Biomedical Sciences 2018; 4(1): 10-17 http://www.sciencepublishinggroup.com/j/bs doi: 10.11648/j.bs.20180401.12 ISSN: 2575-3924 (Print); ISSN: 2575-3932 (Online) In Vitro Effects of Biofield Energy Treated Vitamin D 3 Supplementation on Bone Formation by Osteoblasts Cells Sakina Aleemah Ansari1, Mahendra Kumar Trivedi1, Alice Branton1, Dahryn Trivedi1, Gopal Nayak1, Mayank Gangwar2, Snehasis Jana2, * 1Trivedi Global, Inc., Henderson, USA 2Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, India Email address: *Corresponding author To cite this article: Sakina Aleemah Ansari, Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Mayank Gangwar, Snehasis Jana. In Vitro Effects of Biofield Energy Treated Vitamin D Supplementation on Bone Formation by Osteoblasts Cells. Biomedical Sciences. 3 Vol. 4, No. 1, 2018, pp. 10-17. doi: 10.11648/j.bs.20180401.12 Received: December 26, 2017; Accepted: January 26, 2018; Published: February 8, 2018 Abstract: 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 D and DMEM medium on various bone health parameters such as alkaline phosphatase 3 enzyme (ALP) activity, collagen levels and bone mineralization. Both the test items (TI) i.e. vitamin D and DMEM medium 3 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 D and DMEM would play an important role in the 3 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. Keywords: Biofield Energy, Osteosarcoma Cells, Vitamin D, Osteoporosis, Bone Disorders, Bone Mineralization apoptotic, wound healing, anti-cancer, anti-psychotic, and 1. Introduction anti-fibrotic roles. Vitamin D receptors (VDRs) are widely present in most of the body organs like brain, heart, lungs, Vitamin D has multiple effects which regulate the kidney, liver, pancreas, large and small intestines, muscles, functions in different organs such as brain, lungs, liver, reproductive, nervous system, etc. [1]. VDRs influence cell- kidneys, and heart, immune, skeletal, and reproductive to-cell communication, normal cell growth, cell systems. Moreover, it has significant anti-inflammatory, anti- differentiation, cell cycling and proliferation, hormonal arthritic, anti-osteoporosis, anti-stress, anti-aging, anti- balance, neurotransmission, skin health, immune and Biomedical Sciences 2018; 4(1): 10-17 11 cardiovascular functions. Bone-related health issues become arrays of an organic matrix known as Osteoid [9]. Likewise, a major problem among the population from village to the calcium phosphate is deposited in the Osteoid and gets cities. Vitamin D plays a vital role in preserving a healthy mineralized (combination of calcium phosphate and mineralized skeleton of most of the vertebrates including hydroxyapatite) and provides rigidity to the bone [10]. Thus, humans. Cod liver oil, irradiation of other foods including these parameters are very essential in order to study the bone plants, sunlight, etc. are found to be effective against bone health in cell lines. Authors evaluated the in vitro effect of related disorders, which lead to discovering the active the Biofield Energy Treated vitamin D as a test item, a 3 principle- vitamin D [1]. The role of vitamin D has been well Complementary and Alternative Medicine (CAM) on bone defined not only for improving the bone mineralization but health using MG-63 cell line for major biomarkers. also with increased bone resorption, aging, inflammation and Within the burgeoning ground of CAM therapies, Biofield overall quality of life. Vitamin D is synthesized in the skin Energy Treatment or energy medicine, is emerging with 3 by sunlight and once formed it sequentially metabolized in significant benefits in various scientific fields. The effects of the liver and kidney to 1,25-dihydroxyvitamin D (calcitriol, the CAM therapies have great potential, which include the vitamin D hormone) [2]. Calcitriol play an important role external qigong, Johrei, Reiki, therapeutic touch, yoga, Qi in maintaining the normal level of calcium and phosphorus, Gong, polarity therapy, Tai Chi, pranic healing, deep promotes bone mineralization, induce or repress the genes breathing, chiropractic/osteopathic manipulation, guided responsible for conserving the mineral homeostasis and imagery, meditation, massage, homeopathy, hypnotherapy, skeletal integrity, and inhibit hypertension, kidney damage, progressive relaxation, acupressure, acupuncture, special cardiovascular and immune disorders (such as Lupus, diets, relaxation techniques, Rolfing structural integration, Addison Disease, Graves’ Disease, Hashimoto Thyroiditis, healing touch, movement therapy, pilates, mindfulness, Multiple Sclerosis, Myasthenia Gravis, Anemia, Sjogren Ayurvedic medicine, traditional Chinese herbs and medicines Syndrome, Systemic Lupus Erythematosus, Diabetes, in biological systems both in vitro and in vivo [11]. Biofield Alopecia Areata, Fibromyalgia, Vitiligo, Psoriasis, Energy Healing Treatment (The Trivedi Effect®) contain a Scleroderma, Chronic Fatigue Syndrome and Vasculitis), and putative bioenergy, which is channeled by a renowned the secondary hyperparathyroidism [3]. Vitamin D practitioners from a distance. Biofield Energy Healing as a insufficiency and deficiency is the major health problem, CAM showed a significant results in biological studies [12]. which causes metabolic bone disease in the young and However, the National Center for Complementary and elderly populations [4]. Fortified foods have a variable Alternative Medicine (NCCAM), well-defined Biofield amount of vitamin D and most of the foods do not contain therapies in the subcategory of Energy Therapies [13]. The vitamin D, which can be fulfilled using some supplements. In Trivedi Effect®- Consciousness Energy Healing Treatment order to avoid the bone related disorders such as has been reported with significant revolution in the osteomalacia, exacerbate osteoporosis, hyperparathyroidism, physicochemical properties of metals, chemicals, ceramics immune disorders, etc. calcium 1000-1500 mg/day along and polymers [14-16], improved agricultural crop yield, with vitamin D supplement around 400 IU/day is very productivity, and quality [17, 18], transformed antimicrobial important for maintaining the good bone health [5]. characteristics [19-21], biotechnology [22-23], improved Various in vitro studies have readily demonstrated the role bioavailability [24-26], skin health [27, 28], nutraceuticals of bone health using cell lines and its resorbing effects using [29, 30], cancer research [31, 32], and human health and three important key biomarkers, such as ALP, collagen and wellness. calcium. MG-63 cell line derived from juxtacortical Based on the significant outcomes of Biofield Energy osteosarcoma, which represents an immature osteoblast Treatment and vital role of vitamin D on bone health, 3 phenotype and undergoes temporal development in long term authors sought to evaluate the impact of the Biofield Energy culture. The response of MG-63 cells to 1,25- Treatment (The Trivedi Effect®) on vitamin D as test sample 3 dihydroxyvitamin D (1,25 (OH) 2D ) administration has for bone health activity with respect to the assessment of 3 3 been studied to be similar to normal human osteoblast cells different bone health parameters like ALP, collagen content, [6]. Hence, MG-63 cell line is widely used for studying the and bone mineralization using standard in vitro assays in potential of any test compounds to improve the bone health MG-63 cells. [7]. The formation of new bone involves a complex series of events including the proliferation and differentiation of 2. Material and Methods osteoblasts, and eventually the formation of a mineralized extracellular matrix. ALP is a phenotypic marker for the 2.1. Chemicals and Reagents early differentiation and maturation of osteoblasts. ALP Rutin hydrate was purchased from TCI, Japan, while increases the local concentration of inorganic phosphate for vitamin D (denoted as test item) and L-ascorbic acid were bone mineralization and hence is an important marker for 3 obtained from Sigma-Aldrich, USA. Fetal bovine serum osteogenic activity [8]. Similarly, active osteoblasts (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) synthesize and extrude collagen, which plays an important were purchased from Life Technology, USA. Antibiotics role in the formation of bone extracellular matrix by solution (penicillin-streptomycin) was procured from providing strength and flexibility. Collagen fibrils formed an 12 Sakina Aleemah Ansari et al.: In Vitro Effects of Biofield Energy Treated Vitamin D Supplementation on 3 Bone Formation by Osteoblasts Cells HiMedia, India, while 3-(4, 5-diamethyl-2-thiazolyl)-2, 5- 2.5. Determination of Non-Cytotoxic Concentration diphenyl-2H-tetrazolium) (MTT), Direct Red 80, and The cell viability was performed by MTT assay in human ethylene diamine tetra acetic acid (EDTA) were purchased bone osteosarcoma cell line (MG-63). The cells were counted from Sigma, USA. All the other chemicals used in this and plated in 96 well plates at the density corresponding to 5 experiment were analytical grade procured from India. X 103 to 10 X 103 cells/well/180 µL of cell growth medium. 2.2. Cell Culture The above cells were incubated overnight under growth conditions and allowed the cell recovery and exponential Human bone osteosarcoma cell line -MG-63 was used as growth, which were subjected to serum stripping or test system in the present study. The MG-63 cell line was starvation. The cells were treated with the test item, DMEM, maintained in DMEM growth medium for routine culture and positive control. The untreated cells were served as supplemented with 10% FBS. Growth conditions were baseline control. The cells in the above plate (s) were maintained as 37°C, 5% CO and 95% humidity and 2 incubated for a time point ranging from 24 to 72 hours in subcultured by trypsinisation followed by splitting the cell CO incubator at 37°C, 5% CO , and 95% humidity. 2 2 suspension into fresh flasks and supplementing with fresh Following incubation, the plates were taken out and 20 µL of cell growth medium. Three days before the start of the 5 mg/mL of MTT solution were added to all the wells experiment (i.e., day -3), the growth medium of near- followed by additional incubation for 3 hours at 37°C. The confluent cells was replaced with fresh phenol-free DMEM, supernatant was aspirated and 150 µL of DMSO was added supplemented with 10% charcoal dextran stripped FBS (CD- to each well to dissolve formazan crystals. The absorbance of FBS) and 1% penicillin-streptomycin [33]. each well was read at 540 nm using Synergy HT micro plate reader, BioTek, USA [34]. The percentage cytotoxicity at 2.3. Experimental Design each tested concentrations of the test substance were The experimental groups consisted of cells in baseline calculated using the following equation (1): control, vehicle control groups (0.05% DMSO with Biofield % Cytotoxicity = (1-X/R)*100 (1) Energy Treated and untreated DMEM), positive control group (rutin hydrate) and experimental test groups. The Where, X = Absorbance of treated cells; R = Absorbance experimental groups included the combination of the Biofield of untreated cells Energy Treated and untreated vitamin D /DMEM. It 3 The percentage cell viability corresponding to each consisted of four major treatment groups on specified cells treatment was obtained using the following equation (2): with Untreated-DMEM + Untreated-Test item (UT-TI), UT- DMEM + Biofield Energy Treated test item (BT-TI), BT- % Cell Viability = 100 - % Cytotoxicity (2) DMEM + UT-TI, and BT-DMEM + BT-TI. The concentrations exhibiting ≥70% Cell viability was 2.4. Consciousness Energy Healing Treatment Strategies considered as non-cytotoxic. The test item and DMEM were divided into two parts. 2.6. Assessment of ALP Activity One part each of the test item and DMEM was treated The cells were counted using an hemocytometer and plated with the Biofield Energy by a renowned Biofield Energy in a 24-well plate at the density corresponding 1 x 104 Healer (also known as The Trivedi Effect®) and coded as cells/well in phenol free DMEM supplemented with 10% the Biofield Energy Treated item, while the second part CD-FBS. Following respective treatments, the cells in the did not receive any sort of treatment. This Biofield Energy above plate were incubated for 48 hours in CO incubator at Healing Treatment was provided by Sakina Aleemah 2 37°C, 5% CO , and 95% humidity. After 48 hours of Ansari remotely for ~5 minutes. Biofield Energy Healer 2 incubation, the plate was taken out and processed for the was remotely located in the USA, while the test samples measurement of ALP enzyme activity. The cells were washed were located in the research laboratory of Dabur Research with 1X PBS and lysed by freeze thaw method i.e., Foundation, New Delhi, India. This Biofield Energy incubation at -80°C for 20 minutes followed by incubation at Treatment was administered for 5 minutes through the 37°C for 10 minutes. To the lysed cells, 50 µL of substrate Healer’s unique Energy Transmission process remotely to solution i.e., 5 mM of p-nitrophenyl phosphate (pNPP) in 1M the test samples under laboratory conditions. Sakina diethanolamine and 0.24 mM magnesium chloride (MgCl ) Aleemah Ansari in this study never visited the laboratory 2 solution (pH 10.4) was added to all the wells followed by in person, nor had any contact with the test item and incubation for 1 hour at 37°C. The absorbance of the above medium. Further, the control group was treated with a solution was read at 405 nm using Synergy HT micro plate sham healer for comparative purposes. The sham healer reader (Biotek, USA). The absorbance values obtained were did not have any knowledge about the Biofield Energy normalized with substrate blank (pNPP solution alone) Treatment. After that, the Biofield Energy Treated and absorbance values [33]. The percentage increase in ALP untreated samples were kept in similar sealed conditions enzyme activity with respect to the untreated cells (baseline for experimental study. group) was calculated using equation (3): Biomedical Sciences 2018; 4(1): 10-17 13 % Increase = [(X-R)/R)]*100 (3) 2.8. Assessment of Bone Mineralization by Alizarin Red S Staining Where, X = Absorbance of cells corresponding to positive control and test groups The MG-63 cells were counted using an hemocytometer R = Absorbance of cells corresponding to baseline group and plated in 24-well plate at the density corresponding to 10 (untreated cells) x 103 cells/well in phenol free DMEM supplemented with 10% CD-FBS. Following respective treatments, the cells in 2.7. Assessment of Collagen Synthesis the above plate were incubated for 48 hours in CO incubator 2 at 37°C, 5% CO , and 95% humidity to allow cell recovery The MG-63 cells were counted using an hemocytometer 2 and exponential growth. Following overnight incubation, the and plated in 24-well plate at the density corresponding to 10 x 103 cells/well in phenol free DMEM supplemented with above cells will be subjected to serum stripping for 24 hours. The cells will be then be treated with non-cytotoxic 10% CD-FBS. Following respective treatments, the cells in concentrations of the test samples and positive control. After the above plate were incubated for 48 hours in CO incubator 2 3-7 days of incubation with the test samples and positive at 37°C, 5% CO , and 95% humidity. After 48 hours of 2 control, the plates were taken out cell layers and processed incubation, the plate was taken out and the amount of further for staining with Alizarin Red S dye. The cells were collagen accumulated in MG-63 cells corresponding to each fixed in 70% ethanol for 1 hour, after which Alizarin Red treatment was measured by Direct Sirius red dye binding solution (40 µm; pH 4.2) was added to the samples for 20 assay. In brief, the cell layers were washed with PBS and minutes with shaking. The cells were washed with distilled fixed in Bouin’s solution (5% acetic acid, 9% formaldehyde water to remove unbound dye. For quantitative analysis by and 0.9% picric acid) for 1 hours at room temperature (RT). absorbance evaluation, nodules were solubilized with 10% After 1 hour of incubation, the above wells were washed with cetylpyridinium chloride for 15 minutes with shaking. milliQ water and air dried. The cells were then stained with Absorbance was measured at 562 nm using Biotek Synergy Sirius red dye solution for 1 hour at RT followed by washing HT micro plate reader [33]. The percentage increase in bone in 0.01 N HCl to remove unbound dye. The collagen dye mineralization with respect to the untreated cells (baseline complex obtained in the above step was dissolved in 0.1 N group) was calculated using the following equation (5): NaOH and absorbance was read at 540 nm using Biotek Synergy HT micro plate reader. The level of collagen was % Increase = [(X-R)/R]*100 (5) extrapolated using standard curve obtained from purified Calf Collagen Bornstein and Traub Type I (Sigma Type III) Where, X = Absorbance in cells corresponding to positive [33]. The percentage increase in collagen level with respect control or test groups; R = Absorbance in cells corresponding to the untreated cells (baseline group) was calculated using to baseline (untreated) group. equation (4): 2.9. Statistical Analysis % Increase = [(X-R)/R]*100 (4) All the values were represented as percentage of respective Where, X = Collagen levels in cells corresponding to parameters. For multiple group comparison, one-way positive control and test groups analysis of variance (ANOVA) was used followed by post- R = Collagen levels in cells corresponding to baseline hoc analysis by Dunnett’s test. Statistically significant values group (untreated cells) were set at the level of p≤0.05. 3. Results and Discussion 3.1. Cell Viability Study Using MTT Figure 1. MTT assays of the test formulations on MG-63 cell line after 72 hours. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. 14 Sakina Aleemah Ansari et al.: In Vitro Effects of Biofield Energy Treated Vitamin D Supplementation on 3 Bone Formation by Osteoblasts Cells MTT assay was done to test the cell viability of the item (UT-DMEM+BT-TI) showed a significant increase in Biofield Energy Treated test samples (vitamin D and the ALP level by 119% at 50 µg/mL, while Biofield Treated 3 DMEM medium) in MG-63 cells, which is presented in medium and untreated Test item (BT-DMEM+UT-TI) Figure 1. The percentage of cell viability in different tested showed a significant increased ALP level by 161.5%, 86.4%, groups showed significant improved cell viability. The results and 69.5% at 1, 10, and 50 µg/mL, respectively as compared showed that both the test samples in combination at tested with the untreated test item and DMEM group. However, the concentration ranges were found to have significant cell Biofield Energy Treated medium and Biofield Energy viability with more than 79%. MTT assay for cell viability is Treated Test item (BT-DMEM+BT-TI) showed a significant one of the primary method to find cell growth, proliferation, increased ALP level by 52.5% and 252.3% at 10 and 50 and morphological effects. These data suggests that the test µg/mL, respectively as compared with the untreated test item item along with DMEM groups were found safe up to and DMEM group. Various reports suggested that bone maximum of 100 µg/mL against the tested MG-63 cells. diseases such as post-menopausal women, osteoporosis, bone Thus, different concentrations i.e. safe concentrations are cancers, Paget’s disease of bone, healing fracture, bone used to study the bone health parameters such as on the growth, acromegaly, myelofibrosis, osteogenic sarcoma, or levels of ALP activity, collagen synthesis, and bone bone metastases, leukemia, and rarely myeloma were related mineralization in MG-63 cells. to ALP abnormalities and can be overcome by the supplementation [35-37]. The experimental data suggest that 3.2. Study of ALP Enzyme Activity Biofield Energy Healing Treatment in test samples showed a significant improved level of the ALP, which could be the ALP enzyme activity was evaluated for Biofield Energy best supplementation to treat various bone related diseases Treated test item and DMEM at various concentrations on such as osteoporosis, in middle and old-aged, post- and MG-63 cell line (Figure 2). The results in terms of menopausal women [36]. Thus, overall data concluded that percentage ALP were described and compared with respect The Trivedi Effect®-Energy of Consciousness Healing based to the untreated group. The positive control, rutin showed a vit D and DMEM could be used to improve the ALP significant increased values of ALP by 59.53%, 60.38%, and 3 concentration in many bone disorders. 84.53% with respect to the untreated cells. The experimental test group’s viz. untreated medium and Biofield Treated Test Figure 2. Estimation of ALP enzyme activity of the Biofield Energy Treated test items on MG-63 cell line. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. 3.3. Estimation of Collagen Synthesis with the untreated test item and DMEM group. However, BT-DMEM+BT-TI group showed a significant increased Biofield Energy Treated vit D and DMEM were estimated 3 collagen level by 134.2%, 117.0% and 95.9% at 0.1, 1, and for the level of collagen among various tested concentrations. 10 µg/mL, respectively as compared with the untreated test The results of collagen are presented as % values with item and DMEM group. Collagen in various forms has been respect to the untreated cells in Figure 3. The rutin hydrate reported with increased bone mass and play important role showed a significant increased value of collagen by 40.55%, against many joint diseases. Collagen, a therapeutic agent of 45.70%, and 58.59% at 0.01, 0.1, and 1 µg/mL, respectively. potential utility that is mostly utilized in the treatment of Besides, the experimental test groups such as UT- bone diseases such as osteoarthritis and osteoporosis. DMEM+BT-TI showed a significant increased collagen level Besides, collagen supplements were reported to have high by 65.8%, 70.8%, and 81.0 % at 0.1, 1, and 10 µg/mL, level of safety for long-term use in these chronic bone respectively while BT-DMEM+UT-TI group showed a disorders. With increase in age, the level of collagen significant increased collagen level by 86.1%, 117.9%, and production decreases that can be maintained using collagen 11.6% at 0.1, 1, and 10 µg/mL, respectively as compared supple1mentation and with other nutritional factors [38, 39]. Biomedical Sciences 2018; 4(1): 10-17 15 However, the present experimentation suggested that the Effect®) would be used to improve the collagen level for Biofield Energy Treated vit D and DMEM groups showed a bone health, which can be used to decrease aging process and 3 significant improved level of collagen compared with the inflammation. untreated group. Biofield Energy Treated vit D (The Trivedi 3 Figure 3. Effect of the test item on MG-63 cell line for collagen level. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. 3.4. Study of Bone Mineralization among test group’s viz. UT-DMEM+BT-TI showed a significant increased bone mineralization by 235.2% at 100 Bone mineralization play an important role in the µg/mL, while BT-DMEM+UT-TI group showed a treatment of osteoporosis or other bone diseases. Loss in significantly increased bone mineralization by 152.7%, bone mass results due to the decreased bone mineralization 92.6%, and 17% at 0.1, 1, and 100 µg/mL, respectively as capacity, and poor calcium absorption in bones, which leads compared with the untreated test item and DMEM group. to reduced bone mineral density (BMD) and various However, BT-DMEM+BT-TI group showed a significant structural abnormalities [40, 41]. Biofield Energy Treated vit increased bone mineralization by 158% and 16.4% at 1 and D3 and DMEM groups showed a significant improved bone 100 µg/mL, respectively as compared with the untreated test mineralization on MG-63 cell line. The results are presented item and DMEM group. The experimental test groups in term of percentage change of bone mineralization among showed that Biofield Energy Healing Treatment significantly different experimental groups in Figure 4. The positive improved the rate of bone mineralization compared with the control, rutin group showed a significant increased value of untreated groups, which can be used in various bone related bone mineralization by 47.98%, 59.73%, and 139.02% at 5, disorders and recovery process. 10, and 25 µg/mL, respectively. The experimental data Figure 4. Effect of the test item on MG-63 cell line for bone mineralization. VC: Vehicle control (DMSO-0.05%), UT: Untreated; BT: Biofield Treated; TI: Test Item. increased in the Biofield Energy Treated test by 119% at 50 4. Conclusions 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- The experimental test groups showed a significant cell DMEM+UT-TI group as compared with the untreated test viability using MTT assay with more than 79% among the item and DMEM group. In addition, BT-DMEM+BT-TI tested groups, which suggest that test samples are safe and group showed a significant increased ALP level by 52.5% nontoxic. Bone health parameters such as ALP level was 16 Sakina Aleemah Ansari et al.: In Vitro Effects of Biofield Energy Treated Vitamin D Supplementation on 3 Bone Formation by Osteoblasts Cells and 252.3% at 10 and 50 µg/mL, respectively as compared Fetal bovine serum; EDTA: Ethylene Diamine Tetra Acetic with the untreated group. The level of collagen was Acid, UT: Untreated, BT: Biofield Energy Treated, TI: Test significantly increased by 65.8%, 70.8%, and 81.0 % at 0.1, Item. 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, Acknowledgements respectively in the BT-DMEM+UT-TI group. In addition, 134.2%, 117.0%, and 95.9% increased collagen was reported Authors are grateful to Dabur Research Foundation, at 0.1, 1, and 10 µg/mL, respectively as compared with the Trivedi Global, Inc., Trivedi Science, Trivedi Testimonials, untreated test item and DMEM group. and Trivedi Master Wellness for their support throughout the Similarly, the bone mineralization percent was work. 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 References group as compared with the untreated group. In addition, BT- DMEM+BT-TI group showed a significant increased bone [1] Holick MF (1996) Vitamin D and bone health. J Nutr 126: 1159S-64S. mineralization by 158% and 16.4% at 1 and 100 µg/mL, respectively as compared with the untreated group. 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