mineralization by 47.98%, 59.73%, and 139.02% at 5, 10, and 25 µg/mL, respectively. The experimental data among test group's viz. UT-DMEM+BT-TI showed a significant increased bone mineralization by 235.2% at 100 µg/mL, while BT-DMEM+UT-TI group showed a significantly increased bone mineralization by 152.7%, 92.6%, and 17% at 0.1, 1, and 100 µg/mL, respectively as compared with the untreated test item and DMEM group. However, BT-DMEM+BT-TI group showed a significant increased bone mineralization by 158% and 16.4% at 1 and 100 µg/mL, respectively as compared with the untreated test item and DMEM group. The experimental test groups showed that Biofield Energy Healing Treatment significantly improved the rate of bone mineralization compared with the untreated groups, which can be used in various bone related disorders and recovery process.

Conclusions
The experimental test groups showed a significant cell viability using MTT assay with more than 79% among the tested groups, which suggest that test samples are safe and nontoxic. Bone health parameters such as ALP level was increased in the Biofield Energy Treated test by 119% at 50 µg/mL in UT-DMEM+BT-TI, while 161.5%, 86.4%, and 69.5% at 1, 10, and 50 µg/mL, respectively, in the BT-DMEM+UT-TI group as compared with the untreated test item and DMEM group. In addition, BT-DMEM+BT-TI group showed a significant increased ALP level by 52.5% and 252.3% at 10 and 50 µg/mL, respectively as compared with the untreated group. The level of collagen was significantly increased by 65.8%, 70.8%, and 81.0 % at 0.1, 1, and 10 µg/mL, respectively in the UT-DMEM+BT-TI, while 86.1%, 117.9%, and 11.6% at 0.1, 1, and 10 µg/mL, respectively in the BT-DMEM+UT-TI group. In addition, 134.2%, 117.0%, and 95.9% increased collagen was reported at 0.1, 1, and 10 µg/mL, respectively as compared with the untreated test item and DMEM group.
Similarly, the bone mineralization percent was significantly increased by 235.2% at 100 µg/mL in the UT-DMEM+BT-TI group, while 152.7%, 92.6%, and 17% at 0.1, 1, and 100 µg/mL, respectively in the BT-DMEM+UT-TI group as compared with the untreated group. In addition, BT-DMEM+BT-TI group showed a significant increased bone mineralization by 158% and 16.4% at 1 and 100 µg/mL, respectively as compared with the untreated group. Overall, the Biofield Energy Treated (The Trivedi Effect ® ) test samples were found to have a significant impact on tested bone health parameters viz. collagen, bone mineralization, and ALP, which are very vital to combat the bone disorders. Therefore, the Consciousness Energy Healing based vitamin D 3 might be a suitable alternative nutritional supplement, which could be useful for the management of various bone related disorders viz. osteoporosis, Paget's disease of bone, rickets, deformed bones, osteomalacia, bone and/or joint pain, increased frequency of fractures, osteoma, hormonal imbalance, stress, aging, bone loss and fractures, and other bone diseases that are caused by poor nutrition, genetics, or problems with the rate of bone growth or rebuilding. Biofield Energy Treated Vitamin D 3 can be useful as antiinflammatory, anti-aging, anti-stress, anti-arthritic, antiosteoporosis, anti-cancer, anti-apoptotic, wound healing, antipsychotic and anti-fibrotic roles. It also influence cell-to-cell communication, normal cell growth, cell differentiation, neurotransmission, cell cycling and proliferation, hormonal balance, skin health, immune and cardiovascular functions. Besides, it can also be utilized in organ transplants (for example kidney transplants, liver transplants and heart transplants), hormonal imbalance, aging, and various immune related disease conditions such as Ulcerative Colitis, Alzheimer's Disease, Dermatitis, Irritable Bowel Syndrome, Asthma, Hashimoto Thyroiditis, Pernicious Anemia, Sjogren Syndrome, Multiple Sclerosis, Aplastic Anemia, Hepatitis, Diverticulitis, Graves' Disease, Dermatomyositis, Diabetes, Myasthenia Gravis, Parkinson's Disease, Atherosclerosis, Systemic Lupus Erythematosus, stress, etc. with a safe therapeutic index to improve overall health, and quality of life.